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Lipid sponge phase nanostructures as carriers for enzymes

Valldeperas Badell, Maria LU (2019)
Abstract
Nonlamellar lipid liquid crystalline phases have many potential applications, such as for drug delivery, protein encapsulation or crystallization. Lipid liquid crystalline sponge phase (L3) has so far not been very much considered in these applications, in spite of apparent advantages in terms of its flexibility and capacity of forming large aqueous pores able to encapsulate large bioactive molecules. In this thesis the potential of these L3 phases as carriers of two important enzymes used in food industry was explored.
In the first part of my thesis, a novel lipid system able to form highly swollen L3 phases was investigated. This lipid system was composed of diglycerol monoleate (DGMO), glycerol... (More)
Nonlamellar lipid liquid crystalline phases have many potential applications, such as for drug delivery, protein encapsulation or crystallization. Lipid liquid crystalline sponge phase (L3) has so far not been very much considered in these applications, in spite of apparent advantages in terms of its flexibility and capacity of forming large aqueous pores able to encapsulate large bioactive molecules. In this thesis the potential of these L3 phases as carriers of two important enzymes used in food industry was explored.
In the first part of my thesis, a novel lipid system able to form highly swollen L3 phases was investigated. This lipid system was composed of diglycerol monoleate (DGMO), glycerol monoleate (Capmul GMO-50) and polysorbate 80 (P80). Since the L3 phase is not as well characterized as other lipid liquid crystalline phases, a detailed study was performed using scattering and spectroscopic methods. The results showed that the L3 phase is closely related to other bilayer-type of structures, especially inverse bicontinuous cubic phases. The formed L3 phase was found to be easy to disperse in excess water to form sponge-like nanoparticles (L3-NPs). Their structure, interfacial properties and the role of P80 for particle stabilization were determined. Small angle neutron scattering revealed that P80 was mostly located at the lipid-aqueous interface, but also contributed to form the inner L3 structure. This indicated that the highly extended structures observed on the L3-NPs surface by cryo-TEM were formed thanks to P80. This type of structures stabilises the nanoparticles. The interfacial properties of L3-NPs were studied using different surface techniques. Adsorption of the L3-NPs on hydrophilic silica led to spreading of the particles on the surface to form a bilayer-type of structure.
The second part of this work demonstrates the applicability of these lipid L3 phases as matrices for enzyme encapsulation. The enzymes investigated here were Aspartic protease (34 kDa) and the dimeric form of β-galactosidase (238 kDa). Both of them are commonly used in the dairy industry and therefore, their immobilization is crucial to improve their stability and control their activity. In this thesis, structural changes of the lipid system, the stability of the encapsulated proteins, the location of the enzyme within the L3 phase and the nature of lipid-enzyme interactions were revealed. The results suggest that both enzymes were successfully entrapped into the L3 phase. Both enzymes maintain a higher activity when encapsulated compared to the free enzyme kept under the same conditions. Furthermore, different techniques proved that the enzymes are located within the L3-NPs, with encapsulation efficiencies of 81 % and 100 % for Aspartic protease and β-galactosidase, respectively. Finally, lipid-enzyme interactions were investigated to explain the efficiency of the encapsulation process. The results suggested that there are attractive interactions between enzymes and the lipid bilayer, where hydrophobic interactions play a major role.
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Abstract (Swedish)
Lipids are organic molecules vital for living organisms. They are part of cell membranes and provide an important energy reserve once they are digested by our body. They are most commonly known as fats and oils. Some important so-called polar lipids are amphiphilic molecules, which means they have a head group that is water ‘loving’ (hydrophilic) and a hydrocarbon tail is water ‘hating’ (hydrophobic) as shown in Figure 1. It is well known that oils do not mix well with water. Therefore, when polar lipid molecules are added to water they organize themselves so that the head group is in contact with water while the tail is not. This is known as lipid self-assembly and, for example, that is how cell membranes are formed, where the important... (More)
Lipids are organic molecules vital for living organisms. They are part of cell membranes and provide an important energy reserve once they are digested by our body. They are most commonly known as fats and oils. Some important so-called polar lipids are amphiphilic molecules, which means they have a head group that is water ‘loving’ (hydrophilic) and a hydrocarbon tail is water ‘hating’ (hydrophobic) as shown in Figure 1. It is well known that oils do not mix well with water. Therefore, when polar lipid molecules are added to water they organize themselves so that the head group is in contact with water while the tail is not. This is known as lipid self-assembly and, for example, that is how cell membranes are formed, where the important part of the structure is a lipid bilayer (Figure 1).
Depending on the type of lipid used and the environmental conditions (pH, temperature, salt concentration), they can form a variety of different structures in water. This makes lipids very useful for different applications, especially in medicine and food industry, where they are commonly used as encapsulation systems. In this thesis I have used a lipid self-assembly structure called lipid sponge phase. As the name indicates, it has a resemblance to a bathtub sponge, where the sponge solid material will be the lipid network while the holes with air are filled with water. This sponge phase has a bilayer structure as in the cell membranes. However, in this case the bilayer is bent so that it forms a network of lipid bilayers and water pores (as shown in the cover). These types of structures were studied in detail with different techniques to understand how to use them as encapsulation systems.
The second part of this thesis consists of encapsulating two proteins into these lipid sponge phases. Proteins are large and complex biomolecules sensitive to degradation. Moreover, they are required for the structure, function and regulation of the body’s cells, tissues and organs. Proteins that are in charge of accelerating biochemical reactions are called enzymes. In this work the encapsulation of two enzymes used in food industry was investigated to increase their stability. Aspartic protease is an enzyme used in the cheese ripening process. Its function is to destabilize the milk and, as a result, the milk is aggregated and cheese curd is formed. An encapsulation of this enzyme could give more control over the ripening process and, hence, cheese texture and flavour could be tuned accordingly. The other enzyme studied here, β-galactosidase, is responsible for breaking down lactose and as such, it is used to produce lactose free milk and other dairy products. Encapsulation of β-galactosidase could provide a larger stability to this enzyme, but it could also make it easier to use. This is very important since nowadays an increasing fraction of the population has lactose intolerance problem, i.e. they lack this enzyme. As a consequence, the demand of β-galactosidase in food industry is rising.
The results showed that the lipid sponge phase could successfully encapsulate these two proteins and protect them from degradation. Since proteins are very large biomolecules, this was possible due to the large pores and flexibility of the sponge phase.
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Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Seddon, John M., Imperial College London, UK
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Sponge phase, lipid liquid crystals, DGMO, GMO-50, polysorbate 80, encapsulation of proteins, Aspartic protease, β-galactosidase, X-ray and neutron scattering, Raman spectroscopy, adsorption, cryo-TEM
pages
260 pages
publisher
Lund University, Faculty of Science, Department of Chemistry
defense location
Sal C, Kemicentrum, Naturvetarvägen 14, Lund
defense date
2019-03-15 09:30:00
ISBN
978-91-7422-628-7
language
English
LU publication?
yes
id
9c6f4394-1bb2-4cef-a2b1-ebda85c81810
date added to LUP
2019-02-15 13:39:10
date last changed
2019-02-18 16:40:43
@phdthesis{9c6f4394-1bb2-4cef-a2b1-ebda85c81810,
  abstract     = {Nonlamellar lipid liquid crystalline phases have many potential applications, such as for drug delivery, protein encapsulation or crystallization. Lipid liquid crystalline sponge phase (L<sub>3</sub>) has so far not been very much considered in these applications, in spite of apparent advantages in terms of its flexibility and capacity of forming large aqueous pores able to encapsulate large bioactive molecules. In this thesis the potential of these L<sub>3</sub> phases as carriers of two important enzymes used in food industry was explored.<br/>In the first part of my thesis, a novel lipid system able to form highly swollen L<sub>3</sub> phases was investigated. This lipid system was composed of diglycerol monoleate (DGMO), glycerol monoleate (Capmul GMO-50) and polysorbate 80 (P80). Since the L<sub>3</sub> phase is not as well characterized as other lipid liquid crystalline phases, a detailed study was performed using scattering and spectroscopic methods. The results showed that the L<sub>3</sub> phase is closely related to other bilayer-type of structures, especially inverse bicontinuous cubic phases. The formed L<sub>3</sub> phase was found to be easy to disperse in excess water to form sponge-like nanoparticles (L<sub>3</sub>-NPs). Their structure, interfacial properties and the role of P80 for particle stabilization were determined. Small angle neutron scattering revealed that P80 was mostly located at the lipid-aqueous interface, but also contributed to form the inner L<sub>3</sub> structure. This indicated that the highly extended structures observed on the L<sub>3</sub>-NPs surface by cryo-TEM were formed thanks to P80. This type of structures stabilises the nanoparticles. The interfacial properties of L<sub>3</sub>-NPs were studied using different surface techniques. Adsorption of the L<sub>3</sub>-NPs on hydrophilic silica led to spreading of the particles on the surface to form a bilayer-type of structure.<br/>The second part of this work demonstrates the applicability of these lipid L<sub>3</sub> phases as matrices for enzyme encapsulation. The enzymes investigated here were Aspartic protease (34 kDa) and the dimeric form of β-galactosidase (238 kDa). Both of them are commonly used in the dairy industry and therefore, their immobilization is crucial to improve their stability and control their activity. In this thesis, structural changes of the lipid system, the stability of the encapsulated proteins, the location of the enzyme within the L<sub>3</sub> phase and the nature of lipid-enzyme interactions were revealed. The results suggest that both enzymes were successfully entrapped into the L<sub>3</sub> phase. Both enzymes maintain a higher activity when encapsulated compared to the free enzyme kept under the same conditions. Furthermore, different techniques proved that the enzymes are located within the L<sub>3</sub>-NPs, with encapsulation efficiencies of 81 % and 100 % for Aspartic protease and β-galactosidase, respectively. Finally, lipid-enzyme interactions were investigated to explain the efficiency of the encapsulation process. The results suggested that there are attractive interactions between enzymes and the lipid bilayer, where hydrophobic interactions play a major role.<br/>},
  author       = {Valldeperas Badell, Maria},
  isbn         = {978-91-7422-628-7},
  language     = {eng},
  publisher    = {Lund University, Faculty of Science, Department of Chemistry},
  school       = {Lund University},
  title        = {Lipid sponge phase nanostructures as carriers for enzymes},
  url          = {https://lup.lub.lu.se/search/ws/files/57986977/Complete_thesis_Maria_Valldeperas.pdf},
  year         = {2019},
}