Development of novel nanoemulsions as delivery systems : A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, New Zealand
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Date
2016
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Massey University
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Abstract
In the past decades, emulsions have been widely used as delivery systems for
incorporating bioactive compounds into foods. With the advancing of
nanotechnology, smaller particles in the nanometric range (i.e. nanoemulsions) can
be created with better properties that are more advantageous than conventional
emulsions in terms of their stability to gravitational separation, optical clarity and
better absorption of nutrients in drug delivery (with increased bioavailability). In
particular, emulsification and solvent evaporation method has been used to produce
nanoemulsions with optimum results. However, like conventional emulsions,
protein-stabilised nanoemulsions become unstable when exposed to certain
environmental stresses such as high temperatures, salt addition and extreme pH
changes. Additionally, liquid emulsions are difficult to transport and use in some
food systems while being susceptible to microbial spoilage. To remedy, a dry, stable
emulsion system has to be obtained for their prospective future in food applications.
The objective of this research was to develop nanoemulsions with useful
attributes. The thesis consists of three main parts in which the first part studied the
formation and properties of nanoemulsions using emulsification and solvent
evaporation method; the second part delved into the making of dried nanoemulsion
powders and the third part focused on the structural modifications of nanoemulsions
and encapsulation of a bioactive compound lutein.
To begin, an experimental study to optimise the conditions for producing
nanoemulsions using emulsification and solvent evaporation methodology was
performed under different processing conditions (microfluidisation pressures and
number of passes), organic phase ratios and materials (oil types and emulsifiers). It
was found that smaller oil droplets (around 80 nm in diameter) were achieved when
increasing the microfluidisation pressure up to 12000 psi (80 MPa) for 4 passes at an
organic phase ratio of 10:90. There was a progressive decrease in particle size with
increasing emulsifier concentration up to a 1% (w/w) level for whey protein isolate
(WPI) and lactoferrin but it did not decrease further at higher concentration. On the
other hand, much larger oil droplets were formed in Tween 20 emulsions (120 – 450
nm). The environmental study showed that lactoferrin and Tween 20 emulsions have a better stability to pH changes (pH 2 – 12) and salt addition (0 – 500 mM NaCl or 0
– 90 mM CaCl2) than WPI stabilised nanoemulsions.
After successful preparation of nanoemulsions, liquid nanoemulsions were
converted to dried powders by spray drying or freeze drying. The nanoemulsions
were mixed with different wall materials consisting of maltodextrin alone, trehalose
alone or a 1:1 ratio of maltodextrin and trehalose at 10, 20 or 30% (w/w) solid
concentration. Results showed that the powders containing 20% trehalose have better
powder properties with lower moisture content and water activity, higher bulk
density and good reconstitution in water. The freeze-dried powders showed excellent
wettability and dispersibility in water but lower encapsulation efficiency than spray
dried powders.
In another part of study, nanoemulsions with modified interfacial structure
were used to improve their stability to environmental stresses. The interactions
between WPI and lactoferrin in aqueous solutions were first studied to explore the
feasibility of using these two proteins to form complex interfacial structures at the
droplet surface in the emulsions. Based on ζ-potential and turbidity measurements,
both proteins were shown to interact with each other via electrostatic interactions at
pH values between 6 and 8. The adsorption of protein layers on a gold surface that
mimics the hydrophobic oil surface was also confirmed by a quartz crystal
microbalance with dissipation (QCM-D) study.
Next, a series of bi-layer nanoemulsions at different pH values and lactoferrin
concentrations were prepared so as to determine the best conditions on the overall
emulsion stability. It was shown that the stability of emulsions was dependent on
both pH and lactoferrin concentration. At pH values close to pI of WPI (around pH
5), the nanoemulsions remained unstable regardless of the lactoferrin concentration
used (0.25 – 5% w/w). The nanoemulsions at pH 6 were also unstable at low
concentrations (0.5 – 1% w/w) presumably due to “bridging flocculation” and
exhibited phase separation. Consequently, a lactoferrin concentration of 3% (w/w)
was used to produce bi-layer nanoemulsions at pH 6. At pH 7 – 10, the bi-layer
nanoemulsions were stable at all lactoferrin concentrations and formed a bi-layer
structure at the interface of droplet.
The formulated nanoemulsions (single layer and bi-layer emulsions) were
subjected to a variety of environmental stresses and in vitro digestion under
simulated gastrointestinal conditions. The emulsion stability to pH changes and salt
addition was improved in the bi-layer emulsions containing WPI and lactoferrin
when compared to the single layer nanoemulsions stabilised by WPI alone. However,
the bi-layer emulsions were more susceptible to destabilisation on heating at
temperatures above 60oC. The in vitro digestion of bi-layer nanoemulsions was
similar to single layer nanoemulsions in which the protein hydrolysis of the
interfacial layers results in extensive droplet flocculation.
In subsequent formulations, lutein was incorporated in the emulsions as a
model of bioactive compound for the application of nanoemulsions as a novel
delivery system. The nanoemulsions well encapsulated lutein in their matrices with
an encapsulation efficiency of 80% and contained small oil droplets (70 – 80 nm).
All the emulsions were physically stable under the tested conditions up to 28 days at
different storage temperatures (5, 20 and 40oC). However, there was a significant
decrease in lutein content during storage especially at higher temperatures due to
oxidative degradation. Nevertheless, the bi-layer nanoemulsions showed a better
stability to lutein degradation. Based on in vitro cell toxicity studies on Caco-2 cells
using MTT assay, both nanoemulsions did not show toxicity as the cell viability was
more than 80% at 10 times or more dilution after 24 hours of incubation. The cellular
uptake of lutein was higher in bi-layer nanoemulsions when compared to single layer
emulsions.
The present work demonstrated that nanoemulsions can be formed using
emulsification and a solvent evaporation method. Dried microcapsules of
nanoemulsions were formed with similar properties as their original nanoemulsions
after reconstitution in water. The nanoemulsions with bi-layer interfacial structure
have better stability to environmental changes than single layer emulsions.
Nanoemulsions did not show more toxicity than their corresponding conventional
emulsions with large oil droplets produced without the use of organic solvent. These
have important implications in the use of nanoemulsions for encapsulation lutein or
other bioactive compounds for applications in foods and beverages.
Description
Listed in 2016 Dean's List of Exceptional Theses
Keywords
Research Subject Categories::TECHNOLOGY::Chemical engineering::Food technology, Dean's List of Exceptional Theses