Abstract
bottom-up” approach for innovation of novel food materials. During the last 20 years, several
food grade proteins were found to self-assemble into nanostructures, which have drawn much
attention due to the enormous potential in the innovation of controlling of the properties and
structures of food. The whey protein a-lactalbumin (a-La), with a globular shape, is the only
food protein that can self-assemble, into nanotubes after limited proteolysis. Understanding
the formation and the structure of these novel nanotubes is crucial for their further
application in food systems and in other areas.
Therefore, the aim of this project was to develop a method for obtaining a large quantity of
bovine a-La with a high purity and to explore the formation and structural aspects of
nanotubes and gels formed from partially hydrolysed a-La under various conditions, especially
over a wide range of pH values.
Initially, 1.1 kilograms of a-La with a purity of 97.4% in protein phase was produced by a
method based on anion exchange chromatography (AIEX) in combination with ultrafiltration
(UF) from a-La enriched whey protein concentrate (aWPC). The a-La was recovered with a
low volume of buffer. This large amount of a-La provides the possibility of exploring the
formation of a-La nanotubes and gels therefrom under varying conditions.
The conditions, such as a-La concentration (30 and 10 gL-1), calcium level (molar ratio
between calcium and a-La of 2.4 and 5.4), and pH values (from 7.5 to 4.0) responsible for
generating nanotubes were mapped out beyond what is presently known. The results showed
that nanotubes can form under various conditions, even at pH 4.0, or at very low a-La
concentration (10 gL-1). The pH influences the rate of hydrolysis and self-assembly in different
directions, but has no influence on the hydrolysis pattern. Increasing calcium level enhanced
the effect of pH on self-assembly process, whereas the low level of a-La concentration (10 gL-1)
was shown to limit the self-assembly. By tuning the rate of hydrolysis or self-assembly, via
altering these three factors, one can control the formation of a-La nanotubes and gels.
In addition, by using small and wide angle X-ray scattering techniques, the structure of the a-
La derived nanotubes was characterized. The results showed that the nanotubes formed under
most of the conditions have a similar size with an outer diameter of 19 nm, inner diameter of
6.6 nm and wall thickness of 6 nm. The building fragments were arranged as dimers, 10 of
which were packing in the cross-section of the nanotube via b-sheet stacking. Three smaller
nanotubes were observed at the extreme reaction conditions, however, the b-sheet stacking
was not affected. It is noteworthy that a mathematics model was developed for characterizing
the structure of such nanotubes, which is a powerful tool that can be used for further studies,
as well as for other similar self-assembly systems.
Furthermore, the morphology of gels and the gelation properties were characterised. With a
higher self-assembly rate, which can be regulated by increasing calcium level and lowering the
pH value, the formation of random aggregates and fibrils led to decreased gel transparency
and gel strength, or even sedimentation. The results also revealed the importance of the high
purity and the native form of a-La. Moreover, these gels also showed interesting ability of
rebuilding after physical breakdown, which subsequently developed into stronger gels.
Together, this thesis has provided a novel insight into the formation and structure of
nanotubes from partially hydrolysed a-La over a wide range of pH values. This offers new
opportunities for applying such nanostructures in real food systems and possibly in other
fields such as the pharmaceutical and material science.
food grade proteins were found to self-assemble into nanostructures, which have drawn much
attention due to the enormous potential in the innovation of controlling of the properties and
structures of food. The whey protein a-lactalbumin (a-La), with a globular shape, is the only
food protein that can self-assemble, into nanotubes after limited proteolysis. Understanding
the formation and the structure of these novel nanotubes is crucial for their further
application in food systems and in other areas.
Therefore, the aim of this project was to develop a method for obtaining a large quantity of
bovine a-La with a high purity and to explore the formation and structural aspects of
nanotubes and gels formed from partially hydrolysed a-La under various conditions, especially
over a wide range of pH values.
Initially, 1.1 kilograms of a-La with a purity of 97.4% in protein phase was produced by a
method based on anion exchange chromatography (AIEX) in combination with ultrafiltration
(UF) from a-La enriched whey protein concentrate (aWPC). The a-La was recovered with a
low volume of buffer. This large amount of a-La provides the possibility of exploring the
formation of a-La nanotubes and gels therefrom under varying conditions.
The conditions, such as a-La concentration (30 and 10 gL-1), calcium level (molar ratio
between calcium and a-La of 2.4 and 5.4), and pH values (from 7.5 to 4.0) responsible for
generating nanotubes were mapped out beyond what is presently known. The results showed
that nanotubes can form under various conditions, even at pH 4.0, or at very low a-La
concentration (10 gL-1). The pH influences the rate of hydrolysis and self-assembly in different
directions, but has no influence on the hydrolysis pattern. Increasing calcium level enhanced
the effect of pH on self-assembly process, whereas the low level of a-La concentration (10 gL-1)
was shown to limit the self-assembly. By tuning the rate of hydrolysis or self-assembly, via
altering these three factors, one can control the formation of a-La nanotubes and gels.
In addition, by using small and wide angle X-ray scattering techniques, the structure of the a-
La derived nanotubes was characterized. The results showed that the nanotubes formed under
most of the conditions have a similar size with an outer diameter of 19 nm, inner diameter of
6.6 nm and wall thickness of 6 nm. The building fragments were arranged as dimers, 10 of
which were packing in the cross-section of the nanotube via b-sheet stacking. Three smaller
nanotubes were observed at the extreme reaction conditions, however, the b-sheet stacking
was not affected. It is noteworthy that a mathematics model was developed for characterizing
the structure of such nanotubes, which is a powerful tool that can be used for further studies,
as well as for other similar self-assembly systems.
Furthermore, the morphology of gels and the gelation properties were characterised. With a
higher self-assembly rate, which can be regulated by increasing calcium level and lowering the
pH value, the formation of random aggregates and fibrils led to decreased gel transparency
and gel strength, or even sedimentation. The results also revealed the importance of the high
purity and the native form of a-La. Moreover, these gels also showed interesting ability of
rebuilding after physical breakdown, which subsequently developed into stronger gels.
Together, this thesis has provided a novel insight into the formation and structure of
nanotubes from partially hydrolysed a-La over a wide range of pH values. This offers new
opportunities for applying such nanostructures in real food systems and possibly in other
fields such as the pharmaceutical and material science.
| Original language | English |
|---|
| Publisher | Department of Food Science, Faculty of Science, University of Copenhagen |
|---|---|
| Number of pages | 172 |
| Publication status | Published - 2015 |