Physical and functional properties of whole milk powders prepared from concentrate partially acidified with CO 2 at two temperatures

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1 Accepted Manuscript Physical and functional properties of whole milk powders prepared from concentrate partially acidified with CO 2 at two temperatures Leni Kosasih, Bhesh Bhandari, Sangeeta Prakash, Nidhi Bhansal, Claire Gaiani PII: DOI: S (15) /j.idairyj 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 and functional properties of whole milk powders prepared from concentrate partially acidified with CO 2 at two temperatures, International Dairy Journal (2016), doi: /j.idairyj 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.

2 1 2 Physical and functional properties of whole milk powders prepared from concentrate partially acidified with CO 2 at two temperatures Leni Kosasih a, Bhesh Bhandari a, Sangeeta Prakash a, Nidhi Bhansal a & Claire Gaiani a,b * a University of Queensland, School of Agricultural and Food Science, St. Lucia, Queensland 4072, Australia b Université de Lorraine, LIBio, 2 avenue de la Forêt de Haye, TSA 40602, Vandoeuvre-lès-Nancy, France * Corresponding author. Tel.: +33(0) address: claire.gaiani@univ-lorraine.fr (C. Gaiani) 24 25

3 26 27 ABSTRACT Effects of carbonation of whole milk concentrate on spray dried powder properties were investigated. Concentrate acidification by CO 2 addition (2000 ppm) was found to strongly modify the functional properties (solubility, dispersibility) and structural/physical properties (porosity, free fat) of the resulting powders. For concentrates treated at low temperature (where the majority of emulsified fat is in a solid state at 4 C), colloidal calcium phosphate (CCP) release, casein dissociation and fat coalescence were observed. For warm CO 2 treated concentrates (30 C) only CCP release was observed. The best functional properties (higher solubility and dispersibility) were found for powders produced from the warm treated concentrates, which were possibly due to the high porosity and better fat globule preservation.

4 40 1. Introduction In the last decade, CO2 application in milk has been extensively studied mainly to improve the shelf-life, quality and yield of diverse dairy products, such as raw and pasteurised milk, cheeses, yogurt, and fermented dairy beverages (Hotchkiss, Werner, & Lee, 2006). For example, CO 2 injection in milk before rennet coagulation can be used to reduce the ph of milk and solubilise micellar calcium phosphate, which resulted in cheese containing a different mineral profile (Nelson, Lynch, & Barbano, 2004). Recently, the use of CO 2 on protein concentrates to improve the functional properties of milk protein concentrate (MPC) powders was studied (Marella, Salunke, Biswas, Kommineni, & Metzger, 2015). It was reported that modification in concentrate mineral environment (Marella et al., 2015) or micelle structure (Law& Leaver, 1998) by adding CO 2 may improve the rehydration properties of MPC powders. The improved solubility was attributed to the decrease in micellar interaction and increase in non-micellar casein release caused by partial acidification (Schokker et al., 2011). The addition of glucono-delta-lactone to partially acidify milk concentrates may also reduce the amount of protein protein interactions during drying, which contribute to the loss of solubility of highprotein MPC powders (Eshpari, Tong, & Corredig, 2014). Nevertheless, the use of CO 2 as a replacement for glucono-delta-lactone, which acts as a milk acidulant (through the reaction product gluconic acid) has gained interest because CO 2 can be totally and easily removed by heating or applying vacuum. The current literature regarding the effect of CO 2 in milk has been mainly focused on skim milk and the resulting powders (Lee, 2014). Meanwhile, its effect on whole milk concentrates has never been studied and the effect of CO 2 on fat has been poorly reported. It is generally accepted that CO 2 has higher solubility in nonpolar solvents, such as lipids, than in polar solvents, such as water, because the molecular structure of CO 2 is apolar and it has a dipole moment of zero (Chaix,

5 65 66 Guillaume, & Guillard, 2014; Hotchkiss et al., 2006). Therefore, the overall objective of this research was to carbonate whole milk concentrates to alter the partition of CO 2 in milk components, and analyse the effect of CO2 on the resulting powders, focusing on functional, physical and structural properties. It was expected that solubilisation of colloidal calcium phosphate (CCP) due to CO 2 acidification would affect the structural organisation of casein micelles and consequently alter the rehydration and functional properties of the resulting whole milk powder (WMP). 2. Material and methods 2.1. Material Whole milk (standard, not lecithinated) and skim milk (medium heat) powders for preparing the concentrates were purchased in 25 kg bags from Total Foodtec Pty Ltd. (Brisbane, Australia). The powders were a maximum of 1 month old for the experiments. Carbonation was accomplished by the addition of known amount of solid CO 2, also known as dry ice. Phosphate buffered saline ( 1) was prepared at a final ph of 7.4 with the following composition: 1.42 g L -1 sodium phosphate, 8.0 g L -1 sodium chloride, 0.2 g L -1 potassium chloride 0.24 g L -1 potassium phosphate Concentrate, carbonation and powder preparation Whole milk concentrates (WMC) were prepared by dissolving 25 g of the powder in 100 g of Milli-Q (deionised) water at 25 C with constant stirring at a high speed with an overhead stirrer for 1 h. For each experiment, 3 L of concentrates was prepared. Optical microscopy on the rehydrated

6 89 90 sample was done to check the complete rehydration of the powder. Finally, WMC was poured in the kegs for carbonation Carbonation of the concentrate was done with frozen CO2 (dry ice). The 3 L concentrate was poured into an eleven litre stainless steel keg equipped with manometer. An adequate amount of dry ice was added in the keg allowing a theoretical CO 2 content of 2000 ppm in the concentrate (Lee, 2014). The kegs were then left for 4 h at 4 or 30 C respectively. After overnight storage, the samples were spray-dried; a control samples without CO2 addition was stored overnight at 4 C prior to spray-drying. A single-stage Anhydro Lab S1 spray dryer (Copenhagen, Denmark) dried the carbonated concentrates (without a decarbonation step). The spray dryer fitted with a pneumatic nozzle, supplied compressed air (6.34 bar), and operated at 170 C and 85 C inlet and outlet air temperatures, respectively. The spray dried samples were collected in zipped aluminium bags and analysed soon after (all analysis were done in less than 3 days) Chemical analysis The CO 2 content in the concentrate was determined using a Mettler Toledo CO 2 Transmitter 5100e Electrochemical probe (InPro 5000 CO2 Sensor, Mettler-Toledo AG, Process Analytics, Urdorf, Switzerland). The electrochemical probe was found to be the most accurate and easiest way to measure CO 2 among other tested methods (i.e., infra-red head space analyser, manometric assay) (Chaix et al., 2014; Lee, 2014). The probe was inserted directly into the liquid and final reading was taken when it reached equilibrium. Depending on the CO 2 level in the test sample, the final reading reached to equilibrium in 3 8 min An ionic calcium probe (LAQUAtwin, compact Ca 2+ meter, B751, Horiba Scientific) directly measured the calcium ion concentration in 0.3 ml carbonated concentrates. The probe was capable of measuring ionic calcium concentration in the range mm. A 0.25 mm resolution was possible

7 in the approximate range of calcium found in milk (range 2.5 to 25 mm). The instrument was calibrated daily with 2.5 mm standards Structural properties Scanning electron microscope A field emission scanning electron microscope (SEM) type JEOL JSM-7100F with a hot (Schottky) electron gun (JEOL Ltd., Tokyo, Japan) and a resolution around 1 nm at 30 kv was used for magnifications higher than 10,000. For lower magnifications, a JEOL JSM-6460LA (JEOL Ltd.) with a tungsten filament electron gun was preferred. Both instrumental analyses were conducted at 5 kv to obtain images. Sample preparation was done following the reported method of Mimouni, Deeth, Whittaker, Gidley, and Bhandari, 2010) with some modifications. A drop of milk concentrate was deposited onto a silicon chip wafer (ProSciTech, Kirwan, Australia) coated with poly-l-lysine (Sigma Aldrich, Castle Hill, Australia) which created electrostatic bonding between micelles and the substrate. A few drops of poly-l-lysine solution (1 mg ml -1 in phosphate-buffered saline 1, ph 7.4) were deposited on the silicon wafer and allowed to air-dry overnight at room temperature in a dust-free environment. One drop of concentrate was then deposited and left for 30 min before rinsing with phosphate buffer (ph 7). A solution of 2.5 % glutaraldehyde in phosphate buffer was then applied for 30 min to achieve chemical fixation of the protein material. After fixation, the samples were washed in phosphate buffer and dehydrated using a graded ethanol series: 50%, 60%, 70%, 80%, 90% (1 time), and 100% (3 times). The elapsed time per solution was 2 min. Finally, samples were dried using CO 2 in a Supercritical Autosamdri-815B critical point dryer (Tousimis, Rockville, MD, USA).

8 Both silicon wafers and powders were subsequently mounted onto SEM stubs by placing or sputtering them on a carbon double-sided adhesive tape. Coating was done with platinum (Q150T Turbo-Pumped Sputter Coater, ProSciTech) for 2 min (~ 10 nm thick) confocal laser scanning microscopy Milk concentrate and powders were analysed by confocal laser scanning microscopy (CLSM) using a Zeiss LSM 700 confocal microscope (Carl Ziess Ltd., North Ryde, New South Wales, Australia). Nile red and rhodamin B (Sigma Aldrich) both at a concentration of 0.1 g L -1 in PEG 200 were used to label fat and proteins, respectively. A ratio of 1/100 (dye/concentrate or powder) was used and left for 20 min before imaging. Observations were done with a 63 immersion oil objective. An argon laser operating at excitation wavelengths of 488 nm was used. Each micrograph is a representation of at least 10 images of each sample Particle density measurements True density of powder is defined (GEA Niro, 2006a) as the mass of particles per unit volume. A Quantachrome Multipycnometer (Quantachrome Instruments, Boynton Beach, FL, USA) was used to determine the true density of milk powders. The pycnometer was operated using nitrogen gas at 1.2 kpa. Occluded and interstitial air are defined as the difference between the volume of particles at a given mass and the volume of the same mass of air-free solids and of powders tapped 100 times, respectively (GEA Niro, 2006b). The occluded and interstitial air contents of milk powder were 160 calculated using the formulas described by GEA Niro (2006b) Size analysis

9 Average size distribution of casein micelles in the concentrates was measured at 25 C using a 3000 HSA Malvern Zetasizer (Nano series, Malvern Instruments, Malvern, UK). Before measurement, the samples were filtered (0.45 μm; Millipore) to avoid fat interactions. The concentrates were finally diluted 200 times with Milli-Q deionised water Functional properties of the powder The solubility (ISO, 2005), dispersibility (ISO, 2014) and wettability (ISO, 2014) of the powders was determined per the International Organisation for Standardisation standards with slight modifications due to the limited quantity of powder: the same ratio between water and powder was kept, but the quantity of powder used was reduced to be able to make all repetitions Milk fat analysis Free fat extraction Free fat extraction from milk powder was done following the procedures described elsewhere (Kim, Chen, & Pearce, 2002; Murriera Pazos, Gaiani, Galet, & Scher, 2012; Vignolles, Jeantet, Lopez, & Schuck, 2007) with some modifications. Milk powder (2 g) was weighed and mixed with 50 ml petroleum spirit for 5 min. The solvent was separated by filtration into a round-bottom flask. The powders on the filtrate paper were dried and kept for encapsulated fat analysis. The solvent in the flask was totally evaporated. Then, the solvent-free flask was dried in the oven. The 185 free fat percentage is the ratio between the weight of extracted fat and the weight of powder Encapsulated fat extraction

10 Milk powder recovered from free fat extraction was weighed and warm water (4 ml at C) was added. The mixture was vortexed for 2 min to dissolve the powder and release the encapsulated fat. A solvent mixture made of n-hexane and 2-propanol in 3:1 ratio (v/v) was added and vortexed for 15 min to extract the fat. The solution was then centrifuged at 1000 g for 15 min and the organic phase was filtered into a dry and clean round-bottom flask. The aqueous phase was re-extracted with the solvent mixture and the collected organic phase was totally evaporated. Then, the solvent-free flask was dried in the oven. The encapsulated fat percentage is the ratio between the weight of extracted fat and the weight of initial powder Total fat extraction Total fat was extracted from 2 g of milk powder following the same procedure used for the extraction of encapsulated fat described in sections Statistical analysis All measurements presented in this paper were performed on three independent samples (except that the functional properties were done on two analysis). The KyPlot software version 2.0 was used and a parametric multiple comparisons test (Tukey test) was performed. The significance level was: ***P < 0.001, **P < 0.01, *P < 0.05 and NS P > 0.05 (not significant). 3. Results and discussion Effect of CO 2 and temperature on whole milk concentrate properties ph and ionic calcium evolution in the concentrates as a function of CO 2 concentration

11 Whole milk concentrate consists of an aqueous (skim portion) and a lipid (milk fat) phase. During carbonation, the ph evolution was measured at two temperatures, 4 C and 30 C, to acquire milk fat under different states, mainly solid and liquid states, respectively. As shown in Fig. 1A, different ph profiles between skim and whole milk concentrates were obtained during carbonation at 4 C. Lower ph values were attained for whole milk concentrate in comparison with skim milk concentrate, both containing the same solid content. Similar profiles were obtained after mathematical correction of Fig. 1A as a function of the estimated CO2 content in the skim portion (Fig. 1B). As mentioned previously, CO 2 is more soluble in lipid than in water (Hotchkiss et al., 2006). Thus, it can be assumed that when fat is in a solid state (i.e., 4 C), very little CO 2 dissolves in the fat portion and most is dissolved in the skim portion. At 4 C, some fat fractions remain liquid (Buchheim, 1970). However, it may be contained in within spherical fat globules that are covered by a layer of solid fat on the surface (Buchheim, 1970) that could act as a barrier to prevent CO 2 migration into the fat globules. This hypothesis was confirmed by Fig. 1C, highlighting the importance of the presence of fat fraction and the temperature of carbonation. Therefore, it can now be concluded that, during carbonation at 30 C, CO 2 dissolves both in skim and in the fat fraction, whereas at 4 C, CO 2 dissolves only in the skim fraction. Similar results were also obtained by others (Ma& Barbano, 2003) while studying the effect of temperature during carbonation in cream. Ionic calcium strongly correlates to the ph decrease during carbonation (Table 1). As the ph reduces by CO 2, CCP gradually solubilises in the concentrates. The concentrates without CO 2 treatment presents a classical ionic calcium content around 2.75 mm. Milk at a normal ph presents only around 10% of the total calcium (30 mm) in a ionic form; corresponding to 3 mm (Lewis, 2011). Nevertheless considerable variations were observed by these authors, with the ionic calcium level varying from 1 5 mm Ca 2+ depending on processing, storage, temperature, breed, etc. This value increased significantly (P < 0.001) to 6.75 and 6.25 mm for concentrates carbonated at 4 and 30 C, respectively. Acidification of milk (with CO 2) was previously found to increase the ionic calcium by

12 % (Klandar, Chevalier-Lucia, & Lagaude, 2009) at ph 5.95 and 5 C. No significant differences were observed between the two carbonated concentrates (P > 0.05). Nevertheless, slight differences in ionic calcium content are possibly due to a reduction in CCP solubility with increasing temperature (Lewis, 2011). Another explanation may be that CO 2 in warm concentrate is dissolved in both skim and fat portions. This means that there is slightly less CO 2 dissolved in the skim portion for warm concentrate in comparison with cold concentrate. Since majority of casein micelles and CCP are present in the skim portion. Thus, there is less effect of CO 2 acidification and less CCP release from casein micelle in warm concentrate than cold concentrate. The concentrate without treatment presents a ph around 6.8 whereas the cold and warm concentrates treated at cold and warm conditions with CO2 have a ph of 5.9 and 6.0, respectively. Again, no significant differences were observed between the two carbonated concentrates (P > 0.05). The tendency of a higher ph value of the warm concentrate can be explained by the slightly lower CO 2 level (Table 1), although the difference was not significant Micelle size and shape evolution with carbonation Micelle size in milk concentrates was analysed by dynamic light scattering (DLS). The results described in Table 1 showed that micelle size in the cold treated concentrate was significantly reduced from 180 nm to approximately 150 nm (P < 0.01). Meanwhile, no significant differences were observed between the concentrate without treatment and the warm treated concentrate, in which both have micelle sizes around 180 nm. Micelles were also observed at two magnifications (50,000 and 100,000) by SEM high field after fixation and dehydration. At low magnification, uniformly dispersed micelles were seen in all concentrates. In the untreated concentrate (Fig. 2A1), micelles with a size around 200 nm were found uniformly distributed with the presence of small micelles or dissociated micelles in the

13 background. When the concentrate was acidified with CO 2 at 4 C (Fig. 2B1), micelles were visibly smaller in size and greater amount of dissociated micelles were present in the background Meanwhile, no apparent differences were seen between the standard and high temperature- acidified concentrate (Fig. 2C1). The surface of micelles in milk concentrates was also visualised in depth at higher magnification (Figs. 2A2,B2,C2). Our study confirmed earlier research (Dalgleish, 2011) that suggests casein micelle are far from regular and are not perfectly spherical. Caseins appear to be organised as an entangled network of protein chains that protrude at the surface (mainly the κ-casein). In addition, microscopy observations and DLS measurements are in agreement as micelles in the cold treated concentrate appeared significantly smaller than in the standard concentrate. It was already demonstrated that the solubilisation of CCP is quick and ph dependent, whereas the dissociation of caseins from micelles varies with ph and temperature (Law& Leaver, 1998). A combination of low temperature and low ph was reported to cause the dissociation of CCP and casein monomers from micelles when cold milk ph decreased from ph 6.7 to 5.2 (Law& Leaver, 1998; Post, Arnold, Weiss, & Hinrichs, 2012). Meanwhile, no difference in micelle size was observed between the warm treated concentrate and the untreated milk (Table 1, Fig. 2A,C). This could be due to the solitary dissociation of CCP and lack of casein dissociation from micelles in the warm treated concentrate, which showed that CO 2 does not influence micelle size in milk concentrates at high temperatures Fat evolution with carbonation CLSM images (at ambient temperature) was conducted after fat labelling of milk concentrates stored at 4 C (Fig. 3A1,A2) and 30 C (Fig. 3B1,B2). For each temperature, images were obtained for both CO 2 treated (Fig. 3A2,B2) and untreated concentrates (Fig. 3A1,B1). Small and regularly distributed fat globules were observed in concentrates without CO 2 treatment,

14 regardless of the temperature. Meanwhile, fat globule coalescence was noticed for concentrates carbonated at 4 C (Fig. 3A2). The concentrate treated at 30 C (Fig. 3B2) was also observed to have larger fat globules than the control. However, fat globules as big as at 4 C were never observed at 30 C. These results are in agreement with the static light scattering analysis (data not shown). Therefore, it is envisaged that formation and breaking of CO 2 bubbles during the carbonation process induced coalescence mainly by a surface-mediated mechanism. This mechanism involved the absorption of CO2 at the fat globule membrane interface and subsequently causing coalescence of fat globules, with concomitant release of liquid oil onto the interfaces (Fuller, Considine, Golding, Matia-Merino, & MacGibbon, 2015). This phenomenon may be more pronounced at 4 C where milk fat is mainly in a solid state (El-Loly, 2011). The combination of fat globule membrane protrusion by crystalline solid fat and CO2 interaction with the membrane might have contributed to the coalescence of fat. It is already known that air accelerates the process of fat coalescence, for instance during the manufacture of ice cream (El-Loly, 2011). However, similar results during carbonation were never reported as carbonation on whole milk concentrate was sparsely studied. Therefore, the effect of CO 2 on fat coalescence will need further investigation Effect of CO 2 on spray dried powder properties Particle size, shape and density The microstructures of the powders are presented in Fig. 4. At low magnifications (Fig. 4A1,B1,C1), similar features were observed among the standard and treated powders, with small particles agglomerated into bigger structures of around µm. At intermediate magnifications (Fig. 4A2,B2,C2), again similar round particles were observed. Finally, at high magnifications where the powder surfaces were clearly visualised, no significant modifications were observed in relation to CO 2 and/or temperature treatments (Fig. 4A3,B3,C3).

15 Even if the particle size, shape and surface of the powders seemed similar by CLSM and SEM imaging, the physical properties of the powders were different. As presented in Table 2, the occluded gas/air contents of powders produced from concentrates treated with CO2 was 10 times higher than the powders from untreated concentrates. Meanwhile, no significant differences were noted between powders from concentrates carbonated at 4 and 30 C (P > 0.05). Moreover, the true density and interstitial air content were not significantly lower for CO 2 treated powders even if a tendency to lower values was observed. The presence of CO2 in milk concentrates is responsible for the achievement of internal porosity in the resulting powders (Lee, 2014). Therefore, these results were expected as the concentrates were not degassed prior to spray drying. Additionally, as shown in Fig. 5A1,B1,C1, porous structures were seen in CO 2 treated powders, whereas standard powders presented some pores, but less important. The cut particles imaged by SEM also showed a lack of vacuoles in the untreated powders (Fig. 5A2), whereas huge vacuoles were present in the treated powders (Fig. 5B2,C2). At higher magnification (Fig. 5A3,B3,C3), small internal pores were visible in the standard powders and the envelope thickness of the other two powders was found to be around 2 3 µm, which supported the CLSM results. These microscopy observations supported the occluded air measurements described in Table 2. The CLSM images also showed fat globules (in green) and proteins (in red) distribution in each powder particle (Fig. 4A1,B1,C1). However, no real differences were noticed among the powders, because all particles presented heterogeneous fat globules with a protein layer at the surface. Many authors have reported that greater than 95% of WMP surface was covered with fat, mostly free fat, followed by layers of protein, lactose and fat globules protected by proteins (Fyfe, Kravchuk, Nguyen, Deeth, & Bhandari, 2011; Kim et al., 2002; Kim, Chen, & Pearce, 2009). However, these surface fat layer, generated due to fat globules breakage, cannot be visualised by CLSM due to its low resolution (Vignolles et al., 2007). 337

16 Particle functional properties The analyses of powder functional properties are presented in Table 1. It was found that all powders did not wet within 5 min. This result was expected as WMP particles in this work were not agglomerated and/or lecithinated according to the manufacture of commercial powders. Our results are also different from other researchers on skim milk powder, where CO 2 was found to improve wettability (Lee, 2014). However, comparisons are impossible due to the different natures of skim and whole milk powders. Nonetheless, the powders produced from concentrates carbonated at 30 C showed significant improvements in dispersibility (P < 0.01) and solubility (P < 0.001), while the reconstitution properties of powders produced from concentrates treated with CO 2 at 4 C were decreased (for solubility). The dispersibility of standard and powders treated at 4 and 30 C were 54, 46 and 65%, respectively. A similar trend was observed for the solubility of these powders, with values of 97, 94 and 99%, respectively. These differences cannot be attributed to modifications of particle shape or surface morphology because all powder particles have similar structures as observed in Fig. 4A,B,C. In addition, particles size for the three powders were also similar with a d50 around 15 µm (data not shown). Meanwhile, improved functional properties have been attributed to powders containing high levels of non-micellar casein in high protein content powders (Schokker et al., 2011). Several factors such as calcium chelators, reduced ph, high pressure, and ionic strength have been found responsible for the structural integrity of casein micelles (Law& Leaver, 1998; Marchin, Putaux, Pignon, & Léonil, 2007). In this study, both CO 2 acidification and temperature were found to play a role in the non-micellar casein content. As shown in Fig. 2, the amount of non- micellar casein in the concentrates was strongly increased by the action of CO 2 acidification in combination with low temperature. However, in this research, the protective effect of non-micellar caseins (serum caseins) observed in high protein content powders (Buldo, 2012) was not confirmed as the resulting powders did not provide better reconstitutability. It is evident that fat caused some modifications in concentrates stored at 4 C due to coalescence. As a result, fat emulsions in the

17 concentrate were larger and non-homogenous (Fig. 3). Meanwhile, larger fat globules are known to produce free fat on the surface of powder particles (Table 2), which rendered the surface hydrophobic, therefore reducing solubility in water (Bhandari, 2013; Kim et al., 2009). On the other hand, the concentrates carbonated at high temperature did not show fat coalescence, thus the resulting powders have less amount of free fat (Table 2) and consequently better reconstitution properties (Table 1). Additionally, the elevated porosity of powders produced from concentrates treated at 30 C may contribute to the enhanced functional properties, as similarly reported for skim milk powders (Lee, 2014). Moreover, WMP with poor reconstitution properties were reported to contain more aggregated particles that consisted of mixtures of fat globules and proteins (Singh& Ye, 2010). Therefore, fat destabilisation may mask the positive effect of elevated levels of nonmicellar caseins in milk concentrates carbonated at low temperatures. 4. Conclusion The effect of CO 2 acidification of whole milk concentrate on WMP properties has not been reported in the literature. Results show that CO 2 acidification of whole milk concentrates at various temperatures allow the production of powders with totally different properties due to the alteration of physical states of fat. By acting on both micelles and milk fat, new generation of powders with targeted functional properties may be produced. In the future, it may be interesting to find CO 2 addition and processes conditions that lead to the increase of non-micellar casein levels (as in 2000 ppm, 4 C powders) without the modifications of fat emulsion (as in 2000 ppm, 30 C powders) to greatly improve the reconstitutability of these powders Acknowledgements 387

18 Author Claire Gaiani would like to thank Europe for their financial support towards this project (Milk PEPPER, N , International Outgoing Fellowship grant). The authors acknowledge the facilities, and the scientific and technical assistance provided by the School of Agriculture and Food Sciences (SAFS) at The University of Queensland and the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis (CMM, The University of Queensland). References Bhandari, B. (2013). Introduction to food powders. In B. Bhandari, N. Bansal, M. Zhang & P. Schuck (Eds.), Handbook of food powders processes and properties (pp. 1-25). Cambridge, UK: Woodhead Publishing. Buchheim, W. (1970). The process of fat crystallization in the fat globules of milk: Electron microscope studies by the freeze-etching method. Milchwissenschaft, 25, Buldo, P. (2012). Crystallization of fat in and outside milk fat globules - Effect of processing and storage conditions. Aarhus University, Denmark: Faculty of Science and Technology. Chaix, E., Guillaume, C., & Guillard, V. (2014). Oxygen and carbon dioxide solubility and diffusivity in solid food matrices: A review of past and current knowledge. Comprehensive Reviews in Food Science and Food Safety, 13, Dalgleish, D. G. (2011). On the structural models of bovine casein micelles - review and possible improvements. Soft Matter, 7, El-Loly, M. M. (2011). Composition, properties and nutritional aspects of milk fat globule membrane a review. Poland Journal of Food Nutrition and Science, 61, 7-32.

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22 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,

23 powders from concentrate stored at 4 C with CO2; C, powders from concentrate stored at 30 C with CO2.

24 Table 1 Physico-chemical properties of the concentrates and functional properties of the resulting powders. a Powder Concentrate properties Powder functional properties CO 2 content (actual; ppm) Ionic Ca 2+ (mm) ph Micelle size - Z average (nm) Wettability (min) a Powders are identified by theoretical CO 2 concentration (none and 2000 ppm) and CO 2 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). Dispersibility (%) Solubility (%) 0 ppm 17±9 2.75± ± ±5.8 > ± ± ppm, 4 C 1902±67 c 6.75±0.25 c 5.9±0.1 c 147.0±1.5 b > ±2.5 ns 94.0±0.6 b 2000 ppm, 30 C 1781±44 c,ns 6.25±0.50 c,ns 6.0±0.2 c,ns 182.0±3.2 ns,b > ±2.5 a,b 99.0±0.5 ns,c

25 Table 2 Physical and chemical properties of the powders. a 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 ) a Powders are identified by theoretical CO 2 concentration (none and 2000 ppm) and CO 2 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). Interstitial air content (ml 100g -1 ) 0 ppm 2.45± ± ± ± ± ± ppm, 4 C 3.89±0.49 b 30.38±4.25 ns 26.65±2.02 ns 1.00±0.08 ns 12.99±0.09 c ±0.91 ns 2000 ppm, 30 C 2.73±0.37 nsa 30.29±0.18 ns,ns 29.78±0.61 ns,ns 1.10±0.03 nsns 11.81±0.21 cns ±0.29 nsns

26 Figure 1

27 Figure 2

28 Figure 3

29 Figure 4

30 Figure 5