Methods: Chapter 7

Preparation of emulsions

WPI and NaCas emulsions with 1.0, 4.0 and 7.0 % protein content were created using protein solutions prepared with phosphate buffer (0.1 M, pH 6.0) instead of RO water.

Experimental description

The experimental set-up is shown in Figure 3.1. Freshly prepared emulsion (165 ml) was pipetted into a glass flask with three openings and an approximate volume of 200 ml. The two openings on the side were closed by stoppers, the one in the middle left open. The flask was placed into a water bath at 40 °C and the temperature of the emulsion adjusted to 37 °C (thermometer in emulsion). The emulsion was stirred by the use of a magnetic stirrer. In a separate glass beaker, 10 ml of the emulsion was combined with 750 µl AAPH solution (210 g/l AAPH in RO water) or RO water and adjusted to pH 6.0 with NaOH (10 M). To the 165 ml emulsion, the analogous volumes of AAPH solution or RO water (12.4 ml) and NaOH solution (≈ 1.5 ml) were added. After 1 minute, an oxygen probe was set into the centre opening to close the system. After 2 minutes, the first oxygen reading was taken. About 3.5 ml emulsion were taken out through a syringe at various sampling times (2–10 min with AAPH; 2–45 min without AAPH) through one of the stoppers (glass stopper with rubber septum). For the volume taken out, oxygen-free nitrogen streamed into the flask from a nitrogen reservoir. The emulsion taken out was immediately put on ice. It was distributed into 15 ml plastic tubes in triplicate (3 x 1 ml) for lipid hydroperoxide measurements and put into the freezer (–18 °C) within 10 min. Emulsion samples with AAPH (0 min) were prepared separately and not by sampling out of the glass flask.

Glass cylinder as N2 reservoir N2

N2

N2

Figure 3.1: Experimental set-up

Oxygen meter N2 c yl inde r

Magnetic stirrer plate

Waterbath

≈ 40 °C

Flask with emulsion O2

probe Rubber tubes

N2 into flask on/off

3.6.4 Methods: Chapter 7.2

Oxygen consumption in protein solutions with AAPH

The experimental set-up was similar to Figure 3.1 in Section 3.6.3, Methods: Chapter 7.1, except that the stopper with the syringe was replaced with a normal plastic stopper. The execution of the experiment was also the same as described in Section 3.6.3, Methods: Chapter 7.1, except for the usage of various aqueous solutions instead of emulsions and that no sampling took place.

Obtaining continuous and cream phase protein solutions for determinations of protein hydroperoxides, amino acids and total sulfhydryls

10 ml or 5 ml of emulsion was added into 50 ml plastic centrifuge tubes respectively. Incubation for various times at 50 ˚C followed. The tubes were then centrifuged with a RC 5C centrifuge from Sorvall at 45,000 g for 60 min at 5 °C. The subnatants (continuous phase of the emulsion) were taken out with 10 ml single use plastic syringes with a blunt steel tip screwed on top. Then the same weight of SDS (2.0 %) solution was added as subnatant had been taken out. Afterwards re-emulsification was done by vortexing with a VM-1000 from Digisystem at the highest vortex speed for 35 s and heavy manual shaking for 10 s. Then the tubes were centrifuged again and also subnatants (cream phase protein solution of the emulsion) taken out as described above.

The cream phase protein solution contained SDS whereas the continuous phase protein solution did not. The cream phase protein was therefore denatured and the continuous phase protein was not. However, in the subsequent methods, both proteins were heavily exposed to denaturants anyway, to SDS (determination of total sulfhydryls), to strong acid or base (amino acid determination) or to trichloroacetic acid and an organic solvent mixture (protein hydroperoxide determination). Therefore the initial difference in the status of the proteins in either fraction was thought to be irrelevant.

Protein hydroperoxide determination

Protein solution (1000 µl) with continuous or cream phase protein was transferred into a 50 ml plastic centrifuge tube. Trichloroacetic acid (10 % weight/volume in RO water)

(5000 µl) plus 5000 µl organic solvent mixture (ethanol/ethyl acetate/n-hexane, 1:1:1 (V/V/V)) were added for protein denaturation/precipitation and the extraction of residual lipid. The mixture was vortexed, centrifuged and the supernatant decanted. To the remaining protein precipitate, which was in pellet form, 5000 µl sulphuric acid (25 mM) was added. The protein was then suspended with a glass rod. Afterwards 280 µl xylenol orange solution (23.34 mM xylenol orange) and 280 µl ferrous iron solution (2.80 mM ferrous sulphate 7-hydrate) based on sulphuric acid (25 mM) were added and the mixture was vortexed. For controls, 5000 µl sulphuric acid (25 mM), 280 µl xylenol orange solution and 280 µl ferrous iron solution were added in triplicate into test tubes. The tubes were then put in the dark for 30 min reaction time at room temperature. Afterwards they were centrifuged again. The supernatant was transferred into 4 ml photometric plastic cells and the absorption at 560 nm was measured against sulphuric acid (25 mM). All samples in this experiment were centrifuged using a RC 5C centrifuge from Sorvall at 45,000 g for 60 min at 5 °C. The absorption obtained in the assay was converted into micromoles of hydroperoxides by means of a hydrogen peroxide standard curve. The hydrogen peroxide curve was created by adding 4970 µl of sulphuric acid (25 mM) plus 30 µl of hydrogen peroxide solution of various concentration levels (based on sulphuric acid (25 mM)) plus 280 µl of xylenol orange and ferrous iron solution into test tubes. The procedure already described above was followed without centrifugation. The assay was adapted from the instructions of Gieseg et al. (2000) and Gay et al. (1999).

Principle of the chemical reaction:

Fe2+ Fe3+

Fe3+ + xylenol orange coloured complex (absorption maximum at 560 nm)

Total sulfhydryl determination

Protein solution (500 µl) or RO water for the controls, was pipetted into test tubes; 100 µl phosphate buffer solution (pH 8.1), 1100 µl SDS solution (5.0 %, weight/volume in RO water) and 500 µl DTNB solution were added. The tubes were placed in the dark for

30 min at room temperature and afterwards the absorption at 412 nm was measured against RO water. Also the absorption at 540 nm was measured against RO water to take a slight turbidity into account and the reading deducted from the absorption values at 412 nm. The absorption readings at 540 nm were low (≤ 0.020). There were four samples of each sample type which were each measured with one replicate. A standard curve was made for quantification of total sulfhydryls by using RO-water-based cysteine solution of various concentration levels instead of protein solution in the assay. The method was adapted from Taylor and Richardson (1980b).

Principle of the chemical reaction (Guingamp et al. 1993):

SH + 5,5`-dithio-bis(2-nitrobenzoic acid) 2-nitro-5-mercaptobenzoic acid yellow

(absorption maximum at 412 nm)

Preparation of the phosphate buffer solution (pH 8.1)

Tris(hydroxymethyl)methylamine (1 M) and trisodium phosphate-12-hydrate (1 M) were dissolved in RO water and the pH was adjusted to pH 8.1 with phosphoric acid (85 %).

Preparation of the DTNB solution

Sodium acetate-3-hydrate (50 mM) and DTNB (2 mM) were dissolved in RO water.

mild basic pH

Chapter 4

Factors affecting lipid oxidation