Chemistry Reference
In-Depth Information
The aim of our work is to demonstrate how protein-polysaccharide inter-
actions can be used to control both the adsorption kinetics and the surface
rheological behaviour of mixed adsorbed layers at air-water and oil-water
interfaces, and to determine which parameters are mainly involved. Here, a
general mechanism of mixed protein + polysaccharide adsorption to liquid
interfaces is presented, while focusing particularly on the comparison of
behaviour at air-water and oil-water interfaces.
13.2 Experimental
Acetate buffer (pH
¼
4.5) was prepared from analytical grade chemicals and
deionized water. Bovine b-Lg was purified using a non-denaturing method as
described previously.
29
Stock protein solutions of 0.2 mg mL
1
were prepared
by dissolving the protein in deionized water and subsequently diluting with
concentrated acetate buffer solution to a final acetate concentration of 5 mM
with an ionic strength of 2 mM. The protein solutions were kept at
401C until
further use. Two pectin samples with different degree of methyl esterification
(DM) were used: low-methoxyl pectin (LMP, DM
¼
30, meaning that 30% of
the galacturonic acid subunits are methyl esterified) and high-methoxyl pectin
(HMP, DM
¼
70) supplied by CP Kelko (Copenhagen, Denmark). Only the
non-methylated galacturonic acid sub-units have a free carboxyl group (weak
acid, pK
a
B
4.5). Polysaccharide solutions were prepared by first wetting the
powder with ethanol, then dissolving in buffer, and subsequently heating at 701C
for 30 min. After overnight storage at 41C, the samples were centrifuged at 6000 g
at room temperature for 10 min. Sunflower oil (Reddy, Vandermoortele,
Roosendaal, the Netherlands) was purified by stirring it under vacuum with
silica gel 60 (70-230 mesh, Merck, Germany) as described before.
30
After
performing this procedure twice, the oil was stored at
201C until use.
Second-cumulant diffusion coefficients of b-Lg-pectin complexes were deter-
mined at 23
0.31C by dynamic light scattering
31
using an ALV light-scattering
instrument equipped with a 400 mW argon laser tuned at a wavelength of 514.5
nm.
32
Hydrodynamic radii were calculated according to the Stokes-Einstein
relation, assuming that the pectin molecules and the complexes adopt spherical
conformations. The protein concentration was 0.1 g L
1
for all the samples, and
the pectin concentrations were varied over the protein/pectin mixing ratio range
0-15 (weight basis).
The surface tension at the air-water or oil-water interface was measured as a
function of time for pure protein and mixed solutions of protein + polysaccha-
ride using an automated drop tensiometer (IT
CONCEPT
, Longessaigne, France).
The arrangement has been described in detail elsewhere.
33
Protein + polysaccha-
ride mixtures were freshly prepared for each experiment and equilibrated for
30 min to allow formation of protein-polysaccharide complexes. Mixing ratios
mentioned in the text are on a weight basis. Unless mentioned otherwise, the
protein concentration was always 0.1 g L
1
. Each experiment started with a clean
interface of a newly formed air bubble (7 mL) or oil droplet (21 mL) in a cuvette
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