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: S0308-8146(13)00253-7

DOI: http://dx.doi.org/10.1016/j.foodchem.2013.02.090

Reference: FOCH 13767

To appear in: Food Chemistry

Received Date: 20 November 2012

Revised Date: 16 February 2013

Accepted Date: 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 β-

lactoglobulin – κ –carrageenan aqueous mixtures as affected by polysaccharide sonication, Food Chemistry (2013),

doi: http://dx.doi.org/10.1016/j.foodchem.2013.02.090

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1

Complex coacervation of β-lactoglobulin – κ–carrageenan aqueous 1

mixtures as affected by polysaccharide sonication 2

Seyed Mohammad Hashem Hosseinia,e , Zahra Emam-Djomeha,*, 3

Seyed Hadi Razavia, Ali Akbar Moosavi-Movahedib, Ali 4

Akbar Sabouryb, Mohammad Amin Mohammadifarc, Asgar 5

Farahnakyd, Maliheh Sadat Atrie, Paul Van der Meerenf 6

a Department of Food Science, Technology and Engineering, Faculty of 7

Agricultural Engineering and Technology, Agricultural Campus of the 8

University of Tehran, Karadj, Iran, Postal Code: 31587-11167, P. O. 9

Box: 4111 10

b Institute of Biochemistry and Biophysics (IBB), University of Tehran, 11

Tehran, Iran 12

c Department of Food Science and Technology, Faculty of Nutrition 13

Sciences, Food Science and Technology/National Nutrition and Food 14

Technology Research Institute, Shahid Beheshti University of Medical 15

Sciences, Tehran, Iran, P. O. Box: 19395-4741 16

d Department of Food Science and Technology, School of Agriculture, 17

Shiraz University, Shiraz, Iran 18

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

E-mail address: emamj@ut.ac.ir (Z. Emam-Djomeh).

2

e Molecular and Cell Biology Department, University of Mazandaran, 19

Babolsar, Iran 20

f Particle and Interfacial Technology Group, Faculty of Bioscience 21

Engineering, Ghent University, Coupure Links 653, B-9000 Gent, 22

Belgium 23

3

24

ABSTRACT 25

The influence of κ-carrageenan (KC) depolymerization using 26

ultrasound on its interaction with β-lactoglobulin (BLG) was investigated 27

by isothermal titration calorimetry (ITC), turbidity measurement, 28

dynamic light scattering and zeta-potential analyses. Time and 29

amplitude of the sonication had a direct effect on the viscosity 30

depression, while the sonication temperature had an opposite effect. 31

ITC measurements indicated that the sonication significantly decreased 32

the affinity constant between KC and BLG. The zeta-potential of the 33

nanoparticles produced from ultrasonicated (US) KC-BLG associative 34

interaction was lower than of those produced from intact (IN) KC-BLG 35

interaction. These differences were attributed to the lower charge 36

density of the KC (US) as a result of sonochemical interactions. 37

Polydispersity and particle size measurements showed that the effect of 38

the sonication was the homogenization of the nanoparticles in the mixed 39

dispersion. The nanoparticles formed may therefore be useful as a 40

delivery system for fortification purposes of acidic beverages. 41

42

Keywords: Coacervation; κ–carrageenan; β-lactoglobulin; Ultrasound; 43

Nanoparticle; Isothermal titration calorimetry 44

4

45

1. Introduction 46

Carrageenans are a family of sulfated linear polysaccharides of D-47

galactose and 3,6-anhydro-D-galactose which are isolated from red 48

algae (Gu, Decker, & McClements, 2005). They are widely used as a 49

thickening, gelling and stabilizing agent as well as fat substitutes in the 50

food industry, particularly in milk products (Weinbreck, Nieuwenhuijse, 51

Robijn, & de Kruif, 2004). There are three major types of carrageenan 52

including kappa (κ), iota (ι), and lambda (λ) -carrageenans, which differ 53

in the number of the sulphate groups (1, 2 and 3, respectively) and their 54

position (Gu et al., 2005). κ- and ι-carrageenan in aqueous solution 55

undergo a thermoreversible conformational ordering (transition from coil 56

(unstructured) at elevated temperatures to helix (ordered) at low 57

temperatures followed by aggregation and network formation at high 58

polysaccharide concentration (i.e. 1%)) through sulfate groups and the 59

3,6-anhydro-D-galactopyransyl ring (Ould Eleya & Turgeon, 2000; 60

Uruakpa & Arntfield, 2004; Gu et al., 2005). κ and ι -carrageenans have 61

also gelling properties in the presence of cations, which is influenced by 62

the nature (i.e. K+ and Ca2+, respectively) and concentration of cations 63

present in the solution and by the biopolymer concentration (Uruakpa et 64

al., 2004; Gu et al., 2005). λ-carrageenan has a random coil 65

conformation at all temperatures and is unable to form gels (Gu et al., 66

2005). 67

5

Protein-polyelectrolyte (DNA) complexes play important roles in 68

living structures (Burova, Grinberg, Grinberg, Usov, Tolstoguzov, & de 69

Kruif, 2007). The considerable interest in biopolymer particles 70

(Klemmer, Waldner, Stone, Low, & Nickerson, 2012; Huang, Sun, Xiao, 71

& Yang, 2012) results from the potential applications of engineered 72

novel structures in the protection of bioactive compounds (Jun-xia, Hai-73

yan, & Jian, 2011), interfacial stabilization (Schmitt, da Silva, Bovay, 74

Rami-Shojaei, Frossard, Kolodziejczyk, & Leser, 2005; Dickinson, 2008) 75

and texturizing such as fat replacing by simulating the rheological, 76

optical and sensorial properties of the lipid droplets (Laneuville, Paquin, 77

& Turgeon, 2005). The phase separation of a protein and 78

polysaccharide aqueous mixture can be classified into two main 79

categories: associative and segregative phase separation. In an 80

associative phase separation (also known as thermodynamic 81

compatibility), both biopolymers are enriched in one of the separating 82

phases (coacervate-rich phase), while the other phase contains mostly 83

solvent (Turgeon & Laneuville, 2009). Associative phase separation is 84

mainly driven by the electrostatic attraction between polyelectrolytes 85

under conditions where they have opposite electrical charges (i.e. pHs 86

between the pKa of the polysaccharide and the isoelectric point (Ip) of 87

the protein) (Turgeon et al., 2009; Chang, McLandsborough, & 88

McClements, 2011). Other non-covalent interactions can also occur 89

such as hydrophobic interaction and hydrogen bonding, making the 90

6

complexes more stable (Klemmer et al., 2012). In a segregative phase 91

separation (also known as thermodynamic incompatibility), two 92

biopolymers are separated into two different phases. This is the case 93

mainly for two nonionic biopolymers, two similarly charged biopolymers, 94

or a charged biopolymer plus a nonionic biopolymer (Fang, Li, Inoue, 95

Lundin, & Appelqvist, 2006). Biopolymer size (molecular weight) and 96

type, chain conformation and flexibility, distribution of reactive groups 97

and the charge density, solvent conditions (e.g., pH, ionic strength, and 98

temperature), protein to polysaccharide mixing ratio, total biopolymer 99

concentration, stirring and pressure are important factors controlling the 100

phase separation behaviors of biopolymer mixtures, particularly of 101

charged biopolymer mixtures and could result in either associative or 102

segregative phase separation (Fang et al., 2006; Turgeon et al., 2009). 103

The gel forming property and rheology of the κ-carrageenan (KC) 104

either alone or in combination with globular proteins is well known. 105

However, the study of complex coacervation between KC and proteins 106

in dilute aqueous mixtures has been limited (Fang et al., 2006; Burova 107

et al., 2007). The purpose of the current work is to study the effect of 108

KC depolymerization using high intensity ultrasound on the complex 109

coacervation between KC (at non-gelling concentrations) and β-110

lactoglobulin (BLG). The target pH was chosen to be 4.25, based on the 111

pH of a clear traditional herbal beverage in order to assess the 112

capability of the produced nanoparticles as delivery systems for 113

7

fortification purposes in the future. To the best of our knowledge, the 114

interaction between KC and BLG has not been studied using isothermal 115

titration calorimetry (ITC). 116

117

2. Materials and methods 118

2.1. Materials 119

κ–carrageenan (KC, 504 kDa, composition: 90% (w/w) KC, 8% 120

(w/w) moisture and 2% (w/w) ash), β-lactoglobulin from bovine milk 121

(BLG, 18.4 kDa, composition: 93% (w/w) BLG, 5.4% (w/w) moisture and 122

1.6% (w/w) ash, a mixture of genetic variants A and B) and sodium 123

azide (as a preservative, minimum purity 99.5%) were purchased from 124

Sigma Chemical Co. (St. Louis, MO, USA). Analytical grade 125

hydrochloric acid was obtained from Merck Co. (Darmstadt, Germany). 126

Deionized water (18.2 MΩ cm resistivity) from a Nanopure water system 127

(Nanopure Infinity, Barnstead International, IA, USA) was used for the 128

preparation of all solutions. In this study all materials were used as such 129

received. 130

2.2. Preparation of solutions 131

KC stock solution (0.5% (w/w), pH: 7.47) was prepared by dispersing 132

into Nanopure water containing 0.03% (w/w) sodium azide at room 133

temperature followed by heating to 85 °C for 30 min under magnetic 134

stirring in order to ensure a complete hydration of polysaccharide. BLG 135

8

stock solution (0.4% (w/w0, pH~ 6.94) was prepared by dispersing into 136

Nanopure water containing 0.03% (w/w) sodium azide and stirred 137

overnight at 250 rpm and ambient temperature in order to use on the 138

following day. 139

2.3. Ultrasonic treatment of KC solution 140

KC stock solution (30 g) was treated by an ultrasonic processor 141

(Hielscher UP200S, power 200 W, frequency 24 kHz, Dr Hielscher Co., 142

Teltow, Germany) for different times (10, 20 or 30 min) at different 143

temperatures (25 or 75 °C) and different amplitudes (50 or 100 %). The 144

sample was held in a temperature controlled water bath to prevent the 145

temperature rise by the sonication. A standard tapered horn tip of 5 mm 146

end diameter was immersed 1.5 cm into the solution during 147

ultrasonication. The ultrasound irradiation was produced directly from 148

the horn tip under continuous mode. 149

2.4. Viscosity measurement 150

The apparent viscosity of the unsonicated (control) and sonicated 151

samples was measured at 25 °C using a rotational viscometer (Model 152

LV-DVII+, Brookfield Engineering Laboratories, Middleboro, MA, USA) 153

equipped with spindle number 1 rotated at 10 rpm. 154

2.5. Turbidimetric analysis at different pHs 155

9

Mixtures of BLG and KC were prepared by first mixing and then 156

diluting the stock solutions at a 2:1 (w/w) BLG:KC mixing ratio and a 157

total biopolymer concentration of 0.15% (w/w). The mixture was 158

acidified gradually by the addition of 0.1 M HCl (pH range of 5-7), 0.4 N 159

HCl (pH range of 3-5) and 2 M HCl (pH range of <1-3) with gentle 160

magnetic stirring for 2 min at each pH level before decreasing it to the 161

next pH. Dilution effects were considered to be minimal. The optical 162

density of the biopolymer mixtures with decreasing pH (from pH ~7 to 163

~1) was analyzed using a UV/visible light spectrophotometer at 600 nm 164

(BioQuest CE 2502, Cecil Ins., Cambridge, UK) using plastic cuvettes 165

(1 cm path length). Deionized water was used as a blank reference. 166

Critical pH values (pHc: formation of soluble complexes, pHφ1: formation 167

of insoluble complexes, pHopt: maximum optical density, pHφ2: 168

dissolution of complexes) were measured graphically as the intersection 169

point of two curve tangents. BLG and KC solutions were used as 170

controls at their corresponding concentrations (0.1 and 0.05 % w/w, 171

respectively). 172

2.6. Isothermal titration calorimetry (ITC) 173

ITC measurements were carried out with a VP-ITC calorimeter 174

(Microcal Inc., Northampton, MA, USA) in order to measure the 175

enthalpic and entropic changes due to BLG-KC interactions at 25 °C. 176

Before titration, the biopolymers were separately dissolved in 5 mM 177

sodium citrate buffer solution (pH 4.25). Heating at 85 °C for 30 min 178

10

was required for KC. The buffer was used to remove the experimental 179

errors resulting from pH mismatch. The BLG dispersion containing 180

about 1 mg/ml was filtered through a 0.22-µm low protein binding 181

polyether sulphone (PES) syringe filter (MS®, TX, USA) to obtain 182

aggregate free BLG dispersion. The concentration of BLG dispersion 183

(monomeric equivalent) was measured by UV/visible light spectroscopy 184

using a specific extinction coefficient of 17600 M-1 cm-1 at 278 nm, as 185

reported by Liang, Tajmir-Riahi, & Subirade (2008) and amounted to 186

0.828 mg/ml. The sodium citrate buffer solution was used as blank 187

reference. The dispersions were degassed under vacuum for 3 min by 188

means of a device provided with the ITC apparatus. The injector-stirrer 189

syringe (290 µL) was loaded with KC solution. Portions of 15 μl (except 190

for the first injection which was 5 µl) of KC solution (0.1 and 0.175% 191

w/w for intact (IN) and sonicated (US) for 20 min at 25 °C and amplitude 192

100% polysaccharides, respectively) were injected sequentially into the 193

titration cell (V = 1.408 ml) initially containing either aggregate free BLG 194

dispersion or buffer solution. The duration of each injection was 20 s, 195

and the equilibration time between consecutive injections was 300 s. 196

During the titration, the stirring speed was 310 rpm. The heat of dilution 197

from the blank titration of KC solution into sodium citrate buffer was 198

measured, and the dilution heat was subtracted from the raw data to 199

measure corrected enthalpy changes. The results are reported as the 200

change in enthalpy per gram of KC (IN) and KC (US) injected into the 201

11

reaction cell. The low concentrations of the biopolymer solutions and 202

the mild temperature supplied a low viscosity at any point of titration, 203

which did not affect the mechanical stirring of the microcalorimeter. 204

Calorimetric data analysis was carried out with Microcal ORIGIN 205

software (v.7.0). Thermodynamic parameters including binding 206

stoichiometry (N), affinity constant (K), enthalpy (ΔH) and entropy (ΔS) 207

changes were calculated by iterative curve fitting of the binding 208

isotherms. The Gibbs free energy change (ΔG) was calculated from the 209

equation (ΔG = ΔH - TΔS). 210

2.7. BLG-KC complexation 211

BLG-KC complexes from the mixing of BLG and KC dispersions at 212

different polysaccharide/protein weight ratios were obtained by the post-213

blending acidification method. A series of samples containing a fixed 214

protein concentration of 0.1% (w/w) but different KC concentrations (0–215

0.2 % (w/w)) was prepared by mixing different ratios of 0.4% (w/w) BLG 216

and 0.5% (w/w) KC stock dispersions as well as deionized water. 217

Biopolymer solutions were adjusted to pH 4.25 using 0.4, 0.1 and/or 218

0.01 M HCl solutions. These solutions were stirred for 1 h and then 219

allowed to equilibrate at ambient temperature for 18–24 h prior to 220

analysis. 221

2.8. Characterization of the complexes 222

2.8.1. Turbidity measurement 223

12

The turbidity of samples was quantified by their absorbance 224

measured at 600 nm using plastic cuvettes (1 cm path length). Sample 225

solutions were vortexed for 5 s prior to analysis. Highly turbid samples 226

were diluted before measurement using deionized water pre-adjusted 227

with HCl to pH 4.25. 228

2.8.2. Particle size and zeta- (ζ-) potential analyses 229

Measurements of particle size distribution were carried out using a 230

dynamic light scattering (DLS) instrument (90Plus, Brookhaven 231

Instruments Corp., Vienna, Austria). Analyses were carried out at a 232

scattering angle of 90° at 25 °C. The effective diameter (also called Z-233

average mean diameter) was only measured in samples which have 234

shown no sedimentation after equilibration. The Z-average mean 235

diameter and polydispersity index (PDI) were obtained by cumulant 236

analysis. The ζ-potential was determined by laser Doppler anemometry 237

with palladium electrodes using a ZetaPals instrument (Brookhaven 238

Instruments Corp.) at fixed light scattering angle of 90° at 25 °C. The ζ-239

potential (mV) was calculated from the electrophoretic mobility using the 240

Helmholtz-Smoluchowski equation. During both dynamic light scattering 241

and electrophoretic light scattering measurements, the viscosity of the 242

continuous phase were assumed to correspond to pure water. 243

2.8.3. Phase contrast optical microscopy 244

13

BLG and KC complexed mixtures were microscopically 245

characterized at different magnifications using a phase contrast optical 246

microscope (Olympus CX40, Olympus Optical Co., Tokyo, Japan) 247

equipped with a AxioCam ERc 5s video camera (Carl Zeiss 248

Microimaging Gmbh, Göttingen, Germany) controlled by an image 249

processor (Kappa ImageBase 2.5). Fifteen microliters of the dispersion 250

were placed between glass slides and then examined. A drop of 251

immersion oil (Merck Co., Darmstadt, Germany) was placed on the 252

glass slide before characterization with 1000 × magnification. 253

2.9. Statistical analysis 254

Measurements were performed at least two or three times using 255

freshly prepared samples and analyzed by ANOVA using the MSTATC 256

programs (version 2.10, East Lansing, MI, USA). Results are reported 257

as means and standard deviations. Comparison of means was carried 258

out using Duncan’s multiple range tests at a confidence level of 0.05. 259

260

3. Results and discussion 261

3.1. Changes in viscosity after sonication 262

The effectiveness of the sonication has been evaluated by 263

measuring the changes in apparent viscosity which is shown versus 264

sonication time at different amplitudes and temperatures in Fig. 1. There 265

14

was a severe decrease in the viscosity of the KC solution (0.5% w/w). 266

As an example, the viscosity of 19 mPa.s for the untreated solution 267

decreased to 3 mPa.s after sonication for 30 min at 25 °C and 268

amplitude 100%. This phenomenon is due to the cleavage of the 269

polysaccharide backbone which results in a decrease in the molecular 270

weight of ultrasonically treated polysaccharides and hence decreasing 271

the effective volume of the polysaccharide chains (Weiss, 272

Kristbergsson, & Kjartansson, 2011). The depolymerization process 273

occurs through the effects of acoustic cavitation and can involve two 274

possible mechanisms: mechanical degradation of the polymer from 275

collapsed cavitation bubbles and chemical degradation as a result of the 276

chemical reaction between the polymer and high energy molecules 277

such as hydroxyl radicals produced from cavitation (Chemat, Huma, & 278

Kamran Khan, 2011). According to Iida, Tuziuti, Yasui, Towata, and 279

Kozuka, (2008), the effect of ultrasonication on viscosity depression is 280

extremely dependent on the mechanical and structural properties of the 281

polysaccharides, i.e. whether the polysaccharides have a stiff linear or 282

random coil configuration. For example, pectin showed a rather small 283

change (about 50% decrease) in viscosity, whereas glucomannan 284

showed a much more severe decrease in viscosity by sonication (Iida et 285

al., 2008). Fig. 1 clearly shows that the sonication temperature had an 286

inverse effect on the viscosity depression when the other parameters 287

(time and amplitude) remained constant; however, this effect was less 288

15

pronounced at higher sonication times. Increasing in the sonication 289

temperature may increase the flexibility of the molecular chain. 290

According to Weiss et al. (2011), flexible biopolymer chains are less 291

susceptible to decreases in viscosity upon ultrasonication. An increase 292

in temperature also leads to an increase in water vapor pressure, which 293

penetrates in larger amounts into the cavitation bubbles and weakens 294

the collapse energy by the so-called “cushioning effect” (Kardos & 295

Luche, 2001). The viscosity of the KC solution decreased significantly 296

(*p<0.05) with increasing time and amplitude of the ultrasonication 297

process, and tends to approach a limiting viscosity value, which may 298

correspond to low molecular weight fractions for which the application of 299

high-intensity ultrasound does not lead to further backbone breakdown 300

(Weiss et al., 2011). 301

3.2. Turbidimetric analysis 302

Turbidimetric analysis as a function of pH was used to study the 303

kinetics of associative phase separation within mixed BLG-KC systems 304

(Fig. 2). Indeed, pH affects the ionization degree of the functional 305

groups of the protein and polysaccharide and electrostatic complexing 306

takes place under acidification (Weinbreck, Nieuwenhuijse, Robijn, & de 307

Kruif, 2003). In the absence of protein, KC solutions remained 308

transparent in studied pH range indicating that they did not form 309

particles large enough to scatter light strongly, due to the sulfate groups 310

which were always ionized, giving the molecules an electrostatic 311

16

repulsion. The BLG dispersion showed a broad peak in the measured 312

turbidity versus pH profile with a maximum value around pH 4 to 5 due 313

to self-association around the Ip of BLG which decreased as the pH 314

became more acid or alkaline. Generally, BLG-KC (US) complexed 315

solutions showed lower turbidity than BLG-KC (IN) solutions which can 316

be attributed to the production of smaller polysaccharide chains after 317

sonication. At pH > 5.30-5.50, biopolymers were considered to be co-318

soluble, although a very slight increase in turbidity of the systems can 319

be seen (Fig. 2) which may be the result of non-coulombic interactions 320

such as hydrophobic and hydrogen bindings. Previous researchers 321

have also found little interaction between BLG and pectin at high pH 322

values (Girard, Turgeon, & Gauthier, 2002). Another possibility is that 323

weak local electrostatic interactions may occur between protein and 324

polysaccharide molecules as shown in work by Dickinson & Galazka 325

(1991). They have demonstrated that native BLG and anionic 326

polysaccharides (dextran sulfate and propylene glycol alginate) could 327

form ionic complexes at neutral pH due to charge-induced charge 328

interactions. One beneficial consequence of this complexation is the 329

protection against a loss of solubility due to aggregation induced by 330

heating or high-pressure processing (Dickinson, 2008). Soluble 331

complexes were formed at a pHc (~5.30-5.50) that was independent of 332

the KC type (sonicated or non-sonicated). Weinbreck et al. (2004) 333

reported a pHc value of 5.5 for different mixtures of whey protein isolate 334

17

and non-gelling carrageenan (comprised mainly λ-carrageenan). 335

According to Turgeon et al. (2009) and Weinbreck et al. (2004), this 336

transition occurs at the molecular level (i.e. complexation begins 337

between a single polysaccharide chain and a defined amount of protein) 338

and is independent on the molecular weight and the mixing ratio. 339

Formation of soluble complexes occurred at a pHc above the Ip of the 340

BLG (~4.7-5.2) which is thought to be due to the ability of the globular 341

proteins for charge regulation around the Ip resulting from their 342

electrical capacitance properties (Dickinson, 2008) and/or due to the 343

presence of positive patches (localized regions with higher charge 344

density) on the surface of BLG as a result of low ionic strength 345

conditions which inhibit charge screening (Weinbreck et al., 2003; 346

Turgeon et al., 2009). When the pH decreased further, the critical pHφ1 347

(~4.85) was reached as a result of nucleation and growth-type kinetics 348

(Sanchez, Mekhloufi, & Renard, 2006). At this point, more and more 349

protein molecules become attached to the polysaccharide (due to an 350

increase in charge density of the protein) until electroneutrality was 351

attained yielding neutral interpolymeric complexes that tend to 352

precipitate (Turgeon et al., 2009). It should be noted that the measured 353

optical density is the result of the number and size of the biopolymer 354

complexes. The highest amount of BLG-KC interactions (pHopt) 355

occurred at pH 1-2 with maximum optical densities of 1.8 and 1.4 for 356

BLG-KC (IN) and BLG-KC (US) mixtures, respectively, which is the 357

18

result of various attractive forces (e.g. van der Waals, hydrophobic, and 358

electrostatic interactions between oppositely charged groups). In this 359

work, pHφ2 was absent since the dissociation of KCs’ sulphate groups is 360

not suppressed at low pH and they remain charged (Turgeon et al., 361

2009). 362

3.3. ITC results 363

The heat flow versus time profiles resulting from the titration of 364

BLG with intact and sonicated KCs at 25 °C and pH 4.25 are shown in 365

Fig. 3 a and b, respectively. The area under each peak represented the 366

heat exchange within the cell containing BLG after each KC injection. 367

The injection profiles in the sample cell were exothermic and decreased 368

regularly to a state of thermodynamic stability (about zero) after the 15th 369

and 12th injections of KC (IN) (0.1% w/w) and KC (US) (0.175% w/w), 370

respectively. Exothermicity is associated with the nonspecific 371

electrostatic neutralization of the opposite charges carried by the two 372

biopolymers indicating an enthalpic contribution of complex 373

coacervation (Girard, Turgeon, & Gauthier, 2003; Schmitt et al., 2005), 374

while its regular decrease is attributed to a reduction in free protein 375

remaining in the reaction cell after successive injections, which explains 376

the lowering of the energy released. Girard et al. (2003) reported a 377

similar exothermic sequence for BLG interaction with low- and high-378

methoxyl pectin, while Aberkane, Jasniewski, Gaiani, Scher, & Sanchez 379

(2010) reported an exothermic-endothermic sequence for BLG – gum 380

19

Arabic interaction. To characterize thermodynamic parameters, the 381

binding isotherms obtained by integrating of the isotherm peaks and 382

subtraction of the heats of dilution of KCs into buffer solution were fitted 383

using the one site binding model provided by the Microcal Origin 384

software and plotted against KC/BLG weight ratio (Fig. 4). The first 385

injection was not taken into account for analysis. The calculation gives a 386

typical sigmoidal saturation curve, which can be concluded as a 387

progressive binding of the BLG molecules present in the titration cell to 388

the binding sites along the KC backbone. The isoenthalpic plateau 389

observed in the binding isotherms was reached at KC (IN) and KC (US) 390

to BLG weight ratios of about 0.20 and 0.30, respectively. Calculation of 391

the thermodynamic parameters including binding stoichiometry (N), 392

affinity constant (K), enthalpy (ΔH) and entropy (TΔS) contributions and 393

Gibbs free energy change (ΔG) for the interaction between KC and BLG 394

showed that the binding enthalpy was negative and favorable, whereas 395

the binding entropy was unfavorable (negative) during KC-BLG 396

interaction. According to Ou and Muthukumar (2006) the complexation 397

between weakly charged polyelectrolytes is driven by a negative 398

enthalpy due to the electrostatic interaction between two oppositely 399

charged components, while counterion release entropy plays only a 400

minor role. The unfavorable entropic effects originate mainly from the 401

loss in biopolymer conformational freedom after association (Dickinson, 402

2008). BLG and KC (IN) interacted with a high affinity constant (KIN: 403

20

10476 ± 6032 g-1.l) and a strong ΔHIN of (-2.706 ± 0.042 cal.g-1). 404

Assuming a molecular weight of 504 kDa for KC (IN), about 142 BLG 405

molecules were involved in the interaction process with KC (IN) (NIN: 406

192.3 ± 1.4 mg KC (IN)/ g BLG). Schmitt et al. (2005) and Aberkane et 407

al. (2010) reported enthalpy change (-0.933 ± 0.001 and -1.072 ± 0.014 408

cal.g-1), affinity constant (25.4 ± 13.0 and 896 ± 66 g-1.l) and binding 409

stoichiometry (86 and 90 BLG molecules) values upon complexation of 410

BLG with Acacia gum (MW ~ 540 kDa) at pH 4.2, respectively. The 411

differences can be explained by the higher charge density on KC (IN) 412

molecules than on Acacia gum molecules. The interaction between BLG 413

and KC (US) occurred with significant (*p<0.05) lower affinity constant 414

(KUS: 535 ± 137 g-1.l) as well as higher binding stoichiometry (NUS: 214.1 415

± 3.0 mg KC (US)/ g BLG) and higher enthalpy change (ΔHUS: -2.940 ± 416

0.062 cal.g-1) values. The decrease in the affinity constant of the KC 417

(US)-BLG interaction can be attributed to the lower negative charge 418

density on KC (US) than on KC (IN) (section 3.4.2.) and changes in the 419

helical structure of the polysaccharide after sonication. These results 420

are in good agreement with those of Chang, McLandsborough and 421

McClements (2012). They found that ε-polylysine (an antimicrobial 422

cationic polyelectrolyte) interacted with an anionic polysaccharide 423

(pectin) more strongly when the charge density on the pectin molecules 424

increased (i.e. with decreasing degree of esterification). The 425

unfavorable entropic contribution (TΔS) was relatively in the same 426

21

range (-2.680 ± 0.034 and -2.780 ± 0.038 cal.g-1 for KC (IN) and KC 427

(US), respectively) as the favorable enthalpic contribution, indicating 428

that any change in enthalpy is accompanied by a similar change in 429

entropy, that is, entropy-enthalpy compensation occurred (Aberkane et 430

al., 2010). The changes in Gibbs free energy were negative for the two 431

types of KCs (-0.026 ± 0.008 and -0.160 ± 0.024 cal.g-1 for KC (IN) and 432

KC (US), respectively) indicating the spontaneous nature of the 433

interactions. The difference in Gibbs free energy changes can be 434

attributed to the fact that the loss in polysaccharide conformational 435

freedom after association is more considerable for larger molecules 436

than smaller molecules. 437

3.4. Complex evaluation 438

3.4.1. Turbidity versus KC/BLG weight ratio profiles 439

The turbidity of the BLG-KC solutions was measured as a function 440

of KC/BLG weight ratio at pH 4.25 to provide some deeper insights into 441

the mechanisms of complexed biopolymer nanoparticle formation (Fig. 442

4) and to find the most suitable conditions for forming stable 443

nanoparticles. The initial turbidity of the BLG suspension in the absence 444

of KC was about 0.113, because of some aggregation of proteins 445

around pH 4.25. The KC to BLG weight ratio had a major effect on the 446

solution turbidity and degree of sediment formation in the solutions. At 447

KC (IN)/BLG weight ratios lower than 0.50, complexed biopolymer 448

22

particles are unstable to aggregation because they can achieve 449

electrical neutrality (protein depletion) due to the high protein binding 450

(Weinbreck et al., 2003) leading to high turbidity and aggregation as 451

seen in Fig. 5 a,b (white sediment at the bottom of the glass vials with a 452

clear serum layer on top). BLG/KC (IN) mixture obtained at 1:10 453

polysaccharide:protein weight ratio was microscopically characterized 454

just after acidification to pH 4.25, during precipitation and after 455

precipitation (lower phase) (Fig. 5c-e, respectively). The initial structures 456

are of spherical shape. It seems that complex coacervation in mixed 457

BLG-KC dispersions is a nucleation and growth mechanism. Similar 458

mechanism was reported by Sanchez et al. (2006), in mixture of BLG 459

with gum Arabic (as a polysaccharide with different flexibility and charge 460

density). According to Sanchez et al. (2006), nucleation and growth 461

mechanism is the general mechanism of complexation/coacervation 462

between biological macromolecules. Complexes grew in size during 463

precipitation and their number was reduced. This feature could be due 464

to coalescence of complexes or Ostwald ripening (Sanchez et al., 465

2006). These samples are unsuitable for utilization as stable colloidal 466

delivery systems in the food industry. Particles formed in the BLG/KC 467

(US) mixed system did not markedly differ in structure as compared to 468

the previous ones (data not shown). At higher polysaccharide/protein 469

weight ratios the samples were less turbid and did not exhibit 470

sedimentation, indicating that colloidal dispersions containing small 471

23

stable complexes with higher stability than the protein aggregates 472

themselves were formed, presumably because the electrostatic and 473

steric repulsion resulting from the presence of a polysaccharide shell 474

around the protein core is sufficiently strong to prevent aggregation as 475

revealed by the influence of KC on the ζ-potential of the complexes (Fig. 476

6a). Since the resolution of the phase contrast microscope is not 477

enough to visualize nanoparticles, no structure was detected at KC 478

(IN)/BLG weight ratio of 1:1 (Fig. 5f). These stable colloidal dispersions 479

may have important implications for practical utilization within foods. 480

The turbidity profile of BLG-KC (US) was similar to that of BLG-KC (IN). 481

Generally, the lower charge density of KC (US) may account for the 482

observed differences in the BLG-KC (US) turbidity profile including a 483

slight shift to the right and an increase in the turbidity of the sample 484

representing KC (US)/BLG weight ratio of 0.5. The data are consistent 485

with the ITC data, which also indicated that there was a strong 486

interaction between the two biopolymers at pH 4.25. 487

3.4.2. ζ-potential versus KC/BLG weight ratio profiles 488

The stability of colloidal systems can be studied by measuring the 489

electrophoretic properties of the colloidal particles. Particles with ζ 490

potentials more positive than +30mV or more negative than -30mV are 491

normally considered stable (Mounsey, O’Kennedy, Fenelon, & 492

Brodkorb, 2008). The ζ-potential profiles of the BLG-KC complexed 493

systems as a function of KC/BLG weight ratio at pH 4.25 is shown in 494

24

Fig. 6a. In the absence of KC, the ζ-potential of the BLG suspension 495

was around + 14 mV, which was due to the fact that BLG was below its 496

Ip and therefore had a net positive charge. The ζ-potential depended 497

considerably on the KC concentration. As the KC (IN)/BLG weight ratio 498

increased from 0 to 0.37, the EM values decreased from positive to 499

negative and the Smoluchowski model yielded ζ-potential values that 500

ranged between +14 and -23 mV, indicating low stability systems which 501

resulted in precipitation. According to Aberkane et al. (2010), the 502

requirement of neutrality at phase separation is not a general rule for 503

protein-polyanionic polymers and phase separation may occur with a 504

negative total charge. Beyond this point (KC (IN)/BLG weight ratio = 505

0.37), the ζ-potential values remained rather constant at high limiting 506

values (ranging between -44 and -51 mV) reflecting an excess of 507

polysaccharide. These measurements showed that negatively charged 508

KC molecules associated with the surfaces of the positively charged 509

BLG aggregates and caused charge reversal. The ζ-potential profile of 510

BLG-KC (US) showed a similar trend with lower intensity. This trend is 511

in agreement with the charge densities of the two types of KCs which 512

were -53.67 ± 5.21 and -41.63 ± 3.69 mV for KC (IN) and KC (US), 513

respectively, at a concentration of 0.1% (w/w) and pH 4.25. Tang, 514

Huang, and Lim (2003) reported that the ζ-potential values of chitosan 515

nanoparticles decreased from (47.48 ± 1.32) to (45.51 ± 0.29) after 10 516

min sonication at amplitude 80% (more gentle conditions than those of 517

25

the current work). This phenomenon can be attributed to the reduction 518

of the KC reactivity after sonication, which may be due to some 519

heterogeneous sonochemical interactions and structural changes that 520

took place during the sonication process. Polysaccharide reactivity is 521

governed by the distribution and number of functional groups attached 522

to the polymerized sugar units that form the backbone of the 523

polysaccharide (Weiss et al., 2011). Polysaccharides subjected to high-524

intensity ultrasound can undergo a large number of sonochemical 525

reactions including glycosylation, acetalyzation, oxidation, C–D, C-526

heteroatom, and C–C bond formations (Kardos et al., 2001), which may 527

eliminate the reactive sites present along the KC backbone or may 528

promote KC-KC interactions which reduce the number of binding sites 529

for the BLG molecules resulting in affinity constant reduction. 530

3.4.3. Particle size versus KC/BLG weight ratio profiles 531

The mean hydrodynamic particle diameters of the BLG-KC 532

systems showing no precipitation as a function of the KC/BLG weight 533

ratio at pH 4.25 are shown in Fig. 6b. The complexes were relatively 534

large at KC (IN)/BLG weight ratios of 0.44 and 0.50 possibly resulting 535

from some sharing of KC molecules between protein aggregates at low 536

polysaccharide concentration. The smallest particles were obtained at a 537

KC (IN)/BLG weight ratio of 0.75, presenting a mean diameter of 408 ± 538

9 nm. These complexes showed a lower PDI than the biopolymers 539

themselves. Generally, the shrinkage of the BLG-KC complexes, 540

26

occurring at low ionic strength could be understood as a reduction of the 541

intramolecular repulsion induced by the interaction of the BLG with the 542

sulphate group of the KC. This compaction phenomenon was well 543

predicted by Monte Carlo simulations which showed that at low ionic 544

strengths, a polyelectrolyte chain would wrap around an oppositely 545

charged spherical particle (Girard et al., 2003). There was an 546

appreciable increase in the diameter of the particles in the mixed 547

system when the KC (IN)/BLG weight ratio was increased from 0.75 to 548

2. Zimet & Livney (2009) concluded that an increase in the 549

polysaccharide concentration increases the viscosity, causing 550

decreased particle mobility (lower fluctuations), which is interpreted by 551

the DLS as an apparently increased particle size. This conclusion is in 552

good agreement with our results since for complexed solutions 553

containing KC (IN), the increase in particle size was found to be more 554

dependent on polysaccharide concentration due to its ability to form 555

more viscous systems. Another possibility is that the changes in 556

intramolecular repulsion and conformation (adoption of a more 557

extended structure) resulting from the decreased ratio of protein 558

molecules per polysaccharide chain may cause a bigger particle size. 559

The mean diameters of the BLG-KC (US) particles were significantly 560

(*p<0.05) smaller than the BLG-KC (IN) particles at KC (US)/BLG 561

weight ratios which corresponded to sufficient repulsion between 562

complexed particles. In the presence of KC (US), the effective 563

27

diameters of the biopolymer complexes remained relatively constant as 564

compared to those containing KC (IN). This may be due to the larger 565

flexibility of the KC (US) chains with the reduction of the charge as well 566

as to the lower viscosity of the biopolymer mixtures. Generally, the 567

polydispersity index of the BLG-KC (IN) nanoparticles (0.313) at weight 568

ratio (0.75) corresponding to minimum particle size was significantly 569

(*p<0.05) higher than that of BLG-KC (US) nanoparticles (0.151) at 570

weight ratio of 1 (minimum particle size), indicating the consequence of 571

polysaccharide sonication was the homogenization of particle sizes in 572

the mixed dispersion. One should keep in mind that the measured 573

results are intensity-weighted, which overestimates the contribution of 574

the larger particles to the detriment of the smaller ones. If volume- or 575

number- weighted distributions are considered, much smaller average 576

diameters are obtained. 577

4. Conclusion 578

The present work shows that ultrasound irradiation can effectively 579

depolymerize KC. The rate of depolymerization was dependent on the 580

amplitude, time and temperature of sonication. KC sonication 581

decreased its affinity constant to BLG at pH 4.25 as determined by ITC. 582

The properties of the biopolymer particles formed depended strongly on 583

the polysaccharide type and concentration as shown by DLS and ζ-584

potential analyses. The soluble complexes formed had good stability 585

against aggregation. Findings could aid in the design of nanoscopic 586

28

delivery systems for encapsulation of both hydrophilic and hydrophobic 587

bioactives in liquid food products and for controlled release objectives, 588

which is the focus of our current research. In the future, more detailed 589

information is required on the mechanism of the helix-coil transition in 590

KC after sonication and association with BLG using high-sensitive 591

differential scanning calorimeter and also on the structure of the soluble 592

complexes formed using high-resolution cryo-TEM. 593

Acknowledgment 594

The authors are thankful to University of Tehran, Iranian 595

Nanotechnology Initiative Council and Ghent University for financial 596

support. 597

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34

Figure Captions 703

704

Fig. 1. Effect of sonication on the apparent viscosity reduction of 0.5% 705

w/w KC solution at different amplitudes and temperatures as a function 706

of time: ( ) Amp. 50%, Temp. 75 °C; ( ) Amp. 100%, Temp. 75 °C; ( ) 707

Amp. 50%, Temp. 25 °C; ( ) Amp. 100%, Temp. 25 °C. 708

709

Fig. 2. Turbidimetric analysis as a function of pH: ( ) BLG dispersion 710

(0.1% w/w), (×) KC (IN) and ( ) KC (US) dispersions (0.05% w/w); ( ) 711

BLG-KC (IN) and ( ) BLG-KC (US) at 0.15% w/w total biopolymer 712

concentration and BLG:KC weight ratio of 2:1. (pHc: formation of soluble 713

complexes, pHφ1: formation of interpolymer complexes) 714

715

Fig. 3. Thermograms corresponding to the titration of the BLG 716

dispersion (0.0828% w/v) with (a) KC (IN) and (b) KC (US) dispersions 717

(0.1% and 0.175% w/w, respectively) in 5 mM sodium citrate buffer (pH 718

4.25) at 25 °C. 719

720

Fig. 4. Binding isotherms (solid lines) corresponding to the titration of 721

the BLG dispersion (0.0828% w/v) with ( ) KC (IN) and ( ) KC (US) 722

dispersions (0.1% and 0.175% w/w, respectively) in 5 mM sodium 723

citrate buffer and optical density profiles (dashed lines) of the BLG 724

dispersion (0.1% w/w) mixed with ( ) KC (IN)- and ( ) KC (US)- 725

35

dispersions, then acidified to pH 4.25 at 25 °C as a function of KC/BLG 726

weight ratio (total biopolymer concentration of 0.1% - 0.3% w/w). 727

728

Fig. 5. a and b: optical images of the BLG dispersion (0.1% w/w) mixed 729

with KC (IN)- and KC (US)- dispersions, respectively, then acidified to 730

pH 4.25 at different KC/BLG weight ratios (total biopolymer 731

concentration of 0.1% - 0.3% w/w). c-e: phase contrast optical 732

micrographs of KC (IN)-BLG mixture at weight ratio of 0.1 just after 733

mixing and acidification to pH 4.25, during precipitation and after 734

precipitation (bottom phase), respectively. f: phase contrast 735

micrographs of KC (IN)-BLG mixture at weight ratio of 1. 736

737

Fig. 6. ζ-potential (a) and effective diameter (b) profiles of the BLG 738

dispersion (0.1% w/w) mixed with ( ) KC (IN)- and ( ) KC (US)- 739

dispersions then acidified to pH 4.25 as a function of KC/BLG weight 740

ratio (total biopolymer concentration of 0.1% - 0.3% w/w). 741

742

36

743

744

745

746

747

748

749

750

751

Fig. 1 752

753

Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed 754

Hadi Razavi, Ali Akbar Moosavi-Movahedi, Ali Akbar Saboury, 755

Mohammad Amin Mohammadifar, Asgar Farahnaky, Maliheh Sadat 756

Atri, Paul Van der Meeren 757

37

758

759

760

761

762

763

764

765

766

767

Fig. 2 768

769

770

Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed Hadi Razavi, Ali 771

Akbar Moosavi-Movahedi, Ali Akbar Saboury, Mohammad Amin Mohammadifar, 772

Asgar Farahnaky, Maliheh Sadat Atri, Paul Van der Meeren 773

774

pHc

pHφ1

39

776

777

778

779

780

781

782

783

Fig. 3 784

785

786

Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed Hadi Razavi, Ali 787

Akbar Moosavi-Movahedi, Ali Akbar Saboury, Mohammad Amin Mohammadifar, 788

Asgar Farahnaky, Maliheh Sadat Atri, Paul Van der Meeren 789

790

791

792

793

794

a b

40

795

796

797

798

799

800

801

802

803

804

805

806

807

808

809

810

Fig. 4 811

812

813

Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed Hadi Razavi, Ali 814

Akbar Moosavi-Movahedi, Ali Akbar Saboury, Mohammad Amin Mohammadifar, 815

Asgar Farahnaky, Maliheh Sadat Atri, Paul Van der Meeren 816

817

818

41

1000 X 40 μm 1000 X 40 μm 400 X 100 μm 1000 X 40 μm

819

820

821

822

823

824

825

826

827

828

829

830

831

Fig. 5 832

Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed Hadi Razavi, Ali 833

Akbar Moosavi-Movahedi, Ali Akbar Saboury, Mohammad Amin Mohammadifar, 834

Asgar Farahnaky, Maliheh Sadat Atri, Paul Van der Meeren 835

For in print 836

837

838

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

1000 X 40 μm 400 X 100 μm 1000 X 40 μm 1000 X 40 μm

a

b

c d e f

42

839

840

841

842

843

844

845

846

847

848

849

850

851

852

853

854

Fig. 6 855

a

b

43

Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Seyed Hadi Razavi, Ali 856

Akbar Moosavi-Movahedi, Ali Akbar Saboury, Mohammad Amin Mohammadifar, 857

Asgar Farahnaky, Maliheh Sadat Atri, Paul Van der Meeren 858

44

Sonication decreased the apparent viscosity of the κ–carrageenan solution. 859

Sonication reduced the affinity constant between κ–carrageenan and β-860

lactoglobulin. 861

Sonication downsized nanoparticles formed in the mixed dispersion with protein. 862

Complexation in mixed BLG-KC dispersions is a nucleation and growth 863

mechanism. 864

865