Colloidal-scale self-assembly of microcapsules for food

Abstract

Microencapsulation is the technique of enclosing valuable or delicate materials in small containers for targeted delivery. These containers may consist of a core and a thin shell surrounding it. The core, with a size ranging from a few to hundreds of micrometers, contains the active material, and serves as template for the microcapsule. The shell, with a thickness of no more than a few hundred nanometers, lends mechanical, physical, or chemical protection, and the means to release the active materials in response to a well defined trigger. Microencapsulation is used in a number of industries, e.g. pharmaceutics and food. In the case of medicines, microencapsulation is important for the delivery of drugs at the correct location, in the right dose, and at the right moment, which otherwise would have to be overdosed to reach the desired therapeutic effects. In the case of food, microencapsulation may mask ingredients that would cause off-taste and protect sensitive or volatile materials like flavors, aromas, and antioxidants. In addition, microencapsulation will enable new product concepts, such as products with new sensory experiences (fizzy products from encapsulated CO2) and, more importantly, healthier foods including nutraceuticals or probiotics. The microencapsulation methods that are used nowadays in industry on a fairly large scale allow limited control over the uniformity of the microcapsule size and geometry, and the thickness of the shell, while the harsh operation conditions pose restrictions on the type of ingredients that can be used. This poses a challenge to improve production methods and materials. Besides, specifically for food, the materials should additionally be food grade and inexpensive. In this thesis, microcapsules (1-20 micron) are constructed via self-assembly; i.e. using driving forces already available in nature. From the routes using self-assembly proposed in literature for microencapsulation two called our special attention, both starting from an oil droplet as template which determines the size of the microcapsule. The first route is that of colloidosome preparation, in which colloidal particles adsorb irreversibly and organize at the oil-water interface, creating a sturdy shell with pores defined by the interstices between the particles. The second route is electrostatic layer-by-layer adsorption: polyelectrolytes of opposite charge are sequentially adsorbed on a charged template, creating a thin film of which the thickness can be controlled with precision in the order of few nanometers. We have combined the two methods by using larger building blocks to construct stronger shells with better defined characteristics, using less adsorption cycles and based on food grade materials. These microcapsules are designed to protect an eventual encapsulated material from low-pH conditions and deliver their contents in response to a change in pH, having in mind applications that would require delivery through the stomach into the small intestine. In Chapter 2, the available techniques to produce colloidosomes are reviewed. Microcapsules can be assembled from both, oil-in-water or water-in-oil emulsions, which translates in the potential ability to encapsulate hydrophilic and hydrophobic materials. Since the size of the microcapsules is defined by the droplet used as template the construction of almost perfectly monodisperse microcapsules is within reach, given the current developments in emulsification technology. The choice of size, geometry, and origin of the colloidal particles to be used to assemble the shell, and the means to lock them together, gives control over the targeting and release behavior of the colloidosomes. The requirement that always needs to be met is that particles should have affinity for the both oil and water at the interface, imposing a restriction on the choice of the to-be-used materials. Further, the methods usually applied to lock the colloidal particles to form the shell (e.g. heat up the microcapsule to sinter particles together) were seen as a drawback for the encapsulation of delicate materials such as foods, for which many active components are heat sensitive or volatile. Without significant sintering of the particles the capsule does not have protective properties. The next chapters are therefore devoted to new microencapsulation techniques that complement the colloidosome and the layer-by-layer adsorption routes. In Chapter 3, the first results are presented on microcapsules prepared by sequential electrostatic adsorption of protein fibrils, with approximate dimensions of 1 µm in length and 4 nm in width, and high methoxyl pectin. The encapsulation procedure was carried out at low pH (3.5), which allowed us to use positively charged oil droplets stabilized with whey protein isolate as templates. Confocal scanning laser microscopy showed that the fibrils adsorbed as an open structure on a layer of pectin in quantities much larger than a monolayer. This was confirmed from the shell thickness determined by scanning electron microscopy. The fibrils remained trapped at the surface after the adsorption of another layer of pectin. It could be shown indirectly that the inclusion of fibrils in the shell had a tremendous impact on the mechanical strength when compared to microcapsules made of layers of protein and pectin only. Since the microcapsules were assembled at low pH, under the action of pH- and ionic strength-dependent electrostatic interactions, we envision that these capsules would survive low pH, and increase their permeability, or totally disorganize, when exposed to a neutral pH. This is further elucidated in the next chapter. In Chapter 4 extensive characterization of the fibril-reinforced microcapsules is reported. Each layer (of fibrils or pectin) added approximately 30 nm to the total thickness, which is considerably more than monolayer coverage, as measured by reflectometry. The microcapsules keep their integrity when exposed to pH below 5.2 (showing slow dissolution at pH 2), but disintegrate at pH 7 or higher. The response is non linear for increasing number of layers, as it was the case for the mechanical strength of the microcapsules. While microcapsules with 7 or less layers had a similar Young modulus, microcapsules with 8 or more layers had twice that strength, around 0.6 GPa, comparable with the strength of polymeric microcapsules that are chemically cross-linked. This was related to the defects present in the shells as observed with scanning electron microscope: capsules with 8 or more layers had smooth and defect-free shells, which resulted in high pH stability. The available food grade materials allowed the definition of a second system (to be assembled also at low pH) presented in Chapter 5. A single layer of charged silica particles was adsorbed on sequential layers of whey protein and high methoxyl pectin, therewith reducing the number of adsorption steps. Those pre-adsorbed layers stabilized the adsorbed hydrophilic silica particles that, otherwise, would make a poor colloidosome structure. In this way a highly porous but strong structure that could be easily loaded was obtained. After loading the pores can be closed through the adsorption of additional layers of protein and pectin. The drawback of the two microencapsulation systems described above is that polyelectrolytes needed to be used in excess, and intermediate washing steps to rinse out the non-adsorbed materials were essential. To overcome this we went a step ahead in Chapter 6 using a microfluidic device to carry out layer-by-layer adsorption of up to ten layers of protein fibrils and pectin in continuous mode. The design of the chip is simple and does not require complex infrastructure around it, since it relies only on the right balance of the hydraulic resistances of different sections of the microfluidic circuit to control the dose of the materials for the microcapsules’ assembly. Although issues like surface modification of the chip for long-run operation, and the scale-up of the process to industrially-interesting volumes are still a challenge, we feel that this is an important step forward toward controlled microcapsule formation. The integration of knowledge on self-assembly, of which some examples can be found in this thesis, combined with the search for new food-grade materials that can act synergistically to assemble a smarter and multifunctional shell, and better design of microfluidics for tight control of this process are key to mature microcapsule formation into a real tool for the food industry. Chapter 7 discusses further requirements for the production of reinforced layer-by-layer microcapsules using microfluidics, and closes this thesis with a general discussion of the results, in the light of possible future developments in the area of microencapsulation. <br/