b-Lactoglobulin Aggregates from Heating with Charged Cosolutes: Formation, Characterization and Foaming - Food Colloids: Self-Assembly and Material Science - page 181

scattered light. The samples before heat treatment were filtered in a two-step
process using 0.1 mm and 0.025 mm filters (NC 03 Membrane Filters, Schleicher
& Schuell, Germany) to eliminate dust particles and non-native protein. The
samples were placed in a square quartz cell (Hellma, pathlength 1 cm) and
heated at 801C for 10 min in the Nanosizer. After heat treatment, the solutions
were diluted to different concentrations with water and measured against water
and toluene as a SLS standard. Application of the so-called Debye plot leads to
the determination of M w and A 2 :

:

KC
R y

1
M w

¼ þ 2A 2 C ð 5 Þ

In Equation (5) C is the concentration of protein particles, K the optical
constant (depending on the apparatus), and R y the Rayleigh factor at a given
scattering angle y (1731) using toluene as the reference scattering medium. The
second virial coefficient is a measure of the affinity between the protein and the
solvent. A value of A 2 4 0 indicates high protein-solvent affinity, A 2 ¼ 0 shows
equivalent protein-solvent and protein-protein affinities and A 2 o 0 indicates
low protein-solvent affinity. A negative A 2 value can be considered to be a sign
of protein aggregation due to strong protein-protein interactions. 27

12.2.6 Determination of Protein Aggregate Size and Electro-
phoretic Mobility
Aggregate size was determined in the samples after heat treatment using the
Nanosizer ZS (Malvern Instruments, UK). The apparatus is equipped with a
laser emitting at 633 nm and with 4.0 mW power source. The instrument was
used in the backscattering configuration where detection is done at a scattering
angle of 1731. This allows considerable reduction of the multiple scattering
signals in turbid samples. The heated protein + cosolute solutions were filtered
using 0.22 mm filters (Millipore) to eliminate dust and other insoluble particles.
The filtered solutions were placed in square plastic cuvettes. Depending on the
sample turbidity (attenuation) the pathlength of the light was set automatically
by the apparatus. The autocorrelation function was calculated from the fluc-
tuation of the scattered intensity. From the fit of the correlation function using
the cumulant technique, 28 the particle diffusion coefficient D app was calculated.
The hydrodynamic diameter D h was calculated from the Stokes-Einstein
equation: 29

k B T
3 pZ D h :

D app ¼ ð 6 Þ

The reported hydrodynamic diameter D h was averaged from two runs each of
20 measurements.
For determination of the electrophoretic mobility, protein+cosolute solu-
tions were filled in 22-mL glass vials (with a screw top and solid cap with PTFE
Liner) (Supelco USA, Milian, Switzerland) and heated in a water-bath while