Fecha de observación: 15 de diciembre de 2016 Temporalización: 50 minutos

3.2.1 Composition of whey protein powders

The total protein content of the whey protein powders was determined using the Kjeldahl method (Williams, 1984) with a nitrogen conversion factor of 6.38. The fat content was determined using the Soxhlet extraction method, as described by Russell et al. (1980). The moisture content was determined by oven drying preweighed duplicate samples at 102qC for 5 h, cooling in a desiccator for 2 h, and reweighing. The lactose content was as reported by the manufacturer. The mineral analyses were carried out at the New Zealand Pastoral Agricultural Research Laboratory, Palmerston North, by inductively coupled argon-plasma emission spectrometry using the method described by Lee et al. (1986). All powders were provided by the Fonterra Co-operative Group Ltd, New Zealand and their composition is shown in Table 3.1.

Table 3.1: Compositions of the three whey protein products used in this study.

Component WPI AWPC CWPC

Protein %, w/w 93.1 80.4 83.0

Fat %, w/w 0.45 5.43 5.90

Ash %, w/w 1.62 3.81 2.78

Lactose %, w/w 0.18 4.08 3.89

Moisture %, w/w 4.93 4.96 5.28

Minerals mmol kg-1 protein

Ca 18.775 74.375 111.25 K 13.308 471.15 175.64 Mg 2.4167 7.6667 27.875 Na 259.43 14.043 147.30

3.2.2 Preparation of the whey protein solutions

Whey protein solutions were prepared by reconstituting appropriate quantities of whey protein powders (WPI, AWPC or CWPC) in milli-Q water so that the protein concentration was > 4% (w/w). Appropriate volumes of a 2 M calcium chloride (CaCl2)

or 2 M sodium chloride (NaCl) solution were added to give a range of different added calcium or sodium concentrations (see just below). Then, each solution was topped up

with milli-Q water so that the final protein concentration was 4% (w/w). The different ranges of final ion concentrations prepared depended on the analysis technique used:

- for polyacrylamide gel electrophoresis (PAGE) and reverse-phase high performance liquid chromatography (RP-HPLC), final added calcium concentrations were 0, 2, 4, 6, 8, 11, 14, 17, 20, 50, 80, 110, 140, 170, 200 and 230 mM, and final added sodium concentrations were 0, 6, 12, 18, 24, 33, 42, 51, 60, 150, 240, 330, 420, 600 and 690 mM;

- for circular dichroism (CD), final added calcium concentrations were 0, 4, 10, 20, 80, 140 and 200 mM;

- for 2D nuclear magnetic resonance (NMR), final added calcium concentrations were 0, 10, 20, 80, 140 and 200 mM;

- for differential scanning calorimetry (DSC), final added calcium concentrations were 0, 4, 6, 20, 50, 80, 140 and 200 mM.

When 0 to 230 mM calcium chloride was added to 4% (w/w protein) whey protein solutions the pH decreased. To investigate the effect of pH shifting due to added calcium chloride on whey protein aggregation, three sets of samples (4%, w/w protein) were prepared:

- samples with added calcium chloride but without pH adjustment;

- samples with added calcium chloride but with the pH re-adjusted to the original pH of the solutions without added calcium chloride by the addition of 1 M NaOH;

- samples without added calcium chloride but with the pH adjusted to the corresponding pHs of the samples with added calcium chloride using 1 M HCl.

Initially, the range of calcium concentration was chosen to be broad, 0 to 230 mM, with many intermediate points, to allow a good understanding of the effects of calcium on the whey protein behaviour under heat treatment. PAGE was the first analysis technique used, and applied to the whole range of calcium concentrations with all intermediate points. From the PAGE results, certain key intermediate calcium concentrations were selected to be analysed with the other techniques, while keeping some consistency to be able to correlate the results.

3.2.3 Heat treatment of the whey protein solutions

Aliquots (3 mL for PAGE and RP-HPLC or 5 mL for CD and 2D NMR) of each whey protein solution were heated, in glass test tubes (7.5 cm in length, 1.2 cm in outside diameter and 1.0 cm in inside diameter), at a given temperature for a given time (depending on the experiment) in a water bath. The samples were then immediately immersed in an ice-water bath (~ 0ºC) for 1 h to stop protein denaturation. The control was the unheated sample without any salt added.

The samples were then analysed by polyacrylamide gel electrophoresis (PAGE), reverse-phase high performance liquid chromatography (RP-HPLC), circular dichroism (CD), and 2D nuclear magnetic resonance (2D NMR).

The samples analysed by differential scanning calorimetry (DSC) were not heat-treated before analysis. The heat treatment was done during the analysis (see section 3.2.8). 3.2.4 PolyAcrylamide Gel Electrophoresis (PAGE)

After heat-treatment, a 50 μL sample of the control (unheated and with no added salt)

and heat-treated solutions were mixed with 1 mL native or SDS (dissociating agent) sample buffer. The native sample buffer contained 0.03% (w/v) of bromophenol blue dissolved in 0.5 M Tris-HCl buffer. The SDS sample buffer contained 0.01% (w/v) of bromophenol blue and 2% (w/v) of SDS dissolved in 0.5 M Tris-HCl buffer. The control and heat-treated solutions were analysed using a Mini-Protean II dual cell system (Bio-Rad Laboratories, Richmond, CA, USA) and the PAGE system for PAGE. The native resolving gel contained 12.5% (w/v) acrylamide dissolved in 1.5 M Tris-HCl buffer, pH 8.8, and the native stacking gel was composed of 4% (v/v) acrylamide made up in 0.5 M Tris-HCl buffer, pH 6.8. The SDS resolving and stacking gels were composed of the same chemicals as the native resolving and stacking gels, respectively,

but small quantities (a few μL) of a 10% (w/v) SDS solution were added. 10 μL of the

samples mixed with the sample buffer were injected into the wells of the gels.

The gels were run at 200 V and 70 mA for approximately 1 h 15 min (until the tracking dye seeped out of the bottom of the gel) and then stained with Coomassie Brilliant Blue (R-250) in 25% (v/v) acetic acid for 1 h. This was followed by two destaining steps using a 10% (v/v) acetic acid/2-propanol solution for a total of 20 h. Immediately after

destaining, the gels were scanned using a computing laser densitometer (Molecular Dynamics model P.D., Sunnyvale, CA, USA) and the integrated intensities of the α-

lactalbumin, β-lactoglobulin and bovine serum albumin bands were determined using the Molecular Dynamics ImageQuant software (Anema & McKenna, 1996).

3.2.5 Reverse-Phase High Performance Liquid Chromatography (RP-HPLC)

After heat treatment, the control (unheated and with no added salt) and heat-treated samples were centrifuged at 16,000 g for 3 minutes to remove any insoluble materials. The supernatants were diluted in milli Q water to make 1% (w/w) protein concentration solutions and analysed by RP-HPLC. The RP-HPLC system consisted of a Waters 2690 Alliance Separation Module (Waters, Milford, MA, USA) interfaced with a Waters 486 MS tunable absorbance detector and a Waters Millenium 32 data acquisition and manipulation system. Queued samples were refrigerated at 5ºC.

Acetonitrile (MeCN; far UV grade) and trifluoroacetic acid (TFA; HPLC grade) were from BDH (Poole, UK). Q Sepharose Fast Flow and Sephadex G-75 were from Pharmacia Biotech (Uppsala, Sweden). All other buffers and reagents were analytical

grade or better. Aqueous buffers (eluents) were filtered through 0.45 μm cellulose

acetate membranes (Millipore, Bedford, MA, USA) and degassed prior to use.

A 1 mL Resource RPC column (Pharmacia Biotech) was operated at room temperature and at a flow-rate of 1 mL min-1. The column was equilibrated in 80% solvent A (0.1%, v/v, TFA in milli-Q water) and after sample injection a 1 min isocratic period was applied followed by a series of linear gradients to 100% solvent B (0.09%, v/v, TFA, 90%, v/v, MeCN in milli-Q water) (Elgar, Norris, Ayers, Pritchard, Otter, & Palmano, 2000).

3.2.6 Circular Dichroism (CD)

After heat treatment, the control (unheated and with no added salt) and heat-treated samples were centrifuged at 16,000 g for 3 minutes to remove any insoluble material. The supernatants were diluted 10-fold in milli Q water and then scanned from 250 to 400 nm in a 10 mm quartz cell with a Jasco Model J-720 spectropolarimeter (Jasco, Hachioji City, Tokyo, Japan) to obtain near-UV CD spectra. The samples were scanned at 50 nm/ min, using a 2 s time constant, a 0.2 nm step resolution, a 1 nm bandwidth,

and a sensitivity of 10 mdeg. Five scans were accumulated, and the average spectrum was saved. The same solutions were diluted another 10-fold in milli-Q water then scanned using a 0.5 mm quartz cell from 185 to 250 nm, and 10 scans were averaged and saved as the far-UV spectrum. The sample compartment of the instrument was flushed with oxygen-free dry nitrogen prior to and during measurements (Considine, Patel, Singh, & Creamer, 2007).

3.2.7 2D Nuclear Magnetic Resonance (2D NMR)

After heat treatment, the pH of the control (unheated and with no added salt) and heat- treated samples was adjusted to 2.5 with a 1 M HCL solution and the samples were then centrifuged at 16,000 g for 3 minutes to remove any insoluble material. The supernatants were analysed by NMR. The spectra were recorded on a Bruker Avance 700 MHz spectrometer (Germany). The 2D TOCSY spectra were recorded with a mixing time of 60 ms and a special width of 9.1 kHz centred at the water frequency. Each spectrum was recorded using a data matrix of 2048 x 256 points. Phase discrimination in the indirect dimension was achieved using the States-TPPI method. Excitation sculpting was used to suppress the water signal. Spectra were processed

using Bruker’s Topspin software (v. 2.1) using standard parameters. Spectra were referenced using the residual water peak at 4.7 ppm.

3.2.8 Differential Scanning Calorimetry (DSC)

All DSC scans were made on a Perkin Elmer (Norwalk, CT, USA) DSC 7 differential scanning calorimeter equipped with an Intracooler II mechanical refrigeration unit. The DSC 7 was controlled, and data were collected, using Perkin Elmer Pyris DSC software, version 2.04. A 20 mg sample of each solution was put into an aluminium volatile- sample pan (Pan-sell kit 0319-1525, Cover-sell kit 0319-1526, O-ring set 0319-1535, all QTY-1000). The pan was sealed and placed in the sample holder of the DSC 7. An empty pan was used as a reference. The samples were heated from 20 to 100ºC at a heating rate of 5ºC/min. The control was the sample with no added salt.

The DSC curves obtained were analysed using the Pyris software by the following method. Two anchor points were inserted at the start and the end of the major peak and a baseline was drawn between them. The following parameters were calculated:

- the onset temperature, calculated from the intersection of the baseline with the extrapolated tangent to the inflection point of the leading edge of the major peak;

- the peak temperature, the highest temperature point of the major peak;

- the area, the calculated area under the thermogram between the two anchor points.