Behaviour of milk protein-stabilized oil-in-water emulsions in simulated physiological fluids : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
Emulsions form a major part of processed food formulations, either being the end
products in themselves or as parts of a more complex food system. For the past
few decades, colloid scientists have focussed mainly on the effects of processing
conditions (e.g. heat, high pressure, and shear) on the physicochemical properties
of emulsions (e.g. viscosity, droplet size distribution and phase stability).
However, the information about the behaviour of food structures post
consumption is very limited. Fundamental knowledge of how the food structures
behave in the mouth is critical, as these oral interactions of food components
influence the common sensorial perceptions (e.g. creaminess, smoothness) and
the release of fat-soluble flavours. Initial studies also suggest that the breakdown
of emulsions in the gastrointestinal tract and the generated interfacial structures
impact lipid digestion, which can consequently influence post-prandial metabolic
responses. This area of research needs to be intensively investigated before the
knowledge can be applied to rational design of healthier food structures that
could modulate the rate of lipid metabolism, bioavailability of nutrients, and also
help in providing targeted delivery of flavour molecules and/or bioactive
Hence, the objective of this research was to gain understanding of how emulsions
behave during their passage through the gastrointestinal tract. In vitro digestion
models that mimic the physicochemical processes and biological conditions in
the mouth and gastrointestinal tract were successfully employed. Behaviour of
model protein-stabilized emulsions (both positively charged (lactoferrin) as well
as negatively charged [β-lactoglobulin (β-lg)] oil-in-water emulsions) at each step
of simulated physiological processing (using model oral, gastric and duodenal
fluids individually) were investigated.
In simulated mouth conditions, oil-in-water emulsions stabilized by lactoferrin or
β-lg at the interfacial layers were mixed with artificial saliva at neutral pH that
contained a range of mucin concentrations and salts. The β-lg emulsions did not
interact with the artificial saliva due to the dominant repulsion between mutually
opposite charges of anionic mucin and anionic β-lg interfacial layer at neutral pH.
However, β-lg emulsions underwent some depletion flocculation on addition of
higher concentrations of mucin due to the presence of unadsorbed mucin
molecules in the continuous phase. In contrast, positively charged lactoferrin
emulsions showed considerable salt-induced aggregation in the presence of salts
(from the saliva) alone. Furthermore, lactoferrin emulsions underwent bridging
flocculation because of electrostatic binding of anionic mucin to the positively
charged lactoferrin-stabilized emulsion droplets.
In acidic pH conditions (pH 1.2) of the simulated gastric fluid (SGF), both
protein-stabilized emulsions were positively charged. Addition of pepsin resulted
in extensive droplet flocculation in both emulsions with a greater extent of
droplet instability in lactoferrin emulsions. Coalescence of the droplets was
observed as a result of peptic hydrolysis of the interfacial protein layers.
Conditions such as ionic strength, pH and exposure to mucin were shown to
significantly influence the rate of hydrolysis of β-lg-stabilized emulsion by
Addition of simulated intestinal fluid (SIF) containing physiological
concentrations of bile salts to the emulsions showed competitive interfacial
displacement of β-lg by bile salts. In the case of lactoferrin-stabilized emulsion
droplets, there was considerable aggregation in the presence of intestinal
electrolytes alone (without added bile salts) at pH 7.5. Binding of anionic bile
salts to cationic interfacial lactoferrin layer resulted in re-stabilization of
salt-aggregated lactoferrin emulsions. On mixing with physiological
concentrations of pancreatin (mixture of pancreatic lipase, amylase and protease),
significant degree of coalescence and fatty acid release occurred for both the
emulsions. This was attributed to the interfacial proteolysis by trypsin
(proteolytic fractions of pancreatin) resulting in interfacial film rupturing.
Exchange of initial interfacial materials by bile salts and trypsin-induced film
breakage enhanced the potential for lipolytic fractions of pancreatin to act on the
hydrophobic lipid core. The lipid digestion products (free fatty acids and mono
and/or diglycerides) generated at the droplet surface further removed the residual
intact protein layers from the interface by competitive displacement mechanisms.
The sequential treatment of the cationic and anionic emulsions with artificial
saliva, SGF and SIF, respectively, was determined to understand the impact of
initial protein type during complete physiological processing from mouth to
intestine. Broadly, both the protein-stabilized emulsions underwent charge
reversals, extensive droplet flocculation, and significant coalescence as they
passed through various stages of the in vitro digestion conditions. Except in the
simulated mouth environment, the initial charge of the emulsifiers had relatively
limited influence on droplet behaviour during the simulated digestion.
The results contribute to the knowledge of how structure and charge of the
emulsified lipid droplets impact digestion at various stages of physiology. This
information might have important consequences for developing suitable
microstructures that allow controlled breakdown of droplets in the mouth and
predictable release of lipids in the gastrointestinal tract.