Role of Electrostatic Interactions on Molecular Self-Assembly of Protein + Phospholipid Films at the Air–Water Interface - Food Colloids: Self-Assembly and Material Science - page 228

how the fundamental principles of non-covalent interactions lead to supramo-
lecular structures, and so influence the formation and stabilization of higher
hierarchical structures (droplets or bubbles) present in most food colloids. 8 For
investigating such molecularly assembled materials, there has been rapid
development of advanced equipment, 9-11 such as atomic force microscopy
(AFM), Brewster angle microscopy (BAM), imaging ellipsometry, reflection-
absorption spectrometry, transmission fluorescence spectrometry, 2-D X-ray
reflectometry, surface plasmon resonance, etc. Furthermore, traditional appa-
ratus such as the Langmuir trough has been equipped with new features to
produce constant/dynamic shearing and/or dilatation,

in situ observations,

molecular deposition, and so on.
In this study, we analyse the role of electrostatic interactions on the structure,
topography, and film thickness of dipalmitoylphosphatidyl choline (DPPC),
b-casein, and their mixtures in spread monolayers at the air-water interface.
This involves surface pressure versus area isotherms coupled with BAM and
AFM. Phospholipids and proteins are often used as emulsifiers in emulsions
and foams due to their amphiphilic character. 12 Thus, the capacity of proteins
and phospholipids to form supramolecular structures that self-assemble at a
fluid interface is of practical importance for the manufacture of food formu-
lations. Since phospholipids and proteins can be present at interfaces with
different net charges, depending on the pH, an analysis of the effect of the pH
on structure formation will give insight into the importance of electrostatic
interactions on self-assembly in the pure and mixed monolayers.

15.2 Experimental
DPPC was supplied by Sigma (499%). The b-casein (99% pure) was supplied
and purified from bulk milk from the Hannah Research Institute (Ayr, Scot-
land). To form the surface film, DPPC was spread in the form of a solution,
using chloroform/ethanol (4:1 by volume) as a spreading solvent. Analytical
grade chloroform (Sigma, 99%) and ethanol (Merck, 499.8%) were used
without further purification. Samples for interfacial characteristics of protein
and DPPC films were prepared using Milli-Q ultrapure water at pH 7. The water
used as sub-phase was purified by means of a Millipore filtration device (Mill-
lipore, Milli Q t ). To adjust the sub-phase pH, buffer solutions were used. Acetic
acid+sodium acetate aqueous solution (CH 3 COOH/CH 3 COONa) was used to
achieve pH 5, and a commercial buffer solution called Trizma ((CH 2 OH) 3 CNH 2 /
(CH 2 OH) 3 CNH 3 Cl) for pH 7 and 9. All these products were supplied by Sigma
(499.5%). The ionic strength was 0.05 M in all the experiments.
Measurements of surface pressure ( p ) versus average area per molecule (A)were
performed on fully automated Langmuir-type film balance, as described elsewhere
for pure 13,14 and mixed 15 monolayers. The p -A isotherm was measured at least
three times. The reproducibility of the results was better than 0.5 mN m 1
for surface pressure and 0.05 m 2 mg 1 for area. The sub-phase temperature was
controlled at 20 0.31C by water circulation from a thermostat.