b-Lactoglobulin Aggregates from Heating with Charged Cosolutes: Formation, Characterization and Foaming - Food Colloids: Self-Assembly and Material Science - page 179

hydration. Afterwards the dispersion (natural pH 7.0 0.1) was centrifuged at
1.08 10 4 g (Rotor SLA-3000, Sorvall Instruments RC5C, Kendro,
Switzerland) for 60 min to remove insoluble matter. The protein solution was
then filtered on paper filter (Schleicher & Schuell filter 597 2 , Germany). The
protein concentration was determined by UV/visible spectroscopy ( e 278 ¼ 1.013
cm 1 g 1 L) using an Uvikon 810 spectrophotometer (Kontron, Flowspec,
Switzerland) and adjusted to 40 g L 1 . The cosolute solutions (arginine HCl,
NaCl and GdnHCl; 0-800 mM) were prepared in Millipore water. Mixtures of
protein and cosolute solutions were prepared by mixing equal volumes
of protein and cosolute to a final protein concentration of 10 g L 1 and
0-400 mM of cosolute and were adjusted to pH 7.0 by addition of 1 M HCl
or NaOH. The amount of acid and base (additional Na 1 and Cl ions) needed
to adjust the pH of the solutions was less than 2% of the final molarity
for pH 7. These amounts were neglected in the final cosolute molarity
calculation.
For the sake of clarity, the following nomenclature is used for the sample
coding: A, Arginine HCl; N, NaCl; G, GdnHCl; these letters are followed by a
numerical code, with '0' for b-LG alone, '10' for a cosolute concentration of 10
mM and so forth.

12.2.3 Determination of Denaturation Kinetics by RP-HPLC
To study the heating kinetics, protein+cosolute solutions were heated at 801C
for time intervals between 0 and 30 min. The time to reach the set temperature
was 2.5 min. Following the heat treatment, the samples were cooled in ice

water to 41C. The denaturation degree of the heated b-LG solutions was
determined by RP-HPLC.
To measure the content of soluble protein after heat treatment, the solutions
were adjusted to pH 4.6 and centrifuged at 2.69 10 4 g (Sorvall Instruments
RC5C, rotor Sorvall SS34) for 15 min. 17 After centrifugation, the samples
were filtered for injection through 0.45 mm filters (Orange Scientific, Waterloo,
Belgium) and adjusted to a protein concentration of

B

0.1-0.3 g L 1 .
The injector system was composed of a Hewlett Packard series 1050
apparatus equipped with an autosampler and an UV detector. The stationary
phase was the hydrophobic interaction column PLRP-S3 (150 4.6 mm) of
Polymer Laboratoires (Ercatech AG, Bern, Switzerland). Elution solvent
A was composed of 0.1% TFA (Pierce Rockford, IL, USA) in water.
Eluent B contained 0.1% TFA in 80% acetonitrile and 20% Lichrosolv water
(Merck, Darmstadt, Germany). The chromatography was carried out at
501C and 205 nm, with a nonlinear elution gradient (43% B at t ¼ 0 min to
100% B at t ¼ 28 min) with a flow rate of 1 mL min 1 , and an injection volume
of 20 mL. 22
The kinetic parameters were calculated using the general rate equation

B

dC t

dt ¼ k n C n ;

ð 1 Þ