Complex coacervation of β-lactoglobulin – κ-Carrageenan aqueous mixtures as affected by polysaccharide sonication

Accepted Manuscript Complex coacervation of β-lactoglobulin – κ –carrageenan aqueous mixtures as affected by polysaccharide sonication Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed Hadi Razavi, Ali Akbar Moosavi-Movahedi, Ali Akbar Saboury, Mohammad Amin Mohammadifar, Asgar Farahnaky, Maliheh Sadat Atri, Paul Van der Meeren PII: DOI: Reference:

S0308-8146(13)00253-7 http://dx.doi.org/10.1016/j.foodchem.2013.02.090 FOCH 13767

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

20 November 2012 16 February 2013 20 February 2013

Please cite this article as: Hosseini, S.M.H., Emam-Djomeh, Z., Razavi, S.H., Moosavi-Movahedi, A.A., Akbar Saboury, A., Mohammadifar, M.A., Farahnaky, A., Atri, M.S., Van der Meeren, P., Complex coacervation of βlactoglobulin – κ –carrageenan aqueous mixtures as affected by polysaccharide sonication, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/j.foodchem.2013.02.090

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

1 

Complex coacervation of β-lactoglobulin – κ–carrageenan aqueous

2 

mixtures as affected by polysaccharide sonication

3 

Seyed Mohammad Hashem Hosseinia,e , Zahra Emam-Djomeha,*,

4 

Seyed Hadi Razavia, Ali Akbar Moosavi-Movahedib, Ali

5 

Akbar Sabouryb, Mohammad Amin Mohammadifarc, Asgar

6 

Farahnakyd, Maliheh Sadat Atrie, Paul Van der Meerenf

7 

a

Department of Food Science, Technology and Engineering, Faculty of

8 

Agricultural Engineering and Technology, Agricultural Campus of the

9 

University of Tehran, Karadj, Iran, Postal Code: 31587-11167, P. O.

10  11  12  13 

Box: 4111 b

Institute of Biochemistry and Biophysics (IBB), University of Tehran,

Tehran, Iran c

Department of Food Science and Technology, Faculty of Nutrition

14 

Sciences, Food Science and Technology/National Nutrition and Food

15 

Technology Research Institute, Shahid Beheshti University of Medical

16 

Sciences, Tehran, Iran, P. O. Box: 19395-4741

17  18 

d

Department of Food Science and Technology, School of Agriculture,

Shiraz University, Shiraz, Iran

                                                             *  Corresponding author. Tel.: +98 26 32248804; fax: +98 26 32249453

E-mail address: [email protected] (Z. Emam-Djomeh).  1   

19  20  21 

e

Molecular and Cell Biology Department, University of Mazandaran,

Babolsar, Iran f

Particle and Interfacial Technology Group, Faculty of Bioscience

22 

Engineering, Ghent University, Coupure Links 653, B-9000 Gent,

23 

Belgium

2   

24  25 

ABSTRACT

26 

The influence of κ-carrageenan (KC) depolymerization using

27 

ultrasound on its interaction with β-lactoglobulin (BLG) was investigated

28 

by isothermal titration calorimetry (ITC), turbidity measurement,

29 

dynamic light scattering and zeta-potential analyses. Time and

30 

amplitude of the sonication had a direct effect on the viscosity

31 

depression, while the sonication temperature had an opposite effect.

32 

ITC measurements indicated that the sonication significantly decreased

33 

the affinity constant between KC and BLG. The zeta-potential of the

34 

nanoparticles produced from ultrasonicated (US) KC-BLG associative

35 

interaction was lower than of those produced from intact (IN) KC-BLG

36 

interaction. These differences were attributed to the lower charge

37 

density of the KC (US) as a result of sonochemical interactions.

38 

Polydispersity and particle size measurements showed that the effect of

39 

the sonication was the homogenization of the nanoparticles in the mixed

40 

dispersion. The nanoparticles formed may therefore be useful as a

41 

delivery system for fortification purposes of acidic beverages.

42  43 

Keywords: Coacervation; κ–carrageenan; β-lactoglobulin; Ultrasound;

44 

Nanoparticle; Isothermal titration calorimetry

3   

45  46 

1. Introduction Carrageenans are a family of sulfated linear polysaccharides of D-

47  48 

galactose and 3,6-anhydro-D-galactose which are isolated from red

49 

algae (Gu, Decker, & McClements, 2005). They are widely used as a

50 

thickening, gelling and stabilizing agent as well as fat substitutes in the

51 

food industry, particularly in milk products (Weinbreck, Nieuwenhuijse,

52 

Robijn, & de Kruif, 2004). There are three major types of carrageenan

53 

including kappa (κ), iota (ι), and lambda (λ) -carrageenans, which differ

54 

in the number of the sulphate groups (1, 2 and 3, respectively) and their

55 

position (Gu et al., 2005). κ- and ι-carrageenan in aqueous solution

56 

undergo a thermoreversible conformational ordering (transition from coil

57 

(unstructured) at elevated temperatures to helix (ordered) at low

58 

temperatures followed by aggregation and network formation at high

59 

polysaccharide concentration (i.e. 1%)) through sulfate groups and the

60 

3,6-anhydro-D-galactopyransyl ring (Ould Eleya & Turgeon, 2000;

61 

Uruakpa & Arntfield, 2004; Gu et al., 2005). κ and ι -carrageenans have

62 

also gelling properties in the presence of cations, which is influenced by

63 

the nature (i.e. K+ and Ca2+, respectively) and concentration of cations

64 

present in the solution and by the biopolymer concentration (Uruakpa et

65 

al., 2004; Gu et al., 2005). λ-carrageenan has a random coil

66 

conformation at all temperatures and is unable to form gels (Gu et al.,

67 

2005).

4   

Protein-polyelectrolyte (DNA) complexes play important roles in

68  69 

living structures (Burova, Grinberg, Grinberg, Usov, Tolstoguzov, & de

70 

Kruif, 2007). The considerable interest in biopolymer particles

71 

(Klemmer, Waldner, Stone, Low, & Nickerson, 2012; Huang, Sun, Xiao,

72 

& Yang, 2012) results from the potential applications of engineered

73 

novel structures in the protection of bioactive compounds (Jun-xia, Hai-

74 

yan, & Jian, 2011), interfacial stabilization (Schmitt, da Silva, Bovay,

75 

Rami-Shojaei, Frossard, Kolodziejczyk, & Leser, 2005; Dickinson, 2008)

76 

and texturizing such as fat replacing by simulating the rheological,

77 

optical and sensorial properties of the lipid droplets (Laneuville, Paquin,

78 

& Turgeon, 2005). The phase separation of a protein and

79 

polysaccharide aqueous mixture can be classified into two main

80 

categories: associative and segregative phase separation. In an

81 

associative phase separation (also known as thermodynamic

82 

compatibility), both biopolymers are enriched in one of the separating

83 

phases (coacervate-rich phase), while the other phase contains mostly

84 

solvent (Turgeon & Laneuville, 2009). Associative phase separation is

85 

mainly driven by the electrostatic attraction between polyelectrolytes

86 

under conditions where they have opposite electrical charges (i.e. pHs

87 

between the pKa of the polysaccharide and the isoelectric point (Ip) of

88 

the protein) (Turgeon et al., 2009; Chang, McLandsborough, &

89 

McClements, 2011). Other non-covalent interactions can also occur

90 

such as hydrophobic interaction and hydrogen bonding, making the

5   

91 

complexes more stable (Klemmer et al., 2012). In a segregative phase

92 

separation (also known as thermodynamic incompatibility), two

93 

biopolymers are separated into two different phases. This is the case

94 

mainly for two nonionic biopolymers, two similarly charged biopolymers,

95 

or a charged biopolymer plus a nonionic biopolymer (Fang, Li, Inoue,

96 

Lundin, & Appelqvist, 2006). Biopolymer size (molecular weight) and

97 

type, chain conformation and flexibility, distribution of reactive groups

98 

and the charge density, solvent conditions (e.g., pH, ionic strength, and

99 

temperature), protein to polysaccharide mixing ratio, total biopolymer

100 

concentration, stirring and pressure are important factors controlling the

101 

phase separation behaviors of biopolymer mixtures, particularly of

102 

charged biopolymer mixtures and could result in either associative or

103 

segregative phase separation (Fang et al., 2006; Turgeon et al., 2009).

104 

The gel forming property and rheology of the κ-carrageenan (KC)

105 

either alone or in combination with globular proteins is well known.

106 

However, the study of complex coacervation between KC and proteins

107 

in dilute aqueous mixtures has been limited (Fang et al., 2006; Burova

108 

et al., 2007). The purpose of the current work is to study the effect of

109 

KC depolymerization using high intensity ultrasound on the complex

110 

coacervation between KC (at non-gelling concentrations) and β-

111 

lactoglobulin (BLG). The target pH was chosen to be 4.25, based on the

112 

pH of a clear traditional herbal beverage in order to assess the

113 

capability of the produced nanoparticles as delivery systems for

6   

114 

fortification purposes in the future. To the best of our knowledge, the

115 

interaction between KC and BLG has not been studied using isothermal

116 

titration calorimetry (ITC).

117  118 

2. Materials and methods

119 

2.1. Materials κ–carrageenan (KC, 504 kDa, composition: 90% (w/w) KC, 8%

120  121 

(w/w) moisture and 2% (w/w) ash), β-lactoglobulin from bovine milk

122 

(BLG, 18.4 kDa, composition: 93% (w/w) BLG, 5.4% (w/w) moisture and

123 

1.6% (w/w) ash, a mixture of genetic variants A and B) and sodium

124 

azide (as a preservative, minimum purity 99.5%) were purchased from

125 

Sigma Chemical Co. (St. Louis, MO, USA). Analytical grade

126 

hydrochloric acid was obtained from Merck Co. (Darmstadt, Germany).

127 

Deionized water (18.2 MΩ cm resistivity) from a Nanopure water system

128 

(Nanopure Infinity, Barnstead International, IA, USA) was used for the

129 

preparation of all solutions. In this study all materials were used as such

130 

received.

131 

2.2. Preparation of solutions

132 

KC stock solution (0.5% (w/w), pH: 7.47) was prepared by dispersing

133 

into Nanopure water containing 0.03% (w/w) sodium azide at room

134 

temperature followed by heating to 85 °C for 30 min under magnetic

135 

stirring in order to ensure a complete hydration of polysaccharide. BLG

7   

136 

stock solution (0.4% (w/w0, pH~ 6.94) was prepared by dispersing into

137 

Nanopure water containing 0.03% (w/w) sodium azide and stirred

138 

overnight at 250 rpm and ambient temperature in order to use on the

139 

following day.

140 

2.3. Ultrasonic treatment of KC solution KC stock solution (30 g) was treated by an ultrasonic processor

141  142 

(Hielscher UP200S, power 200 W, frequency 24 kHz, Dr Hielscher Co.,

143 

Teltow, Germany) for different times (10, 20 or 30 min) at different

144 

temperatures (25 or 75 °C) and different amplitudes (50 or 100 %). The

145 

sample was held in a temperature controlled water bath to prevent the

146 

temperature rise by the sonication. A standard tapered horn tip of 5 mm

147 

end diameter was immersed 1.5 cm into the solution during

148 

ultrasonication. The ultrasound irradiation was produced directly from

149 

the horn tip under continuous mode.

150 

2.4. Viscosity measurement The apparent viscosity of the unsonicated (control) and sonicated

151  152 

samples was measured at 25 °C using a rotational viscometer (Model

153 

LV-DVII+, Brookfield Engineering Laboratories, Middleboro, MA, USA)

154 

equipped with spindle number 1 rotated at 10 rpm.

155 

2.5. Turbidimetric analysis at different pHs

8   

Mixtures of BLG and KC were prepared by first mixing and then

156  157 

diluting the stock solutions at a 2:1 (w/w) BLG:KC mixing ratio and a

158 

total biopolymer concentration of 0.15% (w/w). The mixture was

159 

acidified gradually by the addition of 0.1 M HCl (pH range of 5-7), 0.4 N

160 

HCl (pH range of 3-5) and 2 M HCl (pH range of <1-3) with gentle

161 

magnetic stirring for 2 min at each pH level before decreasing it to the

162 

next pH. Dilution effects were considered to be minimal. The optical

163 

density of the biopolymer mixtures with decreasing pH (from pH ~7 to

164 

~1) was analyzed using a UV/visible light spectrophotometer at 600 nm

165 

(BioQuest CE 2502, Cecil Ins., Cambridge, UK) using plastic cuvettes

166 

(1 cm path length). Deionized water was used as a blank reference.

167 

Critical pH values (pHc: formation of soluble complexes, pHφ1: formation

168 

of insoluble complexes, pHopt: maximum optical density, pHφ2:

169 

dissolution of complexes) were measured graphically as the intersection

170 

point of two curve tangents. BLG and KC solutions were used as

171 

controls at their corresponding concentrations (0.1 and 0.05 % w/w,

172 

respectively).

173 

2.6. Isothermal titration calorimetry (ITC) ITC measurements were carried out with a VP-ITC calorimeter

174  175 

(Microcal Inc., Northampton, MA, USA) in order to measure the

176 

enthalpic and entropic changes due to BLG-KC interactions at 25 °C.

177 

Before titration, the biopolymers were separately dissolved in 5 mM

178 

sodium citrate buffer solution (pH 4.25). Heating at 85 °C for 30 min 9   

179 

was required for KC. The buffer was used to remove the experimental

180 

errors resulting from pH mismatch. The BLG dispersion containing

181 

about 1 mg/ml was filtered through a 0.22-µm low protein binding

182 

polyether sulphone (PES) syringe filter (MS®, TX, USA) to obtain

183 

aggregate free BLG dispersion. The concentration of BLG dispersion

184 

(monomeric equivalent) was measured by UV/visible light spectroscopy

185 

using a specific extinction coefficient of 17600 M-1 cm-1 at 278 nm, as

186 

reported by Liang, Tajmir-Riahi, & Subirade (2008) and amounted to

187 

0.828 mg/ml. The sodium citrate buffer solution was used as blank

188 

reference. The dispersions were degassed under vacuum for 3 min by

189 

means of a device provided with the ITC apparatus. The injector-stirrer

190 

syringe (290 µL) was loaded with KC solution. Portions of 15 μl (except

191 

for the first injection which was 5 µl) of KC solution (0.1 and 0.175%

192 

w/w for intact (IN) and sonicated (US) for 20 min at 25 °C and amplitude

193 

100% polysaccharides, respectively) were injected sequentially into the

194 

titration cell (V = 1.408 ml) initially containing either aggregate free BLG

195 

dispersion or buffer solution. The duration of each injection was 20 s,

196 

and the equilibration time between consecutive injections was 300 s.

197 

During the titration, the stirring speed was 310 rpm. The heat of dilution

198 

from the blank titration of KC solution into sodium citrate buffer was

199 

measured, and the dilution heat was subtracted from the raw data to

200 

measure corrected enthalpy changes. The results are reported as the

201 

change in enthalpy per gram of KC (IN) and KC (US) injected into the

10   

202 

reaction cell. The low concentrations of the biopolymer solutions and

203 

the mild temperature supplied a low viscosity at any point of titration,

204 

which did not affect the mechanical stirring of the microcalorimeter.

205 

Calorimetric data analysis was carried out with Microcal ORIGIN

206 

software (v.7.0). Thermodynamic parameters including binding

207 

stoichiometry (N), affinity constant (K), enthalpy (ΔH) and entropy (ΔS)

208 

changes were calculated by iterative curve fitting of the binding

209 

isotherms. The Gibbs free energy change (ΔG) was calculated from the

210 

equation (ΔG = ΔH - TΔS).

211 

2.7. BLG-KC complexation

212 

BLG-KC complexes from the mixing of BLG and KC dispersions at

213 

different polysaccharide/protein weight ratios were obtained by the post-

214 

blending acidification method. A series of samples containing a fixed

215 

protein concentration of 0.1% (w/w) but different KC concentrations (0–

216 

0.2 % (w/w)) was prepared by mixing different ratios of 0.4% (w/w) BLG

217 

and 0.5% (w/w) KC stock dispersions as well as deionized water.

218 

Biopolymer solutions were adjusted to pH 4.25 using 0.4, 0.1 and/or

219 

0.01 M HCl solutions. These solutions were stirred for 1 h and then

220 

allowed to equilibrate at ambient temperature for 18–24 h prior to

221 

analysis.

222 

2.8. Characterization of the complexes

223 

2.8.1. Turbidity measurement 11   

224 

The turbidity of samples was quantified by their absorbance

225 

measured at 600 nm using plastic cuvettes (1 cm path length). Sample

226 

solutions were vortexed for 5 s prior to analysis. Highly turbid samples

227 

were diluted before measurement using deionized water pre-adjusted

228 

with HCl to pH 4.25.

229 

2.8.2. Particle size and zeta- (ζ-) potential analyses Measurements of particle size distribution were carried out using a

230  231 

dynamic light scattering (DLS) instrument (90Plus, Brookhaven

232 

Instruments Corp., Vienna, Austria). Analyses were carried out at a

233 

scattering angle of 90° at 25 °C. The effective diameter (also called Z-

234 

average mean diameter) was only measured in samples which have

235 

shown no sedimentation after equilibration. The Z-average mean

236 

diameter and polydispersity index (PDI) were obtained by cumulant

237 

analysis. The ζ-potential was determined by laser Doppler anemometry

238 

with palladium electrodes using a ZetaPals instrument (Brookhaven

239 

Instruments Corp.) at fixed light scattering angle of 90° at 25 °C. The ζ-

240 

potential (mV) was calculated from the electrophoretic mobility using the

241 

Helmholtz-Smoluchowski equation. During both dynamic light scattering

242 

and electrophoretic light scattering measurements, the viscosity of the

243 

continuous phase were assumed to correspond to pure water.

244 

2.8.3. Phase contrast optical microscopy

12   

BLG and KC complexed mixtures were microscopically

245  246 

characterized at different magnifications using a phase contrast optical

247 

microscope (Olympus CX40, Olympus Optical Co., Tokyo, Japan)

248 

equipped with a AxioCam ERc 5s video camera (Carl Zeiss

249 

Microimaging Gmbh, Göttingen, Germany) controlled by an image

250 

processor (Kappa ImageBase 2.5). Fifteen microliters of the dispersion

251 

were placed between glass slides and then examined. A drop of

252 

immersion oil (Merck Co., Darmstadt, Germany) was placed on the

253 

glass slide before characterization with 1000 × magnification.

254 

2.9. Statistical analysis Measurements were performed at least two or three times using

255  256 

freshly prepared samples and analyzed by ANOVA using the MSTATC

257 

programs (version 2.10, East Lansing, MI, USA). Results are reported

258 

as means and standard deviations. Comparison of means was carried

259 

out using Duncan’s multiple range tests at a confidence level of 0.05.

260  261 

3. Results and discussion

262 

3.1. Changes in viscosity after sonication

263 

The effectiveness of the sonication has been evaluated by

264 

measuring the changes in apparent viscosity which is shown versus

265 

sonication time at different amplitudes and temperatures in Fig. 1. There

13   

266 

was a severe decrease in the viscosity of the KC solution (0.5% w/w).

267 

As an example, the viscosity of 19 mPa.s for the untreated solution

268 

decreased to 3 mPa.s after sonication for 30 min at 25 °C and

269 

amplitude 100%. This phenomenon is due to the cleavage of the

270 

polysaccharide backbone which results in a decrease in the molecular

271 

weight of ultrasonically treated polysaccharides and hence decreasing

272 

the effective volume of the polysaccharide chains (Weiss,

273 

Kristbergsson, & Kjartansson, 2011). The depolymerization process

274 

occurs through the effects of acoustic cavitation and can involve two

275 

possible mechanisms: mechanical degradation of the polymer from

276 

collapsed cavitation bubbles and chemical degradation as a result of the

277 

chemical reaction between the polymer and high energy molecules

278 

such as hydroxyl radicals produced from cavitation (Chemat, Huma, &

279 

Kamran Khan, 2011). According to Iida, Tuziuti, Yasui, Towata, and

280 

Kozuka, (2008), the effect of ultrasonication on viscosity depression is

281 

extremely dependent on the mechanical and structural properties of the

282 

polysaccharides, i.e. whether the polysaccharides have a stiff linear or

283 

random coil configuration. For example, pectin showed a rather small

284 

change (about 50% decrease) in viscosity, whereas glucomannan

285 

showed a much more severe decrease in viscosity by sonication (Iida et

286 

al., 2008). Fig. 1 clearly shows that the sonication temperature had an

287 

inverse effect on the viscosity depression when the other parameters

288 

(time and amplitude) remained constant; however, this effect was less

14   

289 

pronounced at higher sonication times. Increasing in the sonication

290 

temperature may increase the flexibility of the molecular chain.

291 

According to Weiss et al. (2011), flexible biopolymer chains are less

292 

susceptible to decreases in viscosity upon ultrasonication. An increase

293 

in temperature also leads to an increase in water vapor pressure, which

294 

penetrates in larger amounts into the cavitation bubbles and weakens

295 

the collapse energy by the so-called “cushioning effect” (Kardos &

296 

Luche, 2001). The viscosity of the KC solution decreased significantly

297 

(*p<0.05) with increasing time and amplitude of the ultrasonication

298 

process, and tends to approach a limiting viscosity value, which may

299 

correspond to low molecular weight fractions for which the application of

300 

high-intensity ultrasound does not lead to further backbone breakdown

301 

(Weiss et al., 2011).

302 

3.2. Turbidimetric analysis Turbidimetric analysis as a function of pH was used to study the

303  304 

kinetics of associative phase separation within mixed BLG-KC systems

305 

(Fig. 2). Indeed, pH affects the ionization degree of the functional

306 

groups of the protein and polysaccharide and electrostatic complexing

307 

takes place under acidification (Weinbreck, Nieuwenhuijse, Robijn, & de

308 

Kruif, 2003). In the absence of protein, KC solutions remained

309 

transparent in studied pH range indicating that they did not form

310 

particles large enough to scatter light strongly, due to the sulfate groups

311 

which were always ionized, giving the molecules an electrostatic 15   

312 

repulsion. The BLG dispersion showed a broad peak in the measured

313 

turbidity versus pH profile with a maximum value around pH 4 to 5 due

314 

to self-association around the Ip of BLG which decreased as the pH

315 

became more acid or alkaline. Generally, BLG-KC (US) complexed

316 

solutions showed lower turbidity than BLG-KC (IN) solutions which can

317 

be attributed to the production of smaller polysaccharide chains after

318 

sonication. At pH > 5.30-5.50, biopolymers were considered to be co-

319 

soluble, although a very slight increase in turbidity of the systems can

320 

be seen (Fig. 2) which may be the result of non-coulombic interactions

321 

such as hydrophobic and hydrogen bindings. Previous researchers

322 

have also found little interaction between BLG and pectin at high pH

323 

values (Girard, Turgeon, & Gauthier, 2002). Another possibility is that

324 

weak local electrostatic interactions may occur between protein and

325 

polysaccharide molecules as shown in work by Dickinson & Galazka

326 

(1991). They have demonstrated that native BLG and anionic

327 

polysaccharides (dextran sulfate and propylene glycol alginate) could

328 

form ionic complexes at neutral pH due to charge-induced charge

329 

interactions. One beneficial consequence of this complexation is the

330 

protection against a loss of solubility due to aggregation induced by

331 

heating or high-pressure processing (Dickinson, 2008). Soluble

332 

complexes were formed at a pHc (~5.30-5.50) that was independent of

333 

the KC type (sonicated or non-sonicated). Weinbreck et al. (2004)

334 

reported a pHc value of 5.5 for different mixtures of whey protein isolate

16   

335 

and non-gelling carrageenan (comprised mainly λ-carrageenan).

336 

According to Turgeon et al. (2009) and Weinbreck et al. (2004), this

337 

transition occurs at the molecular level (i.e. complexation begins

338 

between a single polysaccharide chain and a defined amount of protein)

339 

and is independent on the molecular weight and the mixing ratio.

340 

Formation of soluble complexes occurred at a pHc above the Ip of the

341 

BLG (~4.7-5.2) which is thought to be due to the ability of the globular

342 

proteins for charge regulation around the Ip resulting from their

343 

electrical capacitance properties (Dickinson, 2008) and/or due to the

344 

presence of positive patches (localized regions with higher charge

345 

density) on the surface of BLG as a result of low ionic strength

346 

conditions which inhibit charge screening (Weinbreck et al., 2003;

347 

Turgeon et al., 2009). When the pH decreased further, the critical pHφ1

348 

(~4.85) was reached as a result of nucleation and growth-type kinetics

349 

(Sanchez, Mekhloufi, & Renard, 2006). At this point, more and more

350 

protein molecules become attached to the polysaccharide (due to an

351 

increase in charge density of the protein) until electroneutrality was

352 

attained yielding neutral interpolymeric complexes that tend to

353 

precipitate (Turgeon et al., 2009). It should be noted that the measured

354 

optical density is the result of the number and size of the biopolymer

355 

complexes. The highest amount of BLG-KC interactions (pHopt)

356 

occurred at pH 1-2 with maximum optical densities of 1.8 and 1.4 for

357 

BLG-KC (IN) and BLG-KC (US) mixtures, respectively, which is the

17   

358 

result of various attractive forces (e.g. van der Waals, hydrophobic, and

359 

electrostatic interactions between oppositely charged groups). In this

360 

work, pHφ2 was absent since the dissociation of KCs’ sulphate groups is

361 

not suppressed at low pH and they remain charged (Turgeon et al.,

362 

2009).

363 

3.3. ITC results The heat flow versus time profiles resulting from the titration of

364  365 

BLG with intact and sonicated KCs at 25 °C and pH 4.25 are shown in

366 

Fig. 3 a and b, respectively. The area under each peak represented the

367 

heat exchange within the cell containing BLG after each KC injection.

368 

The injection profiles in the sample cell were exothermic and decreased

369 

regularly to a state of thermodynamic stability (about zero) after the 15th

370 

and 12th injections of KC (IN) (0.1% w/w) and KC (US) (0.175% w/w),

371 

respectively. Exothermicity is associated with the nonspecific

372 

electrostatic neutralization of the opposite charges carried by the two

373 

biopolymers indicating an enthalpic contribution of complex

374 

coacervation (Girard, Turgeon, & Gauthier, 2003; Schmitt et al., 2005),

375 

while its regular decrease is attributed to a reduction in free protein

376 

remaining in the reaction cell after successive injections, which explains

377 

the lowering of the energy released. Girard et al. (2003) reported a

378 

similar exothermic sequence for BLG interaction with low- and high-

379 

methoxyl pectin, while Aberkane, Jasniewski, Gaiani, Scher, & Sanchez

380 

(2010) reported an exothermic-endothermic sequence for BLG – gum 18   

381 

Arabic interaction. To characterize thermodynamic parameters, the

382 

binding isotherms obtained by integrating of the isotherm peaks and

383 

subtraction of the heats of dilution of KCs into buffer solution were fitted

384 

using the one site binding model provided by the Microcal Origin

385 

software and plotted against KC/BLG weight ratio (Fig. 4). The first

386 

injection was not taken into account for analysis. The calculation gives a

387 

typical sigmoidal saturation curve, which can be concluded as a

388 

progressive binding of the BLG molecules present in the titration cell to

389 

the binding sites along the KC backbone. The isoenthalpic plateau

390 

observed in the binding isotherms was reached at KC (IN) and KC (US)

391 

to BLG weight ratios of about 0.20 and 0.30, respectively. Calculation of

392 

the thermodynamic parameters including binding stoichiometry (N),

393 

affinity constant (K), enthalpy (ΔH) and entropy (TΔS) contributions and

394 

Gibbs free energy change (ΔG) for the interaction between KC and BLG

395 

showed that the binding enthalpy was negative and favorable, whereas

396 

the binding entropy was unfavorable (negative) during KC-BLG

397 

interaction. According to Ou and Muthukumar (2006) the complexation

398 

between weakly charged polyelectrolytes is driven by a negative

399 

enthalpy due to the electrostatic interaction between two oppositely

400 

charged components, while counterion release entropy plays only a

401 

minor role. The unfavorable entropic effects originate mainly from the

402 

loss in biopolymer conformational freedom after association (Dickinson,

403 

2008). BLG and KC (IN) interacted with a high affinity constant (KIN:

19   

404 

10476 ± 6032 g-1.l) and a strong ΔHIN of (-2.706 ± 0.042 cal.g-1).

405 

Assuming a molecular weight of 504 kDa for KC (IN), about 142 BLG

406 

molecules were involved in the interaction process with KC (IN) (NIN:

407 

192.3 ± 1.4 mg KC (IN)/ g BLG). Schmitt et al. (2005) and Aberkane et

408 

al. (2010) reported enthalpy change (-0.933 ± 0.001 and -1.072 ± 0.014

409 

cal.g-1), affinity constant (25.4 ± 13.0 and 896 ± 66 g-1.l) and binding

410 

stoichiometry (86 and 90 BLG molecules) values upon complexation of

411 

BLG with Acacia gum (MW ~ 540 kDa) at pH 4.2, respectively. The

412 

differences can be explained by the higher charge density on KC (IN)

413 

molecules than on Acacia gum molecules. The interaction between BLG

414 

and KC (US) occurred with significant (*p<0.05) lower affinity constant

415 

(KUS: 535 ± 137 g-1.l) as well as higher binding stoichiometry (NUS: 214.1

416 

± 3.0 mg KC (US)/ g BLG) and higher enthalpy change (ΔHUS: -2.940 ±

417 

0.062 cal.g-1) values. The decrease in the affinity constant of the KC

418 

(US)-BLG interaction can be attributed to the lower negative charge

419 

density on KC (US) than on KC (IN) (section 3.4.2.) and changes in the

420 

helical structure of the polysaccharide after sonication. These results

421 

are in good agreement with those of Chang, McLandsborough and

422 

McClements (2012). They found that ε-polylysine (an antimicrobial

423 

cationic polyelectrolyte) interacted with an anionic polysaccharide

424 

(pectin) more strongly when the charge density on the pectin molecules

425 

increased (i.e. with decreasing degree of esterification). The

426 

unfavorable entropic contribution (TΔS) was relatively in the same

20   

427 

range (-2.680 ± 0.034 and -2.780 ± 0.038 cal.g-1 for KC (IN) and KC

428 

(US), respectively) as the favorable enthalpic contribution, indicating

429 

that any change in enthalpy is accompanied by a similar change in

430 

entropy, that is, entropy-enthalpy compensation occurred (Aberkane et

431 

al., 2010). The changes in Gibbs free energy were negative for the two

432 

types of KCs (-0.026 ± 0.008 and -0.160 ± 0.024 cal.g-1 for KC (IN) and

433 

KC (US), respectively) indicating the spontaneous nature of the

434 

interactions. The difference in Gibbs free energy changes can be

435 

attributed to the fact that the loss in polysaccharide conformational

436 

freedom after association is more considerable for larger molecules

437 

than smaller molecules.

438 

3.4. Complex evaluation

439 

3.4.1. Turbidity versus KC/BLG weight ratio profiles

440 

The turbidity of the BLG-KC solutions was measured as a function

441 

of KC/BLG weight ratio at pH 4.25 to provide some deeper insights into

442 

the mechanisms of complexed biopolymer nanoparticle formation (Fig.

443 

4) and to find the most suitable conditions for forming stable

444 

nanoparticles. The initial turbidity of the BLG suspension in the absence

445 

of KC was about 0.113, because of some aggregation of proteins

446 

around pH 4.25. The KC to BLG weight ratio had a major effect on the

447 

solution turbidity and degree of sediment formation in the solutions. At

448 

KC (IN)/BLG weight ratios lower than 0.50, complexed biopolymer

21   

449 

particles are unstable to aggregation because they can achieve

450 

electrical neutrality (protein depletion) due to the high protein binding

451 

(Weinbreck et al., 2003) leading to high turbidity and aggregation as

452 

seen in Fig. 5 a,b (white sediment at the bottom of the glass vials with a

453 

clear serum layer on top). BLG/KC (IN) mixture obtained at 1:10

454 

polysaccharide:protein weight ratio was microscopically characterized

455 

just after acidification to pH 4.25, during precipitation and after

456 

precipitation (lower phase) (Fig. 5c-e, respectively). The initial structures

457 

are of spherical shape. It seems that complex coacervation in mixed

458 

BLG-KC dispersions is a nucleation and growth mechanism. Similar

459 

mechanism was reported by Sanchez et al. (2006), in mixture of BLG

460 

with gum Arabic (as a polysaccharide with different flexibility and charge

461 

density). According to Sanchez et al. (2006), nucleation and growth

462 

mechanism is the general mechanism of complexation/coacervation

463 

between biological macromolecules. Complexes grew in size during

464 

precipitation and their number was reduced. This feature could be due

465 

to coalescence of complexes or Ostwald ripening (Sanchez et al.,

466 

2006). These samples are unsuitable for utilization as stable colloidal

467 

delivery systems in the food industry. Particles formed in the BLG/KC

468 

(US) mixed system did not markedly differ in structure as compared to

469 

the previous ones (data not shown). At higher polysaccharide/protein

470 

weight ratios the samples were less turbid and did not exhibit

471 

sedimentation, indicating that colloidal dispersions containing small

22   

472 

stable complexes with higher stability than the protein aggregates

473 

themselves were formed, presumably because the electrostatic and

474 

steric repulsion resulting from the presence of a polysaccharide shell

475 

around the protein core is sufficiently strong to prevent aggregation as

476 

revealed by the influence of KC on the ζ-potential of the complexes (Fig.

477 

6a). Since the resolution of the phase contrast microscope is not

478 

enough to visualize nanoparticles, no structure was detected at KC

479 

(IN)/BLG weight ratio of 1:1 (Fig. 5f). These stable colloidal dispersions

480 

may have important implications for practical utilization within foods.

481 

The turbidity profile of BLG-KC (US) was similar to that of BLG-KC (IN).

482 

Generally, the lower charge density of KC (US) may account for the

483 

observed differences in the BLG-KC (US) turbidity profile including a

484 

slight shift to the right and an increase in the turbidity of the sample

485 

representing KC (US)/BLG weight ratio of 0.5. The data are consistent

486 

with the ITC data, which also indicated that there was a strong

487 

interaction between the two biopolymers at pH 4.25.

488 

3.4.2. ζ-potential versus KC/BLG weight ratio profiles

489 

The stability of colloidal systems can be studied by measuring the

490 

electrophoretic properties of the colloidal particles. Particles with ζ

491 

potentials more positive than +30mV or more negative than -30mV are

492 

normally considered stable (Mounsey, O’Kennedy, Fenelon, &

493 

Brodkorb, 2008). The ζ-potential profiles of the BLG-KC complexed

494 

systems as a function of KC/BLG weight ratio at pH 4.25 is shown in 23   

495 

Fig. 6a. In the absence of KC, the ζ-potential of the BLG suspension

496 

was around + 14 mV, which was due to the fact that BLG was below its

497 

Ip and therefore had a net positive charge. The ζ-potential depended

498 

considerably on the KC concentration. As the KC (IN)/BLG weight ratio

499 

increased from 0 to 0.37, the EM values decreased from positive to

500 

negative and the Smoluchowski model yielded ζ-potential values that

501 

ranged between +14 and -23 mV, indicating low stability systems which

502 

resulted in precipitation. According to Aberkane et al. (2010), the

503 

requirement of neutrality at phase separation is not a general rule for

504 

protein-polyanionic polymers and phase separation may occur with a

505 

negative total charge. Beyond this point (KC (IN)/BLG weight ratio =

506 

0.37), the ζ-potential values remained rather constant at high limiting

507 

values (ranging between -44 and -51 mV) reflecting an excess of

508 

polysaccharide. These measurements showed that negatively charged

509 

KC molecules associated with the surfaces of the positively charged

510 

BLG aggregates and caused charge reversal. The ζ-potential profile of

511 

BLG-KC (US) showed a similar trend with lower intensity. This trend is

512 

in agreement with the charge densities of the two types of KCs which

513 

were -53.67 ± 5.21 and -41.63 ± 3.69 mV for KC (IN) and KC (US),

514 

respectively, at a concentration of 0.1% (w/w) and pH 4.25. Tang,

515 

Huang, and Lim (2003) reported that the ζ-potential values of chitosan

516 

nanoparticles decreased from (47.48 ± 1.32) to (45.51 ± 0.29) after 10

517 

min sonication at amplitude 80% (more gentle conditions than those of

24   

518 

the current work). This phenomenon can be attributed to the reduction

519 

of the KC reactivity after sonication, which may be due to some

520 

heterogeneous sonochemical interactions and structural changes that

521 

took place during the sonication process. Polysaccharide reactivity is

522 

governed by the distribution and number of functional groups attached

523 

to the polymerized sugar units that form the backbone of the

524 

polysaccharide (Weiss et al., 2011). Polysaccharides subjected to high-

525 

intensity ultrasound can undergo a large number of sonochemical

526 

reactions including glycosylation, acetalyzation, oxidation, C–D, C-

527 

heteroatom, and C–C bond formations (Kardos et al., 2001), which may

528 

eliminate the reactive sites present along the KC backbone or may

529 

promote KC-KC interactions which reduce the number of binding sites

530 

for the BLG molecules resulting in affinity constant reduction.

531 

3.4.3. Particle size versus KC/BLG weight ratio profiles

532 

The mean hydrodynamic particle diameters of the BLG-KC

533 

systems showing no precipitation as a function of the KC/BLG weight

534 

ratio at pH 4.25 are shown in Fig. 6b. The complexes were relatively

535 

large at KC (IN)/BLG weight ratios of 0.44 and 0.50 possibly resulting

536 

from some sharing of KC molecules between protein aggregates at low

537 

polysaccharide concentration. The smallest particles were obtained at a

538 

KC (IN)/BLG weight ratio of 0.75, presenting a mean diameter of 408 ±

539 

9 nm. These complexes showed a lower PDI than the biopolymers

540 

themselves. Generally, the shrinkage of the BLG-KC complexes, 25   

541 

occurring at low ionic strength could be understood as a reduction of the

542 

intramolecular repulsion induced by the interaction of the BLG with the

543 

sulphate group of the KC. This compaction phenomenon was well

544 

predicted by Monte Carlo simulations which showed that at low ionic

545 

strengths, a polyelectrolyte chain would wrap around an oppositely

546 

charged spherical particle (Girard et al., 2003). There was an

547 

appreciable increase in the diameter of the particles in the mixed

548 

system when the KC (IN)/BLG weight ratio was increased from 0.75 to

549 

2. Zimet & Livney (2009) concluded that an increase in the

550 

polysaccharide concentration increases the viscosity, causing

551 

decreased particle mobility (lower fluctuations), which is interpreted by

552 

the DLS as an apparently increased particle size. This conclusion is in

553 

good agreement with our results since for complexed solutions

554 

containing KC (IN), the increase in particle size was found to be more

555 

dependent on polysaccharide concentration due to its ability to form

556 

more viscous systems. Another possibility is that the changes in

557 

intramolecular repulsion and conformation (adoption of a more

558 

extended structure) resulting from the decreased ratio of protein

559 

molecules per polysaccharide chain may cause a bigger particle size.

560 

The mean diameters of the BLG-KC (US) particles were significantly

561 

(*p<0.05) smaller than the BLG-KC (IN) particles at KC (US)/BLG

562 

weight ratios which corresponded to sufficient repulsion between

563 

complexed particles. In the presence of KC (US), the effective

26   

564 

diameters of the biopolymer complexes remained relatively constant as

565 

compared to those containing KC (IN). This may be due to the larger

566 

flexibility of the KC (US) chains with the reduction of the charge as well

567 

as to the lower viscosity of the biopolymer mixtures. Generally, the

568 

polydispersity index of the BLG-KC (IN) nanoparticles (0.313) at weight

569 

ratio (0.75) corresponding to minimum particle size was significantly

570 

(*p<0.05) higher than that of BLG-KC (US) nanoparticles (0.151) at

571 

weight ratio of 1 (minimum particle size), indicating the consequence of

572 

polysaccharide sonication was the homogenization of particle sizes in

573 

the mixed dispersion. One should keep in mind that the measured

574 

results are intensity-weighted, which overestimates the contribution of

575 

the larger particles to the detriment of the smaller ones. If volume- or

576 

number- weighted distributions are considered, much smaller average

577 

diameters are obtained.

578 

4. Conclusion The present work shows that ultrasound irradiation can effectively

579  580 

depolymerize KC. The rate of depolymerization was dependent on the

581 

amplitude, time and temperature of sonication. KC sonication

582 

decreased its affinity constant to BLG at pH 4.25 as determined by ITC.

583 

The properties of the biopolymer particles formed depended strongly on

584 

the polysaccharide type and concentration as shown by DLS and ζ-

585 

potential analyses. The soluble complexes formed had good stability

586 

against aggregation. Findings could aid in the design of nanoscopic 27   

587 

delivery systems for encapsulation of both hydrophilic and hydrophobic

588 

bioactives in liquid food products and for controlled release objectives,

589 

which is the focus of our current research. In the future, more detailed

590 

information is required on the mechanism of the helix-coil transition in

591 

KC after sonication and association with BLG using high-sensitive

592 

differential scanning calorimeter and also on the structure of the soluble

593 

complexes formed using high-resolution cryo-TEM.

594 

Acknowledgment

595 

The authors are thankful to University of Tehran, Iranian

596 

Nanotechnology Initiative Council and Ghent University for financial

597 

support.

598 

References

599 

Aberkane, L., Jasniewski, J., Gaiani, C., Scher, J., & Sanchez, C.

600 

(2010). Thermodynamic characterization of acacia gum-β-

601 

lactoglobulin complex coacervation. Langmuir, 26, 12523–12533.

602 

Burova, T. V., Grinberg, N. V., Grinberg, V. Y., Usov, A. I., Tolstoguzov,

603 

V. B., & de Kruif, C. G. (2007). Conformational changes in ι- and κ-

604 

carrageenans induced by complex formation with bovine β-casein.

605 

Biomacromolecules, 8, 368-375.

606 

Chang, Y. H., McLandsborough, L., & McClements, D. J. (2011).

607 

Interactions of a cationic antimicrobial (ε-polylysine) with an

608 

anionic biopolymer (pectin): an isothermal titration calorimetry, 28   

609 

microelectrophoresis, and turbidity study. Journal of Agricultural

610 

and Food Chemistry, 59, 5579–5588.

611 

Chang, Y. H., McLandsborough, L., & McClements, D. J. (2012).

612 

Cationic antimicrobial (ε-polylysine)-anionic polysaccharide

613 

(pectin) interactions: influence of polymer charge on physical

614 

stability and antimicrobial efficacy. Journal of Agricultural and

615 

Food Chemistry, 60, 1837–1844.

616 

Chemat, F., Huma, Z., & Kamran Khan, M. (2011). Applications of

617 

ultrasound in food technology: Processing, preservation and

618 

extraction. Ultrasonics Sonochemistry, 18, 813–835.

619 

Dickinson, E. (2008). Interfacial structure and stability of food emulsions

620 

as affected by protein–polysaccharide interactions. Soft Matter, 4,

621 

932–942.

622 

Dickinson, E., & Galazka, V. B. (1991). Emulsion stabilization by ionic

623 

and covalent complexes of β-lactoglobulin with polysaccharides,

624 

Food Hydrocolloids, 5, 281– 296.

625 

Fang, Y., Li, L., Inoue, C., Lundin, L., & Appelqvist, I. (2006).

626 

Associative and segregative phase separations of gelatin/κ-

627 

carrageenan aqueous mixtures. Langmuir, 22, 9532-9537.

628 

Girard, M., Turgeon, S. L., & Gauthier, S. F. (2002). Interbiopolymer

629 

complexing between β-lactoglobulin and low- and high-methylated

630 

pectin measured by potentiometric titration and ultrafiltration. Food

631 

Hydrocolloids, 16, 585–591.

29   

632 

Girard, M., Turgeon, S. L., & Gauthier, S. F. (2003). Thermodynamic

633 

parameters of β-lactoglobulin-pectin complexes assessed by

634 

isothermal titration calorimetry. Journal of Agricultural and Food

635 

Chemistry, 51, 4450–4455.

636 

Gu, Y. S., Decker, E. A., & McClements, D. J. (2005). Influence of pH

637 

and carrageenan type on properties of β-lactoglobulin stabilized

638 

oil-in-water emulsions. Food Hydrocolloids, 19, 83–91.

639 

Huang, G-Q., Sun, Y-T., Xiao, J-X., & Yang, J. (2012). Complex

640 

coacervation of soybean protein isolate and chitosan. Food

641 

Chemistry, 135, 534–539.

642 

Iida, Y., Tuziuti, T., Yasui, K., Towata, A., & Kozuka, T. (2008). Control

643 

of viscosity in starch and polysaccharide solutions with ultrasound

644 

after gelatinization. Innovative Food Science and Emerging

645 

Technologies, 9, 140–146.

646 

Jun-xia, X., Hai-yan, Y., & Jian, Y. (2011). Microencapsulation of sweet

647 

orange oil by complex coacervation with soybean protein

648 

isolate/gum Arabic. Food Chemistry, 125, 1267–1272.

649 

Kardos, N., & Luche, J-L. (2001). Sonochemistry of carbohydrate compounds. Carbohydrate Research, 332, 115–131.

650  651 

Klemmer, K. J., Waldner, L., Stone, A., Low, N. H., & Nickerson, M. T.

652 

(2012). Complex coacervation of pea protein isolate and alginate

653 

polysaccharides. Food Chemistry, 130, 710-715.

30   

654 

Laneuville, S. I., Paquin, P., & Turgeon, S. L. (2005). Formula

655 

optimization of a low-fat food system containing whey protein

656 

isolate-xanthan gum complexes as fat replacer. Journal of Food

657 

Science, 70, S513–S519.

658 

Liang, L., Tajmir-Riahi, H. A., & Subirade, M. (2008). Interaction of β-

659 

lactoglobulin with resveratrol and its biological implications.

660 

Biomacromolecules, 9, 50–56.

661 

Mounsey, J. S., O’Kennedy, B. T., Fenelon, M. A., & Brodkorb, A.

662 

(2008). The effect of heating on β-lactoglobulin–chitosan mixtures

663 

as influenced by pH and ionic strength. Food Hydrocolloids, 22,

664 

65–73.

665 

Ou, Z., & Muthukumar, M. (2006). Entropy and enthalpy of

666 

polyelectrolyte complexation: Langevin dynamics simulations.

667 

Journal of Chemical Physics, 124, 154902– 154911.

668 

Ould Eleya, M. M., & Turgeon, S. L. (2000). Rheology of κ-carrageenan and β-lactoglobulin mixed gels. Food Hydrocolloids, 14, 29–40.

669  670 

Sanchez, C., Mekhloufi, G., & Renard, D. (2006). Complex coacervation

671 

between β-lactoglobulin and acacia gum: a nucleation and growth

672 

mechanism. Journal of Colloid and Interface Science, 299, 867–

673 

873.

674 

Schmitt, C., da Silva, T. P., Bovay, C., Rami-Shojaei, S., Frossard, P.,

675 

Kolodziejczyk, E., & Leser, M. E. (2005). Effect of time on the

676 

interfacial and foaming properties of β-lactoglobulin/acacia gum

31   

677 

electrostatic complexes and coacervates at pH 4.2. Langmuir, 21,

678 

7786–7795.

679 

Tang, E. S. K., Huang, M., & Lim, L. Y. (2003). Ultrasonication of

680 

chitosan and chitosan nanoparticles. International Journal of

681 

Pharmaceutics, 265, 103–114.

682 

Turgeon, S. L., & Laneuville, S. I. (2009). Protein + polysaccharide

683 

coacervates and complexes: from scientific background to their

684 

application as functional ingredients in food products. In S.

685 

Kasapis, I. T. Norton, & J. B. Ubbink (Eds.), Modern biopolymer

686 

science (pp. 327–363). New York: Elsevier.

687 

Uruakpa, F. O., & Arntfield, S. D. (2004). Rheological characteristics of

688 

commercial canola protein isolate–κ-carrageenan systems. Food

689 

Hydrocolloids, 18, 419–427.

690 

Weinbreck, F., Nieuwenhuijse, H., Robijn, G. W., & de Kruif, C. G.

691 

(2003). Complex formation of whey proteins: exocellular

692 

polysaccharide EPS B40. Langmuir, 19, 9404–9410.

693 

Weinbreck, F., Nieuwenhuijse, H., Robijn, G. W., & de Kruif, C. G.

694 

(2004). Complexation of whey proteins with carrageenan. Journal

695 

of Agricultural and Food Chemistry, 52, 3550–3555.

696 

Weiss, J., Kristbergsson, K., & Kjartansson, G. T. (2011). Engineering

697 

food ingredients with high-intensity ultrasound. In H. Feng, G. V.

698 

Barbosa-Cánovas, & J. Weiss (Eds.), Ultrasound technologies for

699 

food and bioprocessing (pp. 239–285). New York: Springer. 32   

700 

Zimet, P., & Livney, Y. D. (2009). Beta-lactoglobulin and its

701 

nanocomplexes with pectin as vehicles for ω-3 polyunsaturated

702 

fatty acids. Food Hydrocolloids, 24, 374–383.

33   

703 

Figure Captions

704  705 

Fig. 1. Effect of sonication on the apparent viscosity reduction of 0.5%

706 

w/w KC solution at different amplitudes and temperatures as a function

707 

of time: ( ) Amp. 50%, Temp. 75 °C; ( ) Amp. 100%, Temp. 75 °C; ( )

708 

Amp. 50%, Temp. 25 °C; ( ) Amp. 100%, Temp. 25 °C.

709  710 

Fig. 2. Turbidimetric analysis as a function of pH: ( ) BLG dispersion

711 

(0.1% w/w), (×) KC (IN) and ( ) KC (US) dispersions (0.05% w/w); ( )

712 

BLG-KC (IN) and ( ) BLG-KC (US) at 0.15% w/w total biopolymer

713 

concentration and BLG:KC weight ratio of 2:1. (pHc: formation of soluble

714 

complexes, pHφ1: formation of interpolymer complexes)

715  716 

Fig. 3. Thermograms corresponding to the titration of the BLG

717 

dispersion (0.0828% w/v) with (a) KC (IN) and (b) KC (US) dispersions

718 

(0.1% and 0.175% w/w, respectively) in 5 mM sodium citrate buffer (pH

719 

4.25) at 25 °C.

720  721 

Fig. 4. Binding isotherms (solid lines) corresponding to the titration of

722 

the BLG dispersion (0.0828% w/v) with ( ) KC (IN) and ( ) KC (US)

723 

dispersions (0.1% and 0.175% w/w, respectively) in 5 mM sodium

724 

citrate buffer and optical density profiles (dashed lines) of the BLG

725 

dispersion (0.1% w/w) mixed with ( ) KC (IN)- and ( ) KC (US)-

34   

726 

dispersions, then acidified to pH 4.25 at 25 °C as a function of KC/BLG

727 

weight ratio (total biopolymer concentration of 0.1% - 0.3% w/w).

728  729 

Fig. 5. a and b: optical images of the BLG dispersion (0.1% w/w) mixed

730 

with KC (IN)- and KC (US)- dispersions, respectively, then acidified to

731 

pH 4.25 at different KC/BLG weight ratios (total biopolymer

732 

concentration of 0.1% - 0.3% w/w). c-e: phase contrast optical

733 

micrographs of KC (IN)-BLG mixture at weight ratio of 0.1 just after

734 

mixing and acidification to pH 4.25, during precipitation and after

735 

precipitation (bottom phase), respectively. f: phase contrast

736 

micrographs of KC (IN)-BLG mixture at weight ratio of 1.

737  738 

Fig. 6. ζ-potential (a) and effective diameter (b) profiles of the BLG

739 

dispersion (0.1% w/w) mixed with ( ) KC (IN)- and ( ) KC (US)-

740 

dispersions then acidified to pH 4.25 as a function of KC/BLG weight

741 

ratio (total biopolymer concentration of 0.1% - 0.3% w/w).

742 

35   

743  744  745  746  747  748  749  750  751 

Fig. 1

752  753  754 

Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed

755 

Hadi Razavi, Ali Akbar Moosavi-Movahedi, Ali Akbar Saboury,

756 

Mohammad Amin Mohammadifar, Asgar Farahnaky, Maliheh Sadat

757 

Atri, Paul Van der Meeren

36   

758  759  760  761 

pHφ1

762  pHc

763  764  765  766  767 

Fig. 2

768  769  770  771 

Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed Hadi Razavi, Ali

772 

Akbar Moosavi-Movahedi, Ali Akbar Saboury, Mohammad Amin Mohammadifar,

773 

Asgar Farahnaky, Maliheh Sadat Atri, Paul Van der Meeren

774 

37   

776  777  778  779  a

780 

b

781  782  783 

Fig. 3

784  785  786  787 

Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed Hadi Razavi, Ali

788 

Akbar Moosavi-Movahedi, Ali Akbar Saboury, Mohammad Amin Mohammadifar,

789 

Asgar Farahnaky, Maliheh Sadat Atri, Paul Van der Meeren

790  791  792  793  794  39   

795  796  797  798  799  800  801  802  803  804  805  806  807  808  809  810 

Fig. 4

811  812  813  814 

Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed Hadi Razavi, Ali

815 

Akbar Moosavi-Movahedi, Ali Akbar Saboury, Mohammad Amin Mohammadifar,

816 

Asgar Farahnaky, Maliheh Sadat Atri, Paul Van der Meeren

817  818 

40   

819 

 

820 

 

821 

 

c

822  1000   X 823 

824 

1000 X

e

40 μm

400 X

100 μm

f

1000 X

40 μm

 

0

0.01

0.03

0.05

0.10

0.15

0.20

0.25

0.37

0.50

0.75

1

1.25

1.5

2

0

0.01

0.03

0.05

0.10

0.15

0.20

0.25

0.37

0.50

0.75

1

1.25

1.5

2

 

a

825 

 

826 

 

827 

40 μm

d

 

b

828 

 

829 

 

830 

 

831 

Fig. 5

832  833 

Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed Hadi Razavi, Ali

834 

Akbar Moosavi-Movahedi, Ali Akbar Saboury, Mohammad Amin Mohammadifar,

835 

Asgar Farahnaky, Maliheh Sadat Atri, Paul Van der Meeren

836 

For in print

837  838 

1000 X

40 μm

1000 X

40 μm

400 X

41   

100 μm

1000 X

40 μm

839 

  a

840  841  842  843  844  845  846  b

847  848  849  850  851  852  853  854 

Fig. 6

855 

42   

856 

Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed Hadi Razavi, Ali

857 

Akbar Moosavi-Movahedi, Ali Akbar Saboury, Mohammad Amin Mohammadifar,

858 

Asgar Farahnaky, Maliheh Sadat Atri, Paul Van der Meeren

43   

859 

Sonication decreased the apparent viscosity of the κ–carrageenan solution.

860 

Sonication reduced the affinity constant between κ–carrageenan and β-

861 

lactoglobulin.

862 

Sonication downsized nanoparticles formed in the mixed dispersion with protein.

863 

Complexation in mixed BLG-KC dispersions is a nucleation and growth

864 

mechanism.

865 

44