Accepted Manuscript

Physical and functional properties of whole milk powders prepared from concentratepartially acidified with CO2 at two temperatures

Leni Kosasih, Bhesh Bhandari, Sangeeta Prakash, Nidhi Bhansal, Claire Gaiani

PII: S0958-6946(15)00231-9

DOI: 10.1016/j.idairyj.2015.12.009

Reference: INDA 3914

To appear in: International Dairy Journal

Received Date: 4 October 2015

Revised Date: 19 December 2015

Accepted Date: 19 December 2015

Please cite this article as: Kosasih, L., Bhandari, B., Prakash, S., Bhansal, N., Gaiani, C., Physical andfunctional properties of whole milk powders prepared from concentrate partially acidified with CO2 at twotemperatures, International Dairy Journal (2016), doi: 10.1016/j.idairyj.2015.12.009.

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Physical and functional properties of whole milk powders prepared from concentrate 1

partially acidified with CO2 at two temperatures 2

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Leni Kosasiha, Bhesh Bhandaria, Sangeeta Prakasha, Nidhi Bhansala & Claire Gaiania,b* 8

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a University of Queensland, School of Agricultural and Food Science, St. Lucia, Queensland 4072, 14

Australia 15

b Université de Lorraine, LIBio, 2 avenue de la Forêt de Haye, TSA 40602, 54518 Vandoeuvre-lès-Nancy, 16

France 17

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* Corresponding author. Tel.: +33(0)3 83 59 60 73 22

E-mail address: claire.gaiani@univ-lorraine.fr (C. Gaiani) 23

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ABSTRACT 27

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Effects of carbonation of whole milk concentrate on spray dried powder properties were 29

investigated. Concentrate acidification by CO2 addition (2000 ppm) was found to strongly modify 30

the functional properties (solubility, dispersibility) and structural/physical properties (porosity, 31

free fat) of the resulting powders. For concentrates treated at low temperature (where the majority 32

of emulsified fat is in a solid state at 4 °C), colloidal calcium phosphate (CCP) release, casein 33

dissociation and fat coalescence were observed. For warm CO2 treated concentrates (30 °C) only 34

CCP release was observed. The best functional properties (higher solubility and dispersibility) were 35

found for powders produced from the warm treated concentrates, which were possibly due to the 36

high porosity and better fat globule preservation. 37

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1. Introduction 40

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In the last decade, CO2 application in milk has been extensively studied mainly to improve 42

the shelf-life, quality and yield of diverse dairy products, such as raw and pasteurised milk, cheeses, 43

yogurt, and fermented dairy beverages (Hotchkiss, Werner, & Lee, 2006). For example, CO2 injection 44

in milk before rennet coagulation can be used to reduce the pH of milk and solubilise micellar 45

calcium phosphate, which resulted in cheese containing a different mineral profile (Nelson, Lynch, & 46

Barbano, 2004). 47

Recently, the use of CO2 on protein concentrates to improve the functional properties of milk 48

protein concentrate (MPC) powders was studied (Marella, Salunke, Biswas, Kommineni, & Metzger, 49

2015). It was reported that modification in concentrate mineral environment (Marella et al., 2015) 50

or micelle structure (Law & Leaver, 1998) by adding CO2 may improve the rehydration properties of 51

MPC powders. The improved solubility was attributed to the decrease in micellar interaction and 52

increase in non-micellar casein release caused by partial acidification (Schokker et al., 2011). The 53

addition of glucono-delta-lactone to partially acidify milk concentrates may also reduce the amount 54

of protein–protein interactions during drying, which contribute to the loss of solubility of high-55

protein MPC powders (Eshpari, Tong, & Corredig, 2014). Nevertheless, the use of CO2 as a 56

replacement for glucono-delta-lactone, which acts as a milk acidulant (through the reaction product 57

gluconic acid) has gained interest because CO2 can be totally and easily removed by heating or 58

applying vacuum. 59

The current literature regarding the effect of CO2 in milk has been mainly focused on skim 60

milk and the resulting powders (Lee, 2014). Meanwhile, its effect on whole milk concentrates has 61

never been studied and the effect of CO2 on fat has been poorly reported. It is generally accepted 62

that CO2 has higher solubility in nonpolar solvents, such as lipids, than in polar solvents, such as 63

water, because the molecular structure of CO2 is apolar and it has a dipole moment of zero (Chaix, 64

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Guillaume, & Guillard, 2014; Hotchkiss et al., 2006). Therefore, the overall objective of this research 65

was to carbonate whole milk concentrates to alter the partition of CO2 in milk components, and 66

analyse the effect of CO2 on the resulting powders, focusing on functional, physical and structural 67

properties. It was expected that solubilisation of colloidal calcium phosphate (CCP) due to CO2 68

acidification would affect the structural organisation of casein micelles and consequently alter the 69

rehydration and functional properties of the resulting whole milk powder (WMP). 70

71

2. Material and methods 72

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2.1. Material 74

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Whole milk (standard, not lecithinated) and skim milk (medium heat) powders for 76

preparing the concentrates were purchased in 25 kg bags from Total Foodtec Pty Ltd. (Brisbane, 77

Australia). The powders were a maximum of 1 month old for the experiments. Carbonation was 78

accomplished by the addition of known amount of solid CO2, also known as dry ice. 79

Phosphate buffered saline (×1) was prepared at a final pH of 7.4 with the following 80

composition: 1.42 g L-1 sodium phosphate, 8.0 g L-1 sodium chloride, 0.2 g L-1 potassium chloride 81

0.24 g L-1 potassium phosphate. 82

83

2.2. Concentrate, carbonation and powder preparation 84

85

Whole milk concentrates (WMC) were prepared by dissolving 25 g of the powder in 100 g of 86

Milli-Q (deionised) water at 25 °C with constant stirring at a high speed with an overhead stirrer for 87

1 h. For each experiment, 3 L of concentrates was prepared. Optical microscopy on the rehydrated 88

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sample was done to check the complete rehydration of the powder. Finally, WMC was poured in the 89

kegs for carbonation. 90

Carbonation of the concentrate was done with frozen CO2 (dry ice). The 3 L concentrate was 91

poured into an eleven litre stainless steel keg equipped with manometer. An adequate amount of 92

dry ice was added in the keg allowing a theoretical CO2 content of 2000 ppm in the concentrate (Lee, 93

2014). The kegs were then left for 4 h at 4 or 30 °C respectively. After overnight storage, the 94

samples were spray-dried; a control samples without CO2 addition was stored overnight at 4 °C 95

prior to spray-drying. A single-stage Anhydro Lab S1 spray dryer (Copenhagen, Denmark) dried the 96

carbonated concentrates (without a decarbonation step). The spray dryer fitted with a pneumatic 97

nozzle, supplied compressed air (6.34 bar), and operated at 170 °C and 85 °C inlet and outlet air 98

temperatures, respectively. The spray dried samples were collected in zipped aluminium bags and 99

analysed soon after (all analysis were done in less than 3 days). 100

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2.3. Chemical analysis 102

103

The CO2 content in the concentrate was determined using a Mettler Toledo CO2 Transmitter 104

5100e Electrochemical probe (InPro 5000 CO2 Sensor, Mettler-Toledo AG, Process Analytics, 105

Urdorf, Switzerland). The electrochemical probe was found to be the most accurate and easiest way 106

to measure CO2 among other tested methods (i.e., infra-red head space analyser, manometric assay) 107

(Chaix et al., 2014; Lee, 2014). The probe was inserted directly into the liquid and final reading was 108

taken when it reached equilibrium. Depending on the CO2 level in the test sample, the final reading 109

reached to equilibrium in 3–8 min. 110

An ionic calcium probe (LAQUAtwin, compact Ca2+ meter, B751, Horiba Scientific) directly 111

measured the calcium ion concentration in 0.3 mL carbonated concentrates. The probe was capable 112

of measuring ionic calcium concentration in the range 1–100 mM. A 0.25 mM resolution was possible 113

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in the approximate range of calcium found in milk (range 2.5 to 25 mM). The instrument was 114

calibrated daily with 2.5 mM standards. 115

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2.4. Structural properties 117

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2.4.1. Scanning electron microscope 119

A field emission scanning electron microscope (SEM) type JEOL JSM-7100F with a hot 120

(Schottky) electron gun (JEOL Ltd., Tokyo, Japan) and a resolution around 1 nm at 30 kV was used 121

for magnifications higher than 10,000. For lower magnifications, a JEOL JSM-6460LA (JEOL Ltd.) 122

with a tungsten filament electron gun was preferred. Both instrumental analyses were conducted at 123

5 kV to obtain images. 124

Sample preparation was done following the reported method of Mimouni, Deeth, Whittaker, 125

Gidley, and Bhandari, 2010) with some modifications. A drop of milk concentrate was deposited 126

onto a silicon chip wafer (ProSciTech, Kirwan, Australia) coated with poly-L-lysine (Sigma Aldrich, 127

Castle Hill, Australia) which created electrostatic bonding between micelles and the substrate. A few 128

drops of poly-L-lysine solution (1 mg mL-1 in phosphate-buffered saline ×1, pH 7.4) were deposited 129

on the silicon wafer and allowed to air-dry overnight at room temperature in a dust-free 130

environment. One drop of concentrate was then deposited and left for 30 min before rinsing with 131

phosphate buffer (pH 7). A solution of 2.5 % glutaraldehyde in phosphate buffer was then applied 132

for 30 min to achieve chemical fixation of the protein material. After fixation, the samples were 133

washed in phosphate buffer and dehydrated using a graded ethanol series: 50%, 60%, 70%, 80%, 134

90% (1 time), and 100% (3 times). The elapsed time per solution was 2 min. Finally, samples were 135

dried using CO2 in a Supercritical Autosamdri-815B critical point dryer (Tousimis, Rockville, MD, 136

USA). 137

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Both silicon wafers and powders were subsequently mounted onto SEM stubs by placing or 138

sputtering them on a carbon double-sided adhesive tape. Coating was done with platinum (Q150T 139

Turbo-Pumped Sputter Coater, ProSciTech) for 2 min (~ 10 nm thick). 140

141

2.4.2. confocal laser scanning microscopy 142

Milk concentrate and powders were analysed by confocal laser scanning microscopy (CLSM) 143

using a Zeiss LSM 700 confocal microscope (Carl Ziess Ltd., North Ryde, New South Wales, 144

Australia). Nile red and rhodamin B (Sigma Aldrich) both at a concentration of 0.1 g L-1 in PEG 200 145

were used to label fat and proteins, respectively. A ratio of 1/100 (dye/concentrate or powder) was 146

used and left for 20 min before imaging. Observations were done with a 63× immersion oil 147

objective. An argon laser operating at excitation wavelengths of 488 nm was used. Each micrograph 148

is a representation of at least 10 images of each sample. 149

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2.5. Particle density measurements 151

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True density of powder is defined (GEA Niro, 2006a) as the mass of particles per unit 153

volume . A Quantachrome Multipycnometer (Quantachrome Instruments, Boynton Beach, FL, USA) 154

was used to determine the true density of milk powders. The pycnometer was operated using 155

nitrogen gas at 1.2 kPa. 156

Occluded and interstitial air are defined as the difference between the volume of particles at 157

a given mass and the volume of the same mass of air-free solids and of powders tapped 100 times, 158

respectively (GEA Niro, 2006b). The occluded and interstitial air contents of milk powder were 159

calculated using the formulas described by GEA Niro (2006b). 160

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2.6. Size analysis 162

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163

Average size distribution of casein micelles in the concentrates was measured at 25 °C using 164

a 3000 HSA Malvern Zetasizer (Nano series, Malvern Instruments, Malvern, UK). Before 165

measurement, the samples were filtered (0.45 μm; Millipore) to avoid fat interactions. The 166

concentrates were finally diluted 200 times with Milli-Q deionised water. 167

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2.7. Functional properties of the powder 169

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The solubility (ISO, 2005), dispersibility (ISO, 2014) and wettability (ISO, 2014) of the 171

powders was determined per the International Organisation for Standardisation standards with 172

slight modifications due to the limited quantity of powder: the same ratio between water and 173

powder was kept, but the quantity of powder used was reduced to be able to make all repetitions. 174

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2.8. Milk fat analysis 176

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2.8.1. Free fat extraction 178

Free fat extraction from milk powder was done following the procedures described 179

elsewhere (Kim, Chen, & Pearce, 2002; Murriera Pazos, Gaiani, Galet, & Scher, 2012; Vignolles, 180

Jeantet, Lopez, & Schuck, 2007) with some modifications. Milk powder (2 g) was weighed and mixed 181

with 50 mL petroleum spirit for 5 min. The solvent was separated by filtration into a round-bottom 182

flask. The powders on the filtrate paper were dried and kept for encapsulated fat analysis. The 183

solvent in the flask was totally evaporated. Then, the solvent-free flask was dried in the oven. The 184

free fat percentage is the ratio between the weight of extracted fat and the weight of powder. 185

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2.8.2. Encapsulated fat extraction 187

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Milk powder recovered from free fat extraction was weighed and warm water (4 mL at 50–188

55 °C) was added. The mixture was vortexed for 2 min to dissolve the powder and release the 189

encapsulated fat. A solvent mixture made of n-hexane and 2-propanol in 3:1 ratio (v/v) was added 190

and vortexed for 15 min to extract the fat. The solution was then centrifuged at 1000 × g for 15 min 191

and the organic phase was filtered into a dry and clean round-bottom flask. The aqueous phase was 192

re-extracted with the solvent mixture and the collected organic phase was totally evaporated. Then, 193

the solvent-free flask was dried in the oven. The encapsulated fat percentage is the ratio between 194

the weight of extracted fat and the weight of initial powder. 195

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2.8.3. Total fat extraction 197

Total fat was extracted from 2 g of milk powder following the same procedure used for the 198

extraction of encapsulated fat described in sections 2.7.2. 199

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2.9. Statistical analysis 201

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All measurements presented in this paper were performed on three independent samples 203

(except that the functional properties were done on two analysis). The KyPlot software version 204

2.0 was used and a parametric multiple comparisons test (Tukey test) was performed. The 205

significance level was: ***P < 0.001, **P < 0.01, *P < 0.05 and NSP > 0.05 (not significant). 206

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3. Results and discussion 208

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3.1. Effect of CO2 and temperature on whole milk concentrate properties 210

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3.1.1. pH and ionic calcium evolution in the concentrates as a function of CO2 concentration 212

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Whole milk concentrate consists of an aqueous (skim portion) and a lipid (milk fat) phase. 213

During carbonation, the pH evolution was measured at two temperatures, 4 °C and 30 °C, to acquire 214

milk fat under different states, mainly solid and liquid states, respectively. As shown in Fig. 1A, 215

different pH profiles between skim and whole milk concentrates were obtained during carbonation 216

at 4 °C. Lower pH values were attained for whole milk concentrate in comparison with skim milk 217

concentrate, both containing the same solid content. Similar profiles were obtained after 218

mathematical correction of Fig. 1A as a function of the estimated CO2 content in the skim portion 219

(Fig. 1B). As mentioned previously, CO2 is more soluble in lipid than in water (Hotchkiss et al., 220

2006). Thus, it can be assumed that when fat is in a solid state (i.e., 4 °C), very little CO2 dissolves in 221

the fat portion and most is dissolved in the skim portion. At 4 °C, some fat fractions remain liquid 222

(Buchheim, 1970). However, it may be contained in within spherical fat globules that are covered by 223

a layer of solid fat on the surface (Buchheim, 1970) that could act as a barrier to prevent CO2 224

migration into the fat globules. This hypothesis was confirmed by Fig. 1C, highlighting the 225

importance of the presence of fat fraction and the temperature of carbonation. Therefore, it can now 226

be concluded that, during carbonation at 30 °C, CO2 dissolves both in skim and in the fat fraction, 227

whereas at 4 °C, CO2 dissolves only in the skim fraction. Similar results were also obtained by others 228

(Ma & Barbano, 2003) while studying the effect of temperature during carbonation in cream. 229

Ionic calcium strongly correlates to the pH decrease during carbonation (Table 1). As the pH 230

reduces by CO2, CCP gradually solubilises in the concentrates. The concentrates without CO2 231

treatment presents a classical ionic calcium content around 2.75 mM. Milk at a normal pH presents 232

only around 10% of the total calcium (30 mM) in a ionic form; corresponding to 3 mM (Lewis, 2011). 233

Nevertheless considerable variations were observed by these authors, with the ionic calcium level 234

varying from 1–5 mM Ca2+ depending on processing, storage, temperature, breed, etc. This value 235

increased significantly (P < 0.001) to 6.75 and 6.25 mM for concentrates carbonated at 4 and 30 °C, 236

respectively. Acidification of milk (with CO2) was previously found to increase the ionic calcium by 237

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180% (Klandar, Chevalier-Lucia, & Lagaude, 2009) at pH 5.95 and 5 °C. No significant differences 238

were observed between the two carbonated concentrates (P > 0.05). Nevertheless, slight differences 239

in ionic calcium content are possibly due to a reduction in CCP solubility with increasing 240

temperature (Lewis, 2011). 241

Another explanation may be that CO2 in warm concentrate is dissolved in both skim and fat 242

portions. This means that there is slightly less CO2 dissolved in the skim portion for warm 243

concentrate in comparison with cold concentrate. Since majority of casein micelles and CCP are 244

present in the skim portion. Thus, there is less effect of CO2 acidification and less CCP release from 245

casein micelle in warm concentrate than cold concentrate. The concentrate without treatment 246

presents a pH around 6.8 whereas the cold and warm concentrates treated at cold and warm 247

conditions with CO2 have a pH of 5.9 and 6.0, respectively. Again, no significant differences were 248

observed between the two carbonated concentrates (P > 0.05). The tendency of a higher pH value of 249

the warm concentrate can be explained by the slightly lower CO2 level (Table 1), although the 250

difference was not significant. 251

252

3.1.2. Micelle size and shape evolution with carbonation 253

Micelle size in milk concentrates was analysed by dynamic light scattering (DLS). The results 254

described in Table 1 showed that micelle size in the cold treated concentrate was significantly 255

reduced from 180 nm to approximately 150 nm (P < 0.01). Meanwhile, no significant differences 256

were observed between the concentrate without treatment and the warm treated concentrate, in 257

which both have micelle sizes around 180 nm. 258

Micelles were also observed at two magnifications (50,000 and 100,000) by SEM high field 259

after fixation and dehydration. At low magnification, uniformly dispersed micelles were seen in all 260

concentrates. In the untreated concentrate (Fig. 2A1), micelles with a size around 200 nm were 261

found uniformly distributed with the presence of small micelles or dissociated micelles in the 262

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background. When the concentrate was acidified with CO2 at 4 °C (Fig. 2B1), micelles were visibly 263

smaller in size and greater amount of dissociated micelles were present in the background. 264

Meanwhile, no apparent differences were seen between the standard and high temperature-265

acidified concentrate (Fig. 2C1). The surface of micelles in milk concentrates was also visualised in 266

depth at higher magnification (Figs. 2A2,B2,C2). Our study confirmed earlier research (Dalgleish, 267

2011) that suggests casein micelle are far from regular and are not perfectly spherical. Caseins 268

appear to be organised as an entangled network of protein chains that protrude at the surface 269

(mainly the κ-casein). In addition, microscopy observations and DLS measurements are in 270

agreement as micelles in the cold treated concentrate appeared significantly smaller than in the 271

standard concentrate. 272

It was already demonstrated that the solubilisation of CCP is quick and pH dependent, 273

whereas the dissociation of caseins from micelles varies with pH and temperature (Law & Leaver, 274

1998). A combination of low temperature and low pH was reported to cause the dissociation of CCP 275

and casein monomers from micelles when cold milk pH decreased from pH 6.7 to 5.2 (Law & Leaver, 276

1998; Post, Arnold, Weiss, & Hinrichs, 2012). Meanwhile, no difference in micelle size was observed 277

between the warm treated concentrate and the untreated milk (Table 1, Fig. 2A,C). This could be 278

due to the solitary dissociation of CCP and lack of casein dissociation from micelles in the warm 279

treated concentrate, which showed that CO2 does not influence micelle size in milk concentrates at 280

high temperatures. 281

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3.1.3. Fat evolution with carbonation 283

CLSM images (at ambient temperature) was conducted after fat labelling of milk 284

concentrates stored at 4 °C (Fig. 3A1,A2) and 30 °C (Fig. 3B1,B2). For each temperature, images 285

were obtained for both CO2 treated (Fig. 3A2,B2) and untreated concentrates (Fig. 3A1,B1). Small 286

and regularly distributed fat globules were observed in concentrates without CO2 treatment, 287

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regardless of the temperature. Meanwhile, fat globule coalescence was noticed for concentrates 288

carbonated at 4 °C (Fig. 3A2). The concentrate treated at 30 °C (Fig. 3B2) was also observed to have 289

larger fat globules than the control. However, fat globules as big as at 4 °C were never observed at 290

30 °C. These results are in agreement with the static light scattering analysis (data not shown). 291

Therefore, it is envisaged that formation and breaking of CO2 bubbles during the carbonation 292

process induced coalescence mainly by a surface-mediated mechanism. This mechanism involved 293

the absorption of CO2 at the fat globule membrane interface and subsequently causing coalescence 294

of fat globules, with concomitant release of liquid oil onto the interfaces (Fuller, Considine, Golding, 295

Matia-Merino, & MacGibbon, 2015). This phenomenon may be more pronounced at 4 °C where milk 296

fat is mainly in a solid state (El-Loly, 2011). The combination of fat globule membrane protrusion by 297

crystalline solid fat and CO2 interaction with the membrane might have contributed to the 298

coalescence of fat. It is already known that air accelerates the process of fat coalescence, for instance 299

during the manufacture of ice cream (El-Loly, 2011). However, similar results during carbonation 300

were never reported as carbonation on whole milk concentrate was sparsely studied. Therefore, the 301

effect of CO2 on fat coalescence will need further investigation. 302

303

3.2. Effect of CO2 on spray dried powder properties 304

305

3.2.1. Particle size, shape and density 306

The microstructures of the powders are presented in Fig. 4. At low magnifications (Fig. 307

4A1,B1,C1), similar features were observed among the standard and treated powders, with small 308

particles agglomerated into bigger structures of around 50–100 µm. At intermediate magnifications 309

(Fig. 4A2,B2,C2), again similar round particles were observed. Finally, at high magnifications where 310

the powder surfaces were clearly visualised, no significant modifications were observed in relation 311

to CO2 and/or temperature treatments (Fig. 4A3,B3,C3). 312

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Even if the particle size, shape and surface of the powders seemed similar by CLSM and SEM 313

imaging, the physical properties of the powders were different. As presented in Table 2, the 314

occluded gas/air contents of powders produced from concentrates treated with CO2 was 10 times 315

higher than the powders from untreated concentrates. Meanwhile, no significant differences were 316

noted between powders from concentrates carbonated at 4 and 30 °C (P > 0.05). Moreover, the true 317

density and interstitial air content were not significantly lower for CO2 treated powders even if a 318

tendency to lower values was observed. The presence of CO2 in milk concentrates is responsible for 319

the achievement of internal porosity in the resulting powders (Lee, 2014). Therefore, these results 320

were expected as the concentrates were not degassed prior to spray drying. Additionally, as shown 321

in Fig. 5A1,B1,C1, porous structures were seen in CO2 treated powders, whereas standard powders 322

presented some pores, but less important. The cut particles imaged by SEM also showed a lack of 323

vacuoles in the untreated powders (Fig. 5A2), whereas huge vacuoles were present in the treated 324

powders (Fig. 5B2,C2). At higher magnification (Fig. 5A3,B3,C3), small internal pores were visible in 325

the standard powders and the envelope thickness of the other two powders was found to be around 326

2–3 µm, which supported the CLSM results. These microscopy observations supported the occluded 327

air measurements described in Table 2. 328

The CLSM images also showed fat globules (in green) and proteins (in red) distribution in 329

each powder particle (Fig. 4A1,B1,C1). However, no real differences were noticed among the 330

powders, because all particles presented heterogeneous fat globules with a protein layer at the 331

surface. Many authors have reported that greater than 95% of WMP surface was covered with fat, 332

mostly free fat, followed by layers of protein, lactose and fat globules protected by proteins (Fyfe, 333

Kravchuk, Nguyen, Deeth, & Bhandari, 2011; Kim et al., 2002; Kim, Chen, & Pearce, 2009). However, 334

these surface fat layer, generated due to fat globules breakage, cannot be visualised by CLSM due to 335

its low resolution (Vignolles et al., 2007). 336

337

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3.2.2. Particle functional properties 338

The analyses of powder functional properties are presented in Table 1. It was found that all 339

powders did not wet within 5 min. This result was expected as WMP particles in this work were not 340

agglomerated and/or lecithinated according to the manufacture of commercial powders. Our results 341

are also different from other researchers on skim milk powder, where CO2 was found to improve 342

wettability (Lee, 2014). However, comparisons are impossible due to the different natures of skim 343

and whole milk powders. Nonetheless, the powders produced from concentrates carbonated at 30 344

°C showed significant improvements in dispersibility (P < 0.01) and solubility (P < 0.001), while the 345

reconstitution properties of powders produced from concentrates treated with CO2 at 4 °C were 346

decreased (for solubility). The dispersibility of standard and powders treated at 4 and 30 °C were 347

54, 46 and 65%, respectively. A similar trend was observed for the solubility of these powders, with 348

values of 97, 94 and 99%, respectively. These differences cannot be attributed to modifications of 349

particle shape or surface morphology because all powder particles have similar structures as 350

observed in Fig. 4A,B,C. In addition, particles size for the three powders were also similar with a d50 351

around 15 µm (data not shown). Meanwhile, improved functional properties have been attributed 352

to powders containing high levels of non-micellar casein in high protein content powders (Schokker 353

et al., 2011). Several factors such as calcium chelators, reduced pH, high pressure, and ionic strength 354

have been found responsible for the structural integrity of casein micelles (Law & Leaver, 1998; 355

Marchin, Putaux, Pignon, & Léonil, 2007). In this study, both CO2 acidification and temperature were 356

found to play a role in the non-micellar casein content. As shown in Fig. 2, the amount of non-357

micellar casein in the concentrates was strongly increased by the action of CO2 acidification in 358

combination with low temperature. However, in this research, the protective effect of non-micellar 359

caseins (serum caseins) observed in high protein content powders (Buldo, 2012) was not confirmed 360

as the resulting powders did not provide better reconstitutability. It is evident that fat caused some 361

modifications in concentrates stored at 4 °C due to coalescence. As a result, fat emulsions in the 362

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concentrate were larger and non-homogenous (Fig. 3). Meanwhile, larger fat globules are known to 363

produce free fat on the surface of powder particles (Table 2), which rendered the surface 364

hydrophobic, therefore reducing solubility in water (Bhandari, 2013; Kim et al., 2009). On the other 365

hand, the concentrates carbonated at high temperature did not show fat coalescence, thus the 366

resulting powders have less amount of free fat (Table 2) and consequently better reconstitution 367

properties (Table 1). Additionally, the elevated porosity of powders produced from concentrates 368

treated at 30 °C may contribute to the enhanced functional properties, as similarly reported for 369

skim milk powders (Lee, 2014). Moreover, WMP with poor reconstitution properties were reported 370

to contain more aggregated particles that consisted of mixtures of fat globules and proteins (Singh & 371

Ye, 2010). Therefore, fat destabilisation may mask the positive effect of elevated levels of non-372

micellar caseins in milk concentrates carbonated at low temperatures. 373

374

4. Conclusion 375

376

The effect of CO2 acidification of whole milk concentrate on WMP properties has not been 377

reported in the literature. Results show that CO2 acidification of whole milk concentrates at various 378

temperatures allow the production of powders with totally different properties due to the alteration 379

of physical states of fat. By acting on both micelles and milk fat, new generation of powders with 380

targeted functional properties may be produced. In the future, it may be interesting to find CO2 381

addition and processes conditions that lead to the increase of non-micellar casein levels (as in 2000 382

ppm, 4 °C powders) without the modifications of fat emulsion (as in 2000 ppm, 30 °C powders) to 383

greatly improve the reconstitutability of these powders. 384

385

Acknowledgements 386

387

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Author Claire Gaiani would like to thank Europe for their financial support towards this project 388

(Milk PEPPER, N°621727, International Outgoing Fellowship grant). The authors acknowledge the 389

facilities, and the scientific and technical assistance provided by the School of Agriculture and Food 390

Sciences (SAFS) at The University of Queensland and the Australian Microscopy & Microanalysis 391

Research Facility at the Centre for Microscopy and Microanalysis (CMM, The University of 392

Queensland). 393

394

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Figure legends

Fig. 1. pH evolution with CO2 concentration for whole milk (�) and skim milk (�)

concentrates with CO2 injected at 4 °C (A), after mathematical correction as a function of the

estimated CO2 in the skim portion at 4 °C (B) and with CO2 injected at 30 °C (C).

Fig. 2. Micelles observed by field emission scanning electron microscopy magnifications

50,000× (1) and 100,000× (2): A, concentrate with no CO2; B, concentrate stored at 4 °C

with CO2; C, concentrate stored at 30 °C with CO2.

Fig. 3. Confocal laser scanning microscopy on whole milk concentrates, fat is labelled with

Nile red and appears in green. Concentrates were stored at (A) 4 °C without (A1) and with

CO2 (A2) or at (B) 30 °C without (B1) and with CO2 (B2).

Fig. 4. Scanning electron microscopy images of the resulting powders at magnifications

1000× (1), 3000× (2) and 10,000× (3): A, powders from concentrate with no CO2; B,

powders from concentrate stored at 4 °C with CO2; C, powders from concentrate stored at

30 °C with CO2.

Fig. 5. Confocal laser scanning microscopy with dual labelling of fat (green) and proteins

(red) of the particles (1) and scanning electron microscopy of cut particles at

magnifications 10,000× (2) and 20,000× (3): A, powders from concentrate with no CO2; B,

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powders from concentrate stored at 4 °C with CO2; C, powders from concentrate stored at

30 °C with CO2.

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Table 1

Physico-chemical properties of the concentrates and functional properties of the resulting powders. a

a Powders are identified by theoretical CO2 concentration (none and 2000 ppm) and CO2 treatment temperature (none, 4 °C and 30°C).

Values are means of 3 analyses on 3 independent spray drying trials: significance from sample 0 ppm indicated by superscript lowercase

letters, significance from sample 2000 ppm indicated by superscript uppercase letters: ns,NS, not significant, P > 0.05; a,A P < 0.05; b,B P <

0.01; c,C P < 0.001).

Powder

Concentrate properties Powder functional properties

CO2 content

(actual; ppm)

Ionic Ca2+

(mM)

pH Micelle size

- Z average

(nm)

Wettability

(min)

Dispersibility

(%)

Solubility (%)

0 ppm 17±9 2.75±0.25 6.8±0.0 179.3±5.8 > 5 53.8±1.8 97.5±0.3

2000 ppm, 4 °C 1902±67c 6.75±0.25c 5.9±0.1c 147.0±1.5b > 5 45.6±2.5ns 94.0±0.6b

2000 ppm, 30 °C 1781±44c,NS 6.25±0.50c,NS 6.0±0.2c,NS 182.0±3.2ns,B > 5 64.6±2.5a,B 99.0±0.5ns,C

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Table 2

Physical and chemical properties of the powders. a

a Powders are identified by theoretical CO2 concentration (none and 2000 ppm) and CO2 treatment temperature (none, 4 °C and 30°C).

Values are means of 3 analyses on 3 independent spray drying trials: significance from sample 0 ppm indicated by superscript lowercase

letters, significance from sample 2000 ppm indicated by superscript uppercase letters: ns,NS, not significant, P > 0.05; a,A P < 0.05; b,B P <

0.01; c,C P < 0.001).

Powder Solvent extraction Physical properties

Free fat

(g L-1)

Total fat

(g L-1)

Encapsulated fat

(g L-1)

True density

(g mL-1)

Occluded air content

(mL 100g-1)

Interstitial air content

(mL 100g-1)

0 ppm 2.45±0.78 30.43±3.51 28.92±0.45 1.14±0.04 1.41±0.02 127.35±0.63

2000 ppm, 4 °C 3.89±0.49b 30.38±4.25ns 26.65±2.02ns 1.00±0.08ns 12.99±0.09c 110.81±0.91ns

2000 ppm, 30°C 2.73±0.37nsA 30.29±0.18ns,NS 29.78±0.61ns,NS 1.10±0.03nsNS 11.81±0.21cNS

103.33±0.29nsNS

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5