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Physical and functional properties of whole milk powders prepared from concentrate partially acidified with CO2 at two temperatures
Accepted Manuscript Physical and functional properties of whole milk powders prepared from concentrate partially 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 and functional properties of whole milk powders prepared from concentrate partially acidified with CO2 at two temperatures, International Dairy Journal (2016), doi: 10.1016/j.idairyj.2015.12.009. 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.
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Physical and functional properties of whole milk powders prepared from concentrate
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partially acidified with CO2 at two temperatures
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Leni Kosasiha, Bhesh Bhandaria, Sangeeta Prakasha, Nidhi Bhansala & Claire Gaiania,b*
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Australia
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b Université
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France
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de Lorraine, LIBio, 2 avenue de la Forêt de Haye, TSA 40602, 54518 Vandoeuvre-lès-Nancy,
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of Queensland, School of Agricultural and Food Science, St. Lucia, Queensland 4072,
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a University
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* Corresponding author. Tel.: +33(0)3 83 59 60 73
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E-mail address: [email protected] (C. Gaiani)
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ABSTRACT
28 Effects of carbonation of whole milk concentrate on spray dried powder properties were
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investigated. Concentrate acidification by CO2 addition (2000 ppm) was found to strongly modify
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the functional properties (solubility, dispersibility) and structural/physical properties (porosity,
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free fat) of the resulting powders. For concentrates treated at low temperature (where the majority
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of emulsified fat is in a solid state at 4 °C), colloidal calcium phosphate (CCP) release, casein
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dissociation and fat coalescence were observed. For warm CO2 treated concentrates (30 °C) only
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CCP release was observed. The best functional properties (higher solubility and dispersibility) were
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found for powders produced from the warm treated concentrates, which were possibly due to the
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high porosity and better fat globule preservation.
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1.
Introduction
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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,
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yogurt, and fermented dairy beverages (Hotchkiss, Werner, & Lee, 2006). For example, CO2 injection
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in milk before rennet coagulation can be used to reduce the pH of milk and solubilise micellar
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calcium phosphate, which resulted in cheese containing a different mineral profile (Nelson, Lynch, &
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Barbano, 2004).
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Recently, the use of CO2 on protein concentrates to improve the functional properties of milk
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protein concentrate (MPC) powders was studied (Marella, Salunke, Biswas, Kommineni, & Metzger,
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2015). It was reported that modification in concentrate mineral environment (Marella et al., 2015)
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or micelle structure (Law & Leaver, 1998) by adding CO2 may improve the rehydration properties of
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MPC powders. The improved solubility was attributed to the decrease in micellar interaction and
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increase in non-micellar casein release caused by partial acidification (Schokker et al., 2011). The
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addition of glucono-delta-lactone to partially acidify milk concentrates may also reduce the amount
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of protein–protein interactions during drying, which contribute to the loss of solubility of high-
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protein MPC powders (Eshpari, Tong, & Corredig, 2014). Nevertheless, the use of CO2 as a
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replacement for glucono-delta-lactone, which acts as a milk acidulant (through the reaction product
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gluconic acid) has gained interest because CO2 can be totally and easily removed by heating or
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applying vacuum.
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The current literature regarding the effect of CO2 in milk has been mainly focused on skim
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milk and the resulting powders (Lee, 2014). Meanwhile, its effect on whole milk concentrates has
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never been studied and the effect of CO2 on fat has been poorly reported. It is generally accepted
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that CO2 has higher solubility in nonpolar solvents, such as lipids, than in polar solvents, such as
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water, because the molecular structure of CO2 is apolar and it has a dipole moment of zero (Chaix,
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Guillaume, & Guillard, 2014; Hotchkiss et al., 2006). Therefore, the overall objective of this research
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was to carbonate whole milk concentrates to alter the partition of CO2 in milk components, and
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analyse the effect of CO2 on the resulting powders, focusing on functional, physical and structural
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properties. It was expected that solubilisation of colloidal calcium phosphate (CCP) due to CO2
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acidification would affect the structural organisation of casein micelles and consequently alter the
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rehydration and functional properties of the resulting whole milk powder (WMP).
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Material and methods
2.1.
Material
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Whole milk (standard, not lecithinated) and skim milk (medium heat) powders for
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preparing the concentrates were purchased in 25 kg bags from Total Foodtec Pty Ltd. (Brisbane,
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Australia). The powders were a maximum of 1 month old for the experiments. Carbonation was
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accomplished by the addition of known amount of solid CO2, also known as dry ice.
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Phosphate buffered saline (×1) was prepared at a final pH of 7.4 with the following
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composition: 1.42 g L-1 sodium phosphate, 8.0 g L-1 sodium chloride, 0.2 g L-1 potassium chloride
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0.24 g L-1 potassium phosphate.
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2.2.
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Concentrate, carbonation and powder preparation
Whole milk concentrates (WMC) were prepared by dissolving 25 g of the powder in 100 g of
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Milli-Q (deionised) water at 25 °C with constant stirring at a high speed with an overhead stirrer for
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1 h. For each experiment, 3 L of concentrates was prepared. Optical microscopy on the rehydrated
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sample was done to check the complete rehydration of the powder. Finally, WMC was poured in the
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kegs for carbonation. Carbonation of the concentrate was done with frozen CO2 (dry ice). The 3 L concentrate was
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poured into an eleven litre stainless steel keg equipped with manometer. An adequate amount of
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dry ice was added in the keg allowing a theoretical CO2 content of 2000 ppm in the concentrate (Lee,
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2014). The kegs were then left for 4 h at 4 or 30 °C respectively. After overnight storage, the
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samples were spray-dried; a control samples without CO2 addition was stored overnight at 4 °C
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prior to spray-drying. A single-stage Anhydro Lab S1 spray dryer (Copenhagen, Denmark) dried the
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carbonated concentrates (without a decarbonation step). The spray dryer fitted with a pneumatic
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nozzle, supplied compressed air (6.34 bar), and operated at 170 °C and 85 °C inlet and outlet air
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temperatures, respectively. The spray dried samples were collected in zipped aluminium bags and
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analysed soon after (all analysis were done in less than 3 days).
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2.3.
Chemical analysis
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The CO2 content in the concentrate was determined using a Mettler Toledo CO2 Transmitter 5100e Electrochemical probe (InPro 5000 CO2 Sensor, Mettler-Toledo AG, Process Analytics,
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Urdorf, Switzerland). The electrochemical probe was found to be the most accurate and easiest way
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to measure CO2 among other tested methods (i.e., infra-red head space analyser, manometric assay)
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(Chaix et al., 2014; Lee, 2014). The probe was inserted directly into the liquid and final reading was
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taken when it reached equilibrium. Depending on the CO2 level in the test sample, the final reading
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reached to equilibrium in 3–8 min.
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An ionic calcium probe (LAQUAtwin, compact Ca2+ meter, B751, Horiba Scientific) directly
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measured the calcium ion concentration in 0.3 mL carbonated concentrates. The probe was capable
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of measuring ionic calcium concentration in the range 1–100 mM. A 0.25 mM resolution was possible
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in the approximate range of calcium found in milk (range 2.5 to 25 mM). The instrument was
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calibrated daily with 2.5 mM standards.
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2.4.
Structural properties
2.4.1.
Scanning electron microscope
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A field emission scanning electron microscope (SEM) type JEOL JSM-7100F with a hot
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(Schottky) electron gun (JEOL Ltd., Tokyo, Japan) and a resolution around 1 nm at 30 kV was used
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for magnifications higher than 10,000. For lower magnifications, a JEOL JSM-6460LA (JEOL Ltd.)
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with a tungsten filament electron gun was preferred. Both instrumental analyses were conducted at
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5 kV to obtain images.
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Sample preparation was done following the reported method of Mimouni, Deeth, Whittaker,
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Gidley, and Bhandari, 2010) with some modifications. A drop of milk concentrate was deposited
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onto a silicon chip wafer (ProSciTech, Kirwan, Australia) coated with poly-L-lysine (Sigma Aldrich,
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Castle Hill, Australia) which created electrostatic bonding between micelles and the substrate. A few
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drops of poly-L-lysine solution (1 mg mL-1 in phosphate-buffered saline ×1, pH 7.4) were deposited
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on the silicon wafer and allowed to air-dry overnight at room temperature in a dust-free
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environment. One drop of concentrate was then deposited and left for 30 min before rinsing with
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phosphate buffer (pH 7). A solution of 2.5 % glutaraldehyde in phosphate buffer was then applied
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for 30 min to achieve chemical fixation of the protein material. After fixation, the samples were
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washed in phosphate buffer and dehydrated using a graded ethanol series: 50%, 60%, 70%, 80%,
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90% (1 time), and 100% (3 times). The elapsed time per solution was 2 min. Finally, samples were
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dried using CO2 in a Supercritical Autosamdri-815B critical point dryer (Tousimis, Rockville, MD,
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USA).
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Both silicon wafers and powders were subsequently mounted onto SEM stubs by placing or
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sputtering them on a carbon double-sided adhesive tape. Coating was done with platinum (Q150T
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Turbo-Pumped Sputter Coater, ProSciTech) for 2 min (~ 10 nm thick).
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2.4.2.
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Milk concentrate and powders were analysed by confocal laser scanning microscopy (CLSM)
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using a Zeiss LSM 700 confocal microscope (Carl Ziess Ltd., North Ryde, New South Wales,
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Australia). Nile red and rhodamin B (Sigma Aldrich) both at a concentration of 0.1 g L-1 in PEG 200
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were used to label fat and proteins, respectively. A ratio of 1/100 (dye/concentrate or powder) was
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used and left for 20 min before imaging. Observations were done with a 63× immersion oil
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objective. An argon laser operating at excitation wavelengths of 488 nm was used. Each micrograph
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is a representation of at least 10 images of each sample.
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Particle density measurements
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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)
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was used to determine the true density of milk powders. The pycnometer was operated using
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nitrogen gas at 1.2 kPa.
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Occluded and interstitial air are defined as the difference between the volume of particles at
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a given mass and the volume of the same mass of air-free solids and of powders tapped 100 times,
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respectively (GEA Niro, 2006b). The occluded and interstitial air contents of milk powder were
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calculated using the formulas described by GEA Niro (2006b).
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2.6. Size analysis
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163 Average size distribution of casein micelles in the concentrates was measured at 25 °C using
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a 3000 HSA Malvern Zetasizer (Nano series, Malvern Instruments, Malvern, UK). Before
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measurement, the samples were filtered (0.45 μm; Millipore) to avoid fat interactions. The
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concentrates were finally diluted 200 times with Milli-Q deionised water.
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Functional properties of the powder
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The solubility (ISO, 2005), dispersibility (ISO, 2014) and wettability (ISO, 2014) of the
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powders was determined per the International Organisation for Standardisation standards with
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slight modifications due to the limited quantity of powder: the same ratio between water and
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powder was kept, but the quantity of powder used was reduced to be able to make all repetitions.
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2.8.
Milk fat analysis
2.8.1.
Free fat extraction
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Free fat extraction from milk powder was done following the procedures described
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elsewhere (Kim, Chen, & Pearce, 2002; Murriera Pazos, Gaiani, Galet, & Scher, 2012; Vignolles,
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Jeantet, Lopez, & Schuck, 2007) with some modifications. Milk powder (2 g) was weighed and mixed
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with 50 mL petroleum spirit for 5 min. The solvent was separated by filtration into a round-bottom
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flask. The powders on the filtrate paper were dried and kept for encapsulated fat analysis. The
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solvent in the flask was totally evaporated. Then, the solvent-free flask was dried in the oven. The
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free fat percentage is the ratio between the weight of extracted fat and the weight of powder.
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2.8.2.
Encapsulated fat extraction
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Milk powder recovered from free fat extraction was weighed and warm water (4 mL at 50–
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55 °C) was added. The mixture was vortexed for 2 min to dissolve the powder and release the
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encapsulated fat. A solvent mixture made of n-hexane and 2-propanol in 3:1 ratio (v/v) was added
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and vortexed for 15 min to extract the fat. The solution was then centrifuged at 1000 × g for 15 min
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and the organic phase was filtered into a dry and clean round-bottom flask. The aqueous phase was
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re-extracted with the solvent mixture and the collected organic phase was totally evaporated. Then,
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the solvent-free flask was dried in the oven. The encapsulated fat percentage is the ratio between
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the weight of extracted fat and the weight of initial powder.
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2.8.3.
Total fat extraction
Total fat was extracted from 2 g of milk powder following the same procedure used for the
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extraction of encapsulated fat described in sections 2.7.2.
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Statistical analysis
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All measurements presented in this paper were performed on three independent samples
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(except that the functional properties were done on two analysis). The KyPlot software version
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2.0 was used and a parametric multiple comparisons test (Tukey test) was performed. The
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significance level was: ***P < 0.001, **P < 0.01, *P < 0.05 and NSP > 0.05 (not significant).
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Results and discussion
3.1.
Effect of CO2 and temperature on whole milk concentrate properties
3.1.1.
pH and ionic calcium evolution in the concentrates as a function of CO2 concentration
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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
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milk fat under different states, mainly solid and liquid states, respectively. As shown in Fig. 1A,
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different pH profiles between skim and whole milk concentrates were obtained during carbonation
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at 4 °C. Lower pH values were attained for whole milk concentrate in comparison with skim milk
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concentrate, both containing the same solid content. Similar profiles were obtained after
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mathematical correction of Fig. 1A as a function of the estimated CO2 content in the skim portion
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(Fig. 1B). As mentioned previously, CO2 is more soluble in lipid than in water (Hotchkiss et al.,
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2006). Thus, it can be assumed that when fat is in a solid state (i.e., 4 °C), very little CO2 dissolves in
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the fat portion and most is dissolved in the skim portion. At 4 °C, some fat fractions remain liquid
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(Buchheim, 1970). However, it may be contained in within spherical fat globules that are covered by
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a layer of solid fat on the surface (Buchheim, 1970) that could act as a barrier to prevent CO2
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migration into the fat globules. This hypothesis was confirmed by Fig. 1C, highlighting the
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importance of the presence of fat fraction and the temperature of carbonation. Therefore, it can now
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be concluded that, during carbonation at 30 °C, CO2 dissolves both in skim and in the fat fraction,
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whereas at 4 °C, CO2 dissolves only in the skim fraction. Similar results were also obtained by others
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(Ma & Barbano, 2003) while studying the effect of temperature during carbonation in cream.
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Ionic calcium strongly correlates to the pH decrease during carbonation (Table 1). As the pH reduces by CO2, CCP gradually solubilises in the concentrates. The concentrates without CO2
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treatment presents a classical ionic calcium content around 2.75 mM. Milk at a normal pH presents
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only around 10% of the total calcium (30 mM) in a ionic form; corresponding to 3 mM (Lewis, 2011).
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Nevertheless considerable variations were observed by these authors, with the ionic calcium level
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varying from 1–5 mM Ca2+ depending on processing, storage, temperature, breed, etc. This value
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increased significantly (P < 0.001) to 6.75 and 6.25 mM for concentrates carbonated at 4 and 30 °C,
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respectively. Acidification of milk (with CO2) was previously found to increase the ionic calcium by
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180% (Klandar, Chevalier-Lucia, & Lagaude, 2009) at pH 5.95 and 5 °C. No significant differences
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were observed between the two carbonated concentrates (P > 0.05). Nevertheless, slight differences
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in ionic calcium content are possibly due to a reduction in CCP solubility with increasing
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temperature (Lewis, 2011).
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Another explanation may be that CO2 in warm concentrate is dissolved in both skim and fat
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portions. This means that there is slightly less CO2 dissolved in the skim portion for warm
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concentrate in comparison with cold concentrate. Since majority of casein micelles and CCP are
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present in the skim portion. Thus, there is less effect of CO2 acidification and less CCP release from
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casein micelle in warm concentrate than cold concentrate. The concentrate without treatment
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presents a pH around 6.8 whereas the cold and warm concentrates treated at cold and warm
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conditions with CO2 have a pH of 5.9 and 6.0, respectively. Again, no significant differences were
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observed between the two carbonated concentrates (P > 0.05). The tendency of a higher pH value of
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the warm concentrate can be explained by the slightly lower CO2 level (Table 1), although the
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difference was not significant.
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3.1.2.
Micelle size and shape evolution with carbonation Micelle size in milk concentrates was analysed by dynamic light scattering (DLS). The results
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described in Table 1 showed that micelle size in the cold treated concentrate was significantly
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reduced from 180 nm to approximately 150 nm (P < 0.01). Meanwhile, no significant differences
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were observed between the concentrate without treatment and the warm treated concentrate, in
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which both have micelle sizes around 180 nm.
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Micelles were also observed at two magnifications (50,000 and 100,000) by SEM high field
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after fixation and dehydration. At low magnification, uniformly dispersed micelles were seen in all
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concentrates. In the untreated concentrate (Fig. 2A1), micelles with a size around 200 nm were
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found uniformly distributed with the presence of small micelles or dissociated micelles in the
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background. When the concentrate was acidified with CO2 at 4 °C (Fig. 2B1), micelles were visibly
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smaller in size and greater amount of dissociated micelles were present in the background.
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Meanwhile, no apparent differences were seen between the standard and high temperature-
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acidified concentrate (Fig. 2C1). The surface of micelles in milk concentrates was also visualised in
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depth at higher magnification (Figs. 2A2,B2,C2). Our study confirmed earlier research (Dalgleish,
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2011) that suggests casein micelle are far from regular and are not perfectly spherical. Caseins
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appear to be organised as an entangled network of protein chains that protrude at the surface
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(mainly the κ-casein). In addition, microscopy observations and DLS measurements are in
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agreement as micelles in the cold treated concentrate appeared significantly smaller than in the
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standard concentrate.
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It was already demonstrated that the solubilisation of CCP is quick and pH dependent,
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whereas the dissociation of caseins from micelles varies with pH and temperature (Law & Leaver,
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1998). A combination of low temperature and low pH was reported to cause the dissociation of CCP
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and casein monomers from micelles when cold milk pH decreased from pH 6.7 to 5.2 (Law & Leaver,
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1998; Post, Arnold, Weiss, & Hinrichs, 2012). Meanwhile, no difference in micelle size was observed
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between the warm treated concentrate and the untreated milk (Table 1, Fig. 2A,C). This could be
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due to the solitary dissociation of CCP and lack of casein dissociation from micelles in the warm
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treated concentrate, which showed that CO2 does not influence micelle size in milk concentrates at
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high temperatures.
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3.1.3.
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Fat evolution with carbonation CLSM images (at ambient temperature) was conducted after fat labelling of milk
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concentrates stored at 4 °C (Fig. 3A1,A2) and 30 °C (Fig. 3B1,B2). For each temperature, images
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were obtained for both CO2 treated (Fig. 3A2,B2) and untreated concentrates (Fig. 3A1,B1). Small
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and regularly distributed fat globules were observed in concentrates without CO2 treatment,
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regardless of the temperature. Meanwhile, fat globule coalescence was noticed for concentrates
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carbonated at 4 °C (Fig. 3A2). The concentrate treated at 30 °C (Fig. 3B2) was also observed to have
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larger fat globules than the control. However, fat globules as big as at 4 °C were never observed at
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30 °C. These results are in agreement with the static light scattering analysis (data not shown).
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Therefore, it is envisaged that formation and breaking of CO2 bubbles during the carbonation
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process induced coalescence mainly by a surface-mediated mechanism. This mechanism involved
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the absorption of CO2 at the fat globule membrane interface and subsequently causing coalescence
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of fat globules, with concomitant release of liquid oil onto the interfaces (Fuller, Considine, Golding,
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Matia-Merino, & MacGibbon, 2015). This phenomenon may be more pronounced at 4 °C where milk
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fat is mainly in a solid state (El-Loly, 2011). The combination of fat globule membrane protrusion by
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crystalline solid fat and CO2 interaction with the membrane might have contributed to the
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coalescence of fat. It is already known that air accelerates the process of fat coalescence, for instance
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during the manufacture of ice cream (El-Loly, 2011). However, similar results during carbonation
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were never reported as carbonation on whole milk concentrate was sparsely studied. Therefore, the
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effect of CO2 on fat coalescence will need further investigation.
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Effect of CO2 on spray dried powder properties
3.2.1.
Particle size, shape and density
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The microstructures of the powders are presented in Fig. 4. At low magnifications (Fig.
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4A1,B1,C1), similar features were observed among the standard and treated powders, with small
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particles agglomerated into bigger structures of around 50–100 µm. At intermediate magnifications
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(Fig. 4A2,B2,C2), again similar round particles were observed. Finally, at high magnifications where
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the powder surfaces were clearly visualised, no significant modifications were observed in relation
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to CO2 and/or temperature treatments (Fig. 4A3,B3,C3).
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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
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occluded gas/air contents of powders produced from concentrates treated with CO2 was 10 times
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higher than the powders from untreated concentrates. Meanwhile, no significant differences were
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noted between powders from concentrates carbonated at 4 and 30 °C (P > 0.05). Moreover, the true
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density and interstitial air content were not significantly lower for CO2 treated powders even if a
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tendency to lower values was observed. The presence of CO2 in milk concentrates is responsible for
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the achievement of internal porosity in the resulting powders (Lee, 2014). Therefore, these results
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were expected as the concentrates were not degassed prior to spray drying. Additionally, as shown
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in Fig. 5A1,B1,C1, porous structures were seen in CO2 treated powders, whereas standard powders
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presented some pores, but less important. The cut particles imaged by SEM also showed a lack of
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vacuoles in the untreated powders (Fig. 5A2), whereas huge vacuoles were present in the treated
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powders (Fig. 5B2,C2). At higher magnification (Fig. 5A3,B3,C3), small internal pores were visible in
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the standard powders and the envelope thickness of the other two powders was found to be around
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2–3 µm, which supported the CLSM results. These microscopy observations supported the occluded
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air measurements described in Table 2.
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The CLSM images also showed fat globules (in green) and proteins (in red) distribution in
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each powder particle (Fig. 4A1,B1,C1). However, no real differences were noticed among the
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powders, because all particles presented heterogeneous fat globules with a protein layer at the
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surface. Many authors have reported that greater than 95% of WMP surface was covered with fat,
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mostly free fat, followed by layers of protein, lactose and fat globules protected by proteins (Fyfe,
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Kravchuk, Nguyen, Deeth, & Bhandari, 2011; Kim et al., 2002; Kim, Chen, & Pearce, 2009). However,
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these surface fat layer, generated due to fat globules breakage, cannot be visualised by CLSM due to
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its low resolution (Vignolles et al., 2007).
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3.2.2.
Particle functional properties The analyses of powder functional properties are presented in Table 1. It was found that all
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powders did not wet within 5 min. This result was expected as WMP particles in this work were not
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agglomerated and/or lecithinated according to the manufacture of commercial powders. Our results
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are also different from other researchers on skim milk powder, where CO2 was found to improve
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wettability (Lee, 2014). However, comparisons are impossible due to the different natures of skim
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and whole milk powders. Nonetheless, the powders produced from concentrates carbonated at 30
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°C showed significant improvements in dispersibility (P < 0.01) and solubility (P < 0.001), while the
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reconstitution properties of powders produced from concentrates treated with CO2 at 4 °C were
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decreased (for solubility). The dispersibility of standard and powders treated at 4 and 30 °C were
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54, 46 and 65%, respectively. A similar trend was observed for the solubility of these powders, with
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values of 97, 94 and 99%, respectively. These differences cannot be attributed to modifications of
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particle shape or surface morphology because all powder particles have similar structures as
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observed in Fig. 4A,B,C. In addition, particles size for the three powders were also similar with a d50
352
around 15 µm (data not shown). Meanwhile, improved functional properties have been attributed
353
to powders containing high levels of non-micellar casein in high protein content powders (Schokker
354
et al., 2011). Several factors such as calcium chelators, reduced pH, high pressure, and ionic strength
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have been found responsible for the structural integrity of casein micelles (Law & Leaver, 1998;
356
Marchin, Putaux, Pignon, & Léonil, 2007). In this study, both CO2 acidification and temperature were
357
found to play a role in the non-micellar casein content. As shown in Fig. 2, the amount of non-
358
micellar casein in the concentrates was strongly increased by the action of CO2 acidification in
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combination with low temperature. However, in this research, the protective effect of non-micellar
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caseins (serum caseins) observed in high protein content powders (Buldo, 2012) was not confirmed
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as the resulting powders did not provide better reconstitutability. It is evident that fat caused some
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modifications in concentrates stored at 4 °C due to coalescence. As a result, fat emulsions in the
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concentrate were larger and non-homogenous (Fig. 3). Meanwhile, larger fat globules are known to
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produce free fat on the surface of powder particles (Table 2), which rendered the surface
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hydrophobic, therefore reducing solubility in water (Bhandari, 2013; Kim et al., 2009). On the other
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hand, the concentrates carbonated at high temperature did not show fat coalescence, thus the
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resulting powders have less amount of free fat (Table 2) and consequently better reconstitution
368
properties (Table 1). Additionally, the elevated porosity of powders produced from concentrates
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treated at 30 °C may contribute to the enhanced functional properties, as similarly reported for
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skim milk powders (Lee, 2014). Moreover, WMP with poor reconstitution properties were reported
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to contain more aggregated particles that consisted of mixtures of fat globules and proteins (Singh &
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Ye, 2010). Therefore, fat destabilisation may mask the positive effect of elevated levels of non-
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micellar caseins in milk concentrates carbonated at low temperatures.
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Conclusion
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The effect of CO2 acidification of whole milk concentrate on WMP properties has not been reported in the literature. Results show that CO2 acidification of whole milk concentrates at various
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temperatures allow the production of powders with totally different properties due to the alteration
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of physical states of fat. By acting on both micelles and milk fat, new generation of powders with
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targeted functional properties may be produced. In the future, it may be interesting to find CO2
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addition and processes conditions that lead to the increase of non-micellar casein levels (as in 2000
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ppm, 4 °C powders) without the modifications of fat emulsion (as in 2000 ppm, 30 °C powders) to
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greatly improve the reconstitutability of these powders.
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Acknowledgements
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Author Claire Gaiani would like to thank Europe for their financial support towards this project
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(Milk PEPPER, N°621727, International Outgoing Fellowship grant). The authors acknowledge the
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facilities, and the scientific and technical assistance provided by the School of Agriculture and Food
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Sciences (SAFS) at The University of Queensland and the Australian Microscopy & Microanalysis
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Research Facility at the Centre for Microscopy and Microanalysis (CMM, The University of
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Queensland).
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Chaix, E., Guillaume, C., & Guillard, V. (2014). Oxygen and carbon dioxide solubility and diffusivity in
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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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Eshpari, H., Tong, P. S., & Corredig, M. (2014). Changes in the physical properties, solubility, and heat
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stability of milk protein concentrates prepared from partially acidified milk. Journal of Dairy
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Science, 97, 7394-7401. Fuller, G. T., Considine, T., Golding, M., Matia-Merino, L., & MacGibbon, A. (2015). Aggregation
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Fyfe, K., Kravchuk, O., Nguyen, A. V., Deeth, H., & Bhandari, B. (2011). Influence of dryer type on surface characteristics of milk powders. Drying Technology, 29, 758-769.
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GEA Niro. (2006a). A-2a Powder bulk density. In GEA Niro analytical methods. Soeborg, Denmark: GEA Niro.
GEA Niro. (2006b). A-11a Particle density occluded air and interstitial air (air pycnometer method). In GEA Niro analytical methods. Soeborg, Denmark: GEA Niro.
Hotchkiss, J. H., Werner, B. G., & Lee, E. Y. C. (2006). Addition of carbon dioxide to dairy products to
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improve quality: A comprehensive review. Comprehensive Reviews in Food Science and Food
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wettability (Vol. ISO/TS 17792:2006). Geneva, Switzerland: International Standardisation Organisation.
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Kim, E. H. J., Chen, X. D., & Pearce, D. (2002). Surface characterization of four industrial spray-dried
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dairy powders in relation to chemical composition, structure and wetting property. Colloids
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and Surfaces B: Biointerfaces, 26, 197-212.
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Kim, E. H. J., Chen, X. D., & Pearce, D. (2009). Surface composition of industrial spray-dried milk
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powders. 1. Development of surface composition during manufacture. Journal of Food
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Engineering, 94, 163-168. Klandar, A. H., Chevalier-Lucia, D., & Lagaude, A. (2009). Effects of reverse CO2 acidification cycles,
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calcium supplementation, pH adjustment and chilled storage on physico-chemical and
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rennet coagulation properties of reconstituted low- and medium-heat skim milk powders.
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Law, A. J. R., & Leaver, J. (1998). Effects of acidification and storage of milk on dissociation of bovine casein micelles. Journal of Agricultural and Food Chemistry, 46, 5008-5016.
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Lee, E. Y. C. (2014). The effects of carbon dioxide on the physico-chemical and microbiological
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properties of skim milk and whey protein concentrates. Brisbane, Australia: The University of
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Queensland.
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Lewis, M. J. (2011). The measurement and significance of ionic calcium in milk – A review.
Ma, Y., & Barbano, D. M. (2003). Effect of temperature of CO2 injection on the pH and freezing point of milks and creams. Journal of Dairy Science, 86, 1578-1589. Marchin, S., Putaux, J.-L., Pignon, F., & Léonil, J. (2007). Effects of the environmental factors on the
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casein micelle structure studied by cryo transmission electron microscopy and small-angle
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x-ray scattering/ultrasmall-angle xray scattering. Journal of Chemical Physics, 126, 125-138.
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Marella, C., Salunke, P., Biswas, A. C., Kommineni, A., & Metzger, L. E. (2015). Manufacture of modified milk protein concentrate utilizing injection of carbon dioxide. Journal of Dairy Science, 98, 3577-3589.
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Mimouni, A., Deeth, H. C., Whittaker, A. K., Gidley, M. J., & Bhandari, B. R. (2010). Investigation of the
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microstructure of milk protein concentrate powders during rehydration: Alterations during
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storage. Journal of Dairy Science, 93, 463-472.
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Murriera Pazos, I., Gaiani, C., Galet, L., & Scher, J. (2012). Composition gradient from surface to core in dairy powders: Agglomeration effect. Food Hydrocolloids, 26, 149-158. Nelson, B. K., Lynch, J. M., & Barbano, D. M. (2004). Impact of milk preacidification with CO2 on
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Cheddar cheese composition and yield. Journal of Dairy Science, 87, 3581-3589.
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Post, A. E., Arnold, B., Weiss, J., & Hinrichs, J. (2012). Effect of temperature and pH on the solubility
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of caseins: Environmental influences on the dissociation of α- and β-casein. Journal of Dairy
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Science, 95, 1603-1616.
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Schokker, E. P., Church, J. S., Mata, J. P., Gilbert, E. P., Puvanenthiran, A., & Udabage, P. (2011). Reconstitution properties of micellar casein powder: Effects of composition and storage.
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International Dairy Journal, 21, 877-886.
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Vignolles, M.-L., Jeantet, R., Lopez, C., & Schuck, P. (2007). Free fat, surface fat and dairy powders: interactions between process and product. A review. Lait, 87, 187-236.
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properties of whole milk powders - A minireview. Lait, 90.
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Singh, H., & Ye, A. (2010). Controlling milk protein interactions to enhance the reconstitution
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Figure legends
Fig. 1. pH evolution with CO2 concentration for whole milk () and skim milk ()
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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).
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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
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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
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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
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1000× (1), 3000× (2) and 10,000× (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.
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
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30 °C with CO2.
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Table 1
Powder
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Physico-chemical properties of the concentrates and functional properties of the resulting powders. a Concentrate properties
Powder functional properties
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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CO2 content (actual; ppm)
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).
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Table 2
Powder
Solvent extraction
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Physical and chemical properties of the powders. a
Physical properties
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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Free fat (g L-1)
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).
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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 and functional properties of whole milk powders prepared from concentrate partially acidified with CO2 at two temperatures, International Dairy Journal (2016), doi: 10.1016/j.idairyj.2015.12.009. 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.
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Physical and functional properties of whole milk powders prepared from concentrate
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partially acidified with CO2 at two temperatures
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Leni Kosasiha, Bhesh Bhandaria, Sangeeta Prakasha, Nidhi Bhansala & Claire Gaiania,b*
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Australia
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b Université
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France
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de Lorraine, LIBio, 2 avenue de la Forêt de Haye, TSA 40602, 54518 Vandoeuvre-lès-Nancy,
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of Queensland, School of Agricultural and Food Science, St. Lucia, Queensland 4072,
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a University
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* Corresponding author. Tel.: +33(0)3 83 59 60 73
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E-mail address: [email protected] (C. Gaiani)
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__________________________________________________________________________________________________________________
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ABSTRACT
28 Effects of carbonation of whole milk concentrate on spray dried powder properties were
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investigated. Concentrate acidification by CO2 addition (2000 ppm) was found to strongly modify
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the functional properties (solubility, dispersibility) and structural/physical properties (porosity,
32
free fat) of the resulting powders. For concentrates treated at low temperature (where the majority
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of emulsified fat is in a solid state at 4 °C), colloidal calcium phosphate (CCP) release, casein
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dissociation and fat coalescence were observed. For warm CO2 treated concentrates (30 °C) only
35
CCP release was observed. The best functional properties (higher solubility and dispersibility) were
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found for powders produced from the warm treated concentrates, which were possibly due to the
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high porosity and better fat globule preservation.
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1.
Introduction
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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,
44
yogurt, and fermented dairy beverages (Hotchkiss, Werner, & Lee, 2006). For example, CO2 injection
45
in milk before rennet coagulation can be used to reduce the pH of milk and solubilise micellar
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calcium phosphate, which resulted in cheese containing a different mineral profile (Nelson, Lynch, &
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Barbano, 2004).
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Recently, the use of CO2 on protein concentrates to improve the functional properties of milk
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protein concentrate (MPC) powders was studied (Marella, Salunke, Biswas, Kommineni, & Metzger,
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2015). It was reported that modification in concentrate mineral environment (Marella et al., 2015)
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or micelle structure (Law & Leaver, 1998) by adding CO2 may improve the rehydration properties of
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MPC powders. The improved solubility was attributed to the decrease in micellar interaction and
53
increase in non-micellar casein release caused by partial acidification (Schokker et al., 2011). The
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addition of glucono-delta-lactone to partially acidify milk concentrates may also reduce the amount
55
of protein–protein interactions during drying, which contribute to the loss of solubility of high-
56
protein MPC powders (Eshpari, Tong, & Corredig, 2014). Nevertheless, the use of CO2 as a
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replacement for glucono-delta-lactone, which acts as a milk acidulant (through the reaction product
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gluconic acid) has gained interest because CO2 can be totally and easily removed by heating or
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applying vacuum.
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The current literature regarding the effect of CO2 in milk has been mainly focused on skim
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milk and the resulting powders (Lee, 2014). Meanwhile, its effect on whole milk concentrates has
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never been studied and the effect of CO2 on fat has been poorly reported. It is generally accepted
63
that CO2 has higher solubility in nonpolar solvents, such as lipids, than in polar solvents, such as
64
water, because the molecular structure of CO2 is apolar and it has a dipole moment of zero (Chaix,
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Guillaume, & Guillard, 2014; Hotchkiss et al., 2006). Therefore, the overall objective of this research
66
was to carbonate whole milk concentrates to alter the partition of CO2 in milk components, and
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analyse the effect of CO2 on the resulting powders, focusing on functional, physical and structural
68
properties. It was expected that solubilisation of colloidal calcium phosphate (CCP) due to CO2
69
acidification would affect the structural organisation of casein micelles and consequently alter the
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rehydration and functional properties of the resulting whole milk powder (WMP).
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2.
Material and methods
2.1.
Material
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Whole milk (standard, not lecithinated) and skim milk (medium heat) powders for
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preparing the concentrates were purchased in 25 kg bags from Total Foodtec Pty Ltd. (Brisbane,
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Australia). The powders were a maximum of 1 month old for the experiments. Carbonation was
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accomplished by the addition of known amount of solid CO2, also known as dry ice.
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Phosphate buffered saline (×1) was prepared at a final pH of 7.4 with the following
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composition: 1.42 g L-1 sodium phosphate, 8.0 g L-1 sodium chloride, 0.2 g L-1 potassium chloride
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0.24 g L-1 potassium phosphate.
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2.2.
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Concentrate, carbonation and powder preparation
Whole milk concentrates (WMC) were prepared by dissolving 25 g of the powder in 100 g of
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Milli-Q (deionised) water at 25 °C with constant stirring at a high speed with an overhead stirrer for
88
1 h. For each experiment, 3 L of concentrates was prepared. Optical microscopy on the rehydrated
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sample was done to check the complete rehydration of the powder. Finally, WMC was poured in the
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kegs for carbonation. Carbonation of the concentrate was done with frozen CO2 (dry ice). The 3 L concentrate was
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poured into an eleven litre stainless steel keg equipped with manometer. An adequate amount of
93
dry ice was added in the keg allowing a theoretical CO2 content of 2000 ppm in the concentrate (Lee,
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2014). The kegs were then left for 4 h at 4 or 30 °C respectively. After overnight storage, the
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samples were spray-dried; a control samples without CO2 addition was stored overnight at 4 °C
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prior to spray-drying. A single-stage Anhydro Lab S1 spray dryer (Copenhagen, Denmark) dried the
97
carbonated concentrates (without a decarbonation step). The spray dryer fitted with a pneumatic
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nozzle, supplied compressed air (6.34 bar), and operated at 170 °C and 85 °C inlet and outlet air
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temperatures, respectively. The spray dried samples were collected in zipped aluminium bags and
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analysed soon after (all analysis were done in less than 3 days).
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2.3.
Chemical analysis
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The CO2 content in the concentrate was determined using a Mettler Toledo CO2 Transmitter 5100e Electrochemical probe (InPro 5000 CO2 Sensor, Mettler-Toledo AG, Process Analytics,
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Urdorf, Switzerland). The electrochemical probe was found to be the most accurate and easiest way
107
to measure CO2 among other tested methods (i.e., infra-red head space analyser, manometric assay)
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(Chaix et al., 2014; Lee, 2014). The probe was inserted directly into the liquid and final reading was
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taken when it reached equilibrium. Depending on the CO2 level in the test sample, the final reading
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reached to equilibrium in 3–8 min.
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An ionic calcium probe (LAQUAtwin, compact Ca2+ meter, B751, Horiba Scientific) directly
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measured the calcium ion concentration in 0.3 mL carbonated concentrates. The probe was capable
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of measuring ionic calcium concentration in the range 1–100 mM. A 0.25 mM resolution was possible
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in the approximate range of calcium found in milk (range 2.5 to 25 mM). The instrument was
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calibrated daily with 2.5 mM standards.
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2.4.
Structural properties
2.4.1.
Scanning electron microscope
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A field emission scanning electron microscope (SEM) type JEOL JSM-7100F with a hot
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(Schottky) electron gun (JEOL Ltd., Tokyo, Japan) and a resolution around 1 nm at 30 kV was used
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for magnifications higher than 10,000. For lower magnifications, a JEOL JSM-6460LA (JEOL Ltd.)
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with a tungsten filament electron gun was preferred. Both instrumental analyses were conducted at
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5 kV to obtain images.
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Sample preparation was done following the reported method of Mimouni, Deeth, Whittaker,
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Gidley, and Bhandari, 2010) with some modifications. A drop of milk concentrate was deposited
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onto a silicon chip wafer (ProSciTech, Kirwan, Australia) coated with poly-L-lysine (Sigma Aldrich,
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Castle Hill, Australia) which created electrostatic bonding between micelles and the substrate. A few
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drops of poly-L-lysine solution (1 mg mL-1 in phosphate-buffered saline ×1, pH 7.4) were deposited
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on the silicon wafer and allowed to air-dry overnight at room temperature in a dust-free
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environment. One drop of concentrate was then deposited and left for 30 min before rinsing with
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phosphate buffer (pH 7). A solution of 2.5 % glutaraldehyde in phosphate buffer was then applied
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for 30 min to achieve chemical fixation of the protein material. After fixation, the samples were
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washed in phosphate buffer and dehydrated using a graded ethanol series: 50%, 60%, 70%, 80%,
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90% (1 time), and 100% (3 times). The elapsed time per solution was 2 min. Finally, samples were
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dried using CO2 in a Supercritical Autosamdri-815B critical point dryer (Tousimis, Rockville, MD,
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USA).
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Both silicon wafers and powders were subsequently mounted onto SEM stubs by placing or
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sputtering them on a carbon double-sided adhesive tape. Coating was done with platinum (Q150T
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Turbo-Pumped Sputter Coater, ProSciTech) for 2 min (~ 10 nm thick).
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2.4.2.
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Milk concentrate and powders were analysed by confocal laser scanning microscopy (CLSM)
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using a Zeiss LSM 700 confocal microscope (Carl Ziess Ltd., North Ryde, New South Wales,
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Australia). Nile red and rhodamin B (Sigma Aldrich) both at a concentration of 0.1 g L-1 in PEG 200
146
were used to label fat and proteins, respectively. A ratio of 1/100 (dye/concentrate or powder) was
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used and left for 20 min before imaging. Observations were done with a 63× immersion oil
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objective. An argon laser operating at excitation wavelengths of 488 nm was used. Each micrograph
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is a representation of at least 10 images of each sample.
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Particle density measurements
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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)
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was used to determine the true density of milk powders. The pycnometer was operated using
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nitrogen gas at 1.2 kPa.
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Occluded and interstitial air are defined as the difference between the volume of particles at
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a given mass and the volume of the same mass of air-free solids and of powders tapped 100 times,
159
respectively (GEA Niro, 2006b). The occluded and interstitial air contents of milk powder were
160
calculated using the formulas described by GEA Niro (2006b).
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2.6. Size analysis
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163 Average size distribution of casein micelles in the concentrates was measured at 25 °C using
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a 3000 HSA Malvern Zetasizer (Nano series, Malvern Instruments, Malvern, UK). Before
166
measurement, the samples were filtered (0.45 μm; Millipore) to avoid fat interactions. The
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concentrates were finally diluted 200 times with Milli-Q deionised water.
168 2.7.
Functional properties of the powder
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The solubility (ISO, 2005), dispersibility (ISO, 2014) and wettability (ISO, 2014) of the
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powders was determined per the International Organisation for Standardisation standards with
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slight modifications due to the limited quantity of powder: the same ratio between water and
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powder was kept, but the quantity of powder used was reduced to be able to make all repetitions.
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2.8.
Milk fat analysis
2.8.1.
Free fat extraction
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Free fat extraction from milk powder was done following the procedures described
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elsewhere (Kim, Chen, & Pearce, 2002; Murriera Pazos, Gaiani, Galet, & Scher, 2012; Vignolles,
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Jeantet, Lopez, & Schuck, 2007) with some modifications. Milk powder (2 g) was weighed and mixed
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with 50 mL petroleum spirit for 5 min. The solvent was separated by filtration into a round-bottom
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flask. The powders on the filtrate paper were dried and kept for encapsulated fat analysis. The
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solvent in the flask was totally evaporated. Then, the solvent-free flask was dried in the oven. The
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free fat percentage is the ratio between the weight of extracted fat and the weight of powder.
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2.8.2.
Encapsulated fat extraction
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Milk powder recovered from free fat extraction was weighed and warm water (4 mL at 50–
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55 °C) was added. The mixture was vortexed for 2 min to dissolve the powder and release the
190
encapsulated fat. A solvent mixture made of n-hexane and 2-propanol in 3:1 ratio (v/v) was added
191
and vortexed for 15 min to extract the fat. The solution was then centrifuged at 1000 × g for 15 min
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and the organic phase was filtered into a dry and clean round-bottom flask. The aqueous phase was
193
re-extracted with the solvent mixture and the collected organic phase was totally evaporated. Then,
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the solvent-free flask was dried in the oven. The encapsulated fat percentage is the ratio between
195
the weight of extracted fat and the weight of initial powder.
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2.8.3.
Total fat extraction
Total fat was extracted from 2 g of milk powder following the same procedure used for the
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extraction of encapsulated fat described in sections 2.7.2.
200 2.9.
Statistical analysis
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All measurements presented in this paper were performed on three independent samples
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(except that the functional properties were done on two analysis). The KyPlot software version
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2.0 was used and a parametric multiple comparisons test (Tukey test) was performed. The
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significance level was: ***P < 0.001, **P < 0.01, *P < 0.05 and NSP > 0.05 (not significant).
207 208
3.
209 210
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Results and discussion
3.1.
Effect of CO2 and temperature on whole milk concentrate properties
3.1.1.
pH and ionic calcium evolution in the concentrates as a function of CO2 concentration
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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
215
milk fat under different states, mainly solid and liquid states, respectively. As shown in Fig. 1A,
216
different pH profiles between skim and whole milk concentrates were obtained during carbonation
217
at 4 °C. Lower pH values were attained for whole milk concentrate in comparison with skim milk
218
concentrate, both containing the same solid content. Similar profiles were obtained after
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mathematical correction of Fig. 1A as a function of the estimated CO2 content in the skim portion
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(Fig. 1B). As mentioned previously, CO2 is more soluble in lipid than in water (Hotchkiss et al.,
221
2006). Thus, it can be assumed that when fat is in a solid state (i.e., 4 °C), very little CO2 dissolves in
222
the fat portion and most is dissolved in the skim portion. At 4 °C, some fat fractions remain liquid
223
(Buchheim, 1970). However, it may be contained in within spherical fat globules that are covered by
224
a layer of solid fat on the surface (Buchheim, 1970) that could act as a barrier to prevent CO2
225
migration into the fat globules. This hypothesis was confirmed by Fig. 1C, highlighting the
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importance of the presence of fat fraction and the temperature of carbonation. Therefore, it can now
227
be concluded that, during carbonation at 30 °C, CO2 dissolves both in skim and in the fat fraction,
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whereas at 4 °C, CO2 dissolves only in the skim fraction. Similar results were also obtained by others
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(Ma & Barbano, 2003) while studying the effect of temperature during carbonation in cream.
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Ionic calcium strongly correlates to the pH decrease during carbonation (Table 1). As the pH reduces by CO2, CCP gradually solubilises in the concentrates. The concentrates without CO2
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treatment presents a classical ionic calcium content around 2.75 mM. Milk at a normal pH presents
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only around 10% of the total calcium (30 mM) in a ionic form; corresponding to 3 mM (Lewis, 2011).
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Nevertheless considerable variations were observed by these authors, with the ionic calcium level
235
varying from 1–5 mM Ca2+ depending on processing, storage, temperature, breed, etc. This value
236
increased significantly (P < 0.001) to 6.75 and 6.25 mM for concentrates carbonated at 4 and 30 °C,
237
respectively. Acidification of milk (with CO2) was previously found to increase the ionic calcium by
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180% (Klandar, Chevalier-Lucia, & Lagaude, 2009) at pH 5.95 and 5 °C. No significant differences
239
were observed between the two carbonated concentrates (P > 0.05). Nevertheless, slight differences
240
in ionic calcium content are possibly due to a reduction in CCP solubility with increasing
241
temperature (Lewis, 2011).
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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
244
concentrate in comparison with cold concentrate. Since majority of casein micelles and CCP are
245
present in the skim portion. Thus, there is less effect of CO2 acidification and less CCP release from
246
casein micelle in warm concentrate than cold concentrate. The concentrate without treatment
247
presents a pH around 6.8 whereas the cold and warm concentrates treated at cold and warm
248
conditions with CO2 have a pH of 5.9 and 6.0, respectively. Again, no significant differences were
249
observed between the two carbonated concentrates (P > 0.05). The tendency of a higher pH value of
250
the warm concentrate can be explained by the slightly lower CO2 level (Table 1), although the
251
difference was not significant.
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3.1.2.
Micelle size and shape evolution with carbonation Micelle size in milk concentrates was analysed by dynamic light scattering (DLS). The results
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described in Table 1 showed that micelle size in the cold treated concentrate was significantly
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reduced from 180 nm to approximately 150 nm (P < 0.01). Meanwhile, no significant differences
257
were observed between the concentrate without treatment and the warm treated concentrate, in
258
which both have micelle sizes around 180 nm.
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Micelles were also observed at two magnifications (50,000 and 100,000) by SEM high field
260
after fixation and dehydration. At low magnification, uniformly dispersed micelles were seen in all
261
concentrates. In the untreated concentrate (Fig. 2A1), micelles with a size around 200 nm were
262
found uniformly distributed with the presence of small micelles or dissociated micelles in the
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background. When the concentrate was acidified with CO2 at 4 °C (Fig. 2B1), micelles were visibly
264
smaller in size and greater amount of dissociated micelles were present in the background.
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Meanwhile, no apparent differences were seen between the standard and high temperature-
266
acidified concentrate (Fig. 2C1). The surface of micelles in milk concentrates was also visualised in
267
depth at higher magnification (Figs. 2A2,B2,C2). Our study confirmed earlier research (Dalgleish,
268
2011) that suggests casein micelle are far from regular and are not perfectly spherical. Caseins
269
appear to be organised as an entangled network of protein chains that protrude at the surface
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(mainly the κ-casein). In addition, microscopy observations and DLS measurements are in
271
agreement as micelles in the cold treated concentrate appeared significantly smaller than in the
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standard concentrate.
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It was already demonstrated that the solubilisation of CCP is quick and pH dependent,
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whereas the dissociation of caseins from micelles varies with pH and temperature (Law & Leaver,
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1998). A combination of low temperature and low pH was reported to cause the dissociation of CCP
276
and casein monomers from micelles when cold milk pH decreased from pH 6.7 to 5.2 (Law & Leaver,
277
1998; Post, Arnold, Weiss, & Hinrichs, 2012). Meanwhile, no difference in micelle size was observed
278
between the warm treated concentrate and the untreated milk (Table 1, Fig. 2A,C). This could be
279
due to the solitary dissociation of CCP and lack of casein dissociation from micelles in the warm
280
treated concentrate, which showed that CO2 does not influence micelle size in milk concentrates at
281
high temperatures.
283 284
3.1.3.
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Fat evolution with carbonation CLSM images (at ambient temperature) was conducted after fat labelling of milk
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concentrates stored at 4 °C (Fig. 3A1,A2) and 30 °C (Fig. 3B1,B2). For each temperature, images
286
were obtained for both CO2 treated (Fig. 3A2,B2) and untreated concentrates (Fig. 3A1,B1). Small
287
and regularly distributed fat globules were observed in concentrates without CO2 treatment,
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regardless of the temperature. Meanwhile, fat globule coalescence was noticed for concentrates
289
carbonated at 4 °C (Fig. 3A2). The concentrate treated at 30 °C (Fig. 3B2) was also observed to have
290
larger fat globules than the control. However, fat globules as big as at 4 °C were never observed at
291
30 °C. These results are in agreement with the static light scattering analysis (data not shown).
292
Therefore, it is envisaged that formation and breaking of CO2 bubbles during the carbonation
293
process induced coalescence mainly by a surface-mediated mechanism. This mechanism involved
294
the absorption of CO2 at the fat globule membrane interface and subsequently causing coalescence
295
of fat globules, with concomitant release of liquid oil onto the interfaces (Fuller, Considine, Golding,
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Matia-Merino, & MacGibbon, 2015). This phenomenon may be more pronounced at 4 °C where milk
297
fat is mainly in a solid state (El-Loly, 2011). The combination of fat globule membrane protrusion by
298
crystalline solid fat and CO2 interaction with the membrane might have contributed to the
299
coalescence of fat. It is already known that air accelerates the process of fat coalescence, for instance
300
during the manufacture of ice cream (El-Loly, 2011). However, similar results during carbonation
301
were never reported as carbonation on whole milk concentrate was sparsely studied. Therefore, the
302
effect of CO2 on fat coalescence will need further investigation.
303 3.2.
Effect of CO2 on spray dried powder properties
3.2.1.
Particle size, shape and density
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The microstructures of the powders are presented in Fig. 4. At low magnifications (Fig.
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4A1,B1,C1), similar features were observed among the standard and treated powders, with small
309
particles agglomerated into bigger structures of around 50–100 µm. At intermediate magnifications
310
(Fig. 4A2,B2,C2), again similar round particles were observed. Finally, at high magnifications where
311
the powder surfaces were clearly visualised, no significant modifications were observed in relation
312
to CO2 and/or temperature treatments (Fig. 4A3,B3,C3).
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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
315
occluded gas/air contents of powders produced from concentrates treated with CO2 was 10 times
316
higher than the powders from untreated concentrates. Meanwhile, no significant differences were
317
noted between powders from concentrates carbonated at 4 and 30 °C (P > 0.05). Moreover, the true
318
density and interstitial air content were not significantly lower for CO2 treated powders even if a
319
tendency to lower values was observed. The presence of CO2 in milk concentrates is responsible for
320
the achievement of internal porosity in the resulting powders (Lee, 2014). Therefore, these results
321
were expected as the concentrates were not degassed prior to spray drying. Additionally, as shown
322
in Fig. 5A1,B1,C1, porous structures were seen in CO2 treated powders, whereas standard powders
323
presented some pores, but less important. The cut particles imaged by SEM also showed a lack of
324
vacuoles in the untreated powders (Fig. 5A2), whereas huge vacuoles were present in the treated
325
powders (Fig. 5B2,C2). At higher magnification (Fig. 5A3,B3,C3), small internal pores were visible in
326
the standard powders and the envelope thickness of the other two powders was found to be around
327
2–3 µm, which supported the CLSM results. These microscopy observations supported the occluded
328
air measurements described in Table 2.
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The CLSM images also showed fat globules (in green) and proteins (in red) distribution in
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each powder particle (Fig. 4A1,B1,C1). However, no real differences were noticed among the
331
powders, because all particles presented heterogeneous fat globules with a protein layer at the
332
surface. Many authors have reported that greater than 95% of WMP surface was covered with fat,
333
mostly free fat, followed by layers of protein, lactose and fat globules protected by proteins (Fyfe,
334
Kravchuk, Nguyen, Deeth, & Bhandari, 2011; Kim et al., 2002; Kim, Chen, & Pearce, 2009). However,
335
these surface fat layer, generated due to fat globules breakage, cannot be visualised by CLSM due to
336
its low resolution (Vignolles et al., 2007).
337
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3.2.2.
Particle functional properties The analyses of powder functional properties are presented in Table 1. It was found that all
340
powders did not wet within 5 min. This result was expected as WMP particles in this work were not
341
agglomerated and/or lecithinated according to the manufacture of commercial powders. Our results
342
are also different from other researchers on skim milk powder, where CO2 was found to improve
343
wettability (Lee, 2014). However, comparisons are impossible due to the different natures of skim
344
and whole milk powders. Nonetheless, the powders produced from concentrates carbonated at 30
345
°C showed significant improvements in dispersibility (P < 0.01) and solubility (P < 0.001), while the
346
reconstitution properties of powders produced from concentrates treated with CO2 at 4 °C were
347
decreased (for solubility). The dispersibility of standard and powders treated at 4 and 30 °C were
348
54, 46 and 65%, respectively. A similar trend was observed for the solubility of these powders, with
349
values of 97, 94 and 99%, respectively. These differences cannot be attributed to modifications of
350
particle shape or surface morphology because all powder particles have similar structures as
351
observed in Fig. 4A,B,C. In addition, particles size for the three powders were also similar with a d50
352
around 15 µm (data not shown). Meanwhile, improved functional properties have been attributed
353
to powders containing high levels of non-micellar casein in high protein content powders (Schokker
354
et al., 2011). Several factors such as calcium chelators, reduced pH, high pressure, and ionic strength
355
have been found responsible for the structural integrity of casein micelles (Law & Leaver, 1998;
356
Marchin, Putaux, Pignon, & Léonil, 2007). In this study, both CO2 acidification and temperature were
357
found to play a role in the non-micellar casein content. As shown in Fig. 2, the amount of non-
358
micellar casein in the concentrates was strongly increased by the action of CO2 acidification in
359
combination with low temperature. However, in this research, the protective effect of non-micellar
360
caseins (serum caseins) observed in high protein content powders (Buldo, 2012) was not confirmed
361
as the resulting powders did not provide better reconstitutability. It is evident that fat caused some
362
modifications in concentrates stored at 4 °C due to coalescence. As a result, fat emulsions in the
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concentrate were larger and non-homogenous (Fig. 3). Meanwhile, larger fat globules are known to
364
produce free fat on the surface of powder particles (Table 2), which rendered the surface
365
hydrophobic, therefore reducing solubility in water (Bhandari, 2013; Kim et al., 2009). On the other
366
hand, the concentrates carbonated at high temperature did not show fat coalescence, thus the
367
resulting powders have less amount of free fat (Table 2) and consequently better reconstitution
368
properties (Table 1). Additionally, the elevated porosity of powders produced from concentrates
369
treated at 30 °C may contribute to the enhanced functional properties, as similarly reported for
370
skim milk powders (Lee, 2014). Moreover, WMP with poor reconstitution properties were reported
371
to contain more aggregated particles that consisted of mixtures of fat globules and proteins (Singh &
372
Ye, 2010). Therefore, fat destabilisation may mask the positive effect of elevated levels of non-
373
micellar caseins in milk concentrates carbonated at low temperatures.
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4.
Conclusion
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The effect of CO2 acidification of whole milk concentrate on WMP properties has not been reported in the literature. Results show that CO2 acidification of whole milk concentrates at various
379
temperatures allow the production of powders with totally different properties due to the alteration
380
of physical states of fat. By acting on both micelles and milk fat, new generation of powders with
381
targeted functional properties may be produced. In the future, it may be interesting to find CO2
382
addition and processes conditions that lead to the increase of non-micellar casein levels (as in 2000
383
ppm, 4 °C powders) without the modifications of fat emulsion (as in 2000 ppm, 30 °C powders) to
384
greatly improve the reconstitutability of these powders.
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Acknowledgements
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Author Claire Gaiani would like to thank Europe for their financial support towards this project
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(Milk PEPPER, N°621727, International Outgoing Fellowship grant). The authors acknowledge the
390
facilities, and the scientific and technical assistance provided by the School of Agriculture and Food
391
Sciences (SAFS) at The University of Queensland and the Australian Microscopy & Microanalysis
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Research Facility at the Centre for Microscopy and Microanalysis (CMM, The University of
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Queensland).
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394 References
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Mimouni, A., Deeth, H. C., Whittaker, A. K., Gidley, M. J., & Bhandari, B. R. (2010). Investigation of the
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microstructure of milk protein concentrate powders during rehydration: Alterations during
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storage. Journal of Dairy Science, 93, 463-472.
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Murriera Pazos, I., Gaiani, C., Galet, L., & Scher, J. (2012). Composition gradient from surface to core in dairy powders: Agglomeration effect. Food Hydrocolloids, 26, 149-158. Nelson, B. K., Lynch, J. M., & Barbano, D. M. (2004). Impact of milk preacidification with CO2 on
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Cheddar cheese composition and yield. Journal of Dairy Science, 87, 3581-3589.
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Post, A. E., Arnold, B., Weiss, J., & Hinrichs, J. (2012). Effect of temperature and pH on the solubility
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of caseins: Environmental influences on the dissociation of α- and β-casein. Journal of Dairy
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Schokker, E. P., Church, J. S., Mata, J. P., Gilbert, E. P., Puvanenthiran, A., & Udabage, P. (2011). Reconstitution properties of micellar casein powder: Effects of composition and storage.
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International Dairy Journal, 21, 877-886.
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Vignolles, M.-L., Jeantet, R., Lopez, C., & Schuck, P. (2007). Free fat, surface fat and dairy powders: interactions between process and product. A review. Lait, 87, 187-236.
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Singh, H., & Ye, A. (2010). Controlling milk protein interactions to enhance the reconstitution
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Figure legends
Fig. 1. pH evolution with CO2 concentration for whole milk () and skim milk ()
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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).
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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
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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
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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
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1000× (1), 3000× (2) and 10,000× (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.
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
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30 °C with CO2.
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Table 1
Powder
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Physico-chemical properties of the concentrates and functional properties of the resulting powders. a Concentrate properties
Powder functional properties
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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CO2 content (actual; ppm)
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).
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Table 2
Powder
Solvent extraction
RI PT
Physical and chemical properties of the powders. a
Physical properties
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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Free fat (g L-1)
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).
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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
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