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Raw materials for Encapsulation – Proteins

Prof. Dr.-Ing. Erich J. Windhab (ETH Zürich)

Raw materials for Encapsulation II - Proteins , with focus on protein structuring in film-/gel bead format
Hello, everybody, and welcome for the second course part on raw materials for encapsulation, in which I will focus on protein systems. I’ll introduce briefly, again, S-Pro Squared Scheme, then go into proteins in encapsulation processing, followed by exemplary description of milk proteins and gelatin. And then have a fourth subchapter comparative testing of protein film/gel properties for encapsulation, followed by summary and conclusion. S-Pro Squared Scheme process makes structure, structure codes the property. Again, started from the consumer perspective. Controlled release of valuable components, active components, and aroma and flavour, as well as nutritive components. As second aspects, we have the protection of functional components against unwanted reactions. This is the reason why we encapsulate the structure.
Certainly on a macro scale, there’s capsules and carriers; meso scale macromolecular networks. And on the micro scale, the molecules of the proteins as well as of encapsulated functional component molecules. On the processing side, encapsulation as it’s meant to be applied in technologies like spray drawing, spray chilling, fluidized bed coating, and extrusion. When we look again to a listing of the materials suited for micro encapsulation, we have the carbohydrates, the proteins, and the lipids. And we can see under the protein category, we have not that many as we had seen for the carbohydrates. It’s mainly gluten. There is isolates from some plant systems, algal proteins, caseins, whey proteins from milk, and gelatin.
All should be related to the technologies mentioned before in their application for encapsulation. The specialities of proteins compared to the polysaccharides. So, they are quite reactive. There are amphiphilic. Accordingly, they have surface active, means orientation that interfaces emulsifying and foaming is a consequence, what they are very good in, and temperature sensitivity as the major aspects extracted here in the comparison to the polysaccharides and carbohydrates treated before. Looking at the possibilities to go for some analysis of the proteins and the related technologies they are used in already. So we have this little table here.
Of importance is always a checklist when we want to orient in the jungle of all these possibilities with proteins and other materials for encapsulation, that we have a good checklist for orienting towards the needs with respect to a specific process or to a specific functionality encapsulation, like increase of shelf life, masking of taste, simplification of handling, controlled or targeted release, improvement of appearance. And to be clear, this then, aspects of functionality of encapsulating product restrictions for the coating material. Concentration of the encapsulate necessary type of release intended. Stability requirements. And certainly, economic aspects, cost constraints.
Looking at the individual properties of proteins and other characteristics, also the gel forming behaviour, network forming behaviour, we can see that there is a lot of impact from different sides– the protein structure, stability of the protein in the bulk, isoelectric point of these amphiphelic molecules, pH of the solution, the other solutes around, and the nature of the other phases. Here, we can see just a sketch of lysozyme as a typical protein. We also find in human saliva, for example, and the beta-lactoglobulin from milk. So it’s a huge diversity. And the question is, how can we get, let’s say, access to these huge diversity in order to manage handling it in a tailor made way?
For this, we need a good tool box. And I would just like to introduce the tool box here, analytical tool box, which goes for properties like interfacial properties. This is why like absorption kinetics, measurements, as an example, pendant drop tensiometer, interfacial rheology, with an interfacial stress rheometer. But also for the processing like droplets and capsule processing, the deformation characteristics, maybe breakup characteristics in, let’s say, model channels where such encapsulated or coated structures are treated fluid mechanically and observed. To look a bit more closely to the structure, so there is certainly x-ray and neutron scattering, very interesting. So interfacial layer morphology can also be investigated by interfacial reflectometry with neutrons as well as with x-ray.
So quite a sophisticated tool box to go into a structural and related property details. I emphasise a bit more this interfacial properties because they are really unique and most relevant for encapsulation and coating of interfaces. Exemplarily, I will look at milk proteins here, beta-lactoglobulin. Beta-lactoglobulin, how it absorbs as an interface as a first indicator in the fascial tension. Depending on the concentration of the beta-lactoglobulin, goes pretty far down. And when we have an exchange with a bulk after the interfacial film has formed, we can see that it’s not completely recovering. That means we have some irreversibility or some skin formation by the beta-lactoglobulin.
To go for, let’s say, interfacial rheometry, so there is two different ways to do this in compression and dilatation with a dynamic pendant drop tensiometer, which makes such a droplet growing and shrinking. And with a interfacial shear rheometer like with such a twin cone where the interface between the two liquids, like oil and water, can be formed in these regions of the tips of these twin cone. And then any type of shear in these 2D layer or in these plane generated, so elastic and viscus properties can be measured. You can even go more sophisticated with having the finalised capsule and shearing it in shear fields between two bands which are moving relative to each other.
If this is the skin, there is a skin formation, you may have these fluctuations, kind of a wobbling of an ellipsoidally deformed skin containing capsule. If there is no skin, you may have just a rotation, which is more smooth. So you can also derive from such, let’s say, more applied conditions an elastic modulus, a Yang’s modulus of the skins formed around these capsules. The theory behind Bartes-Bissell in 1985 started to go already into some details. And in the meantime, there is a lot of simulation also possible in order to get these complex flow behaviour and derive some material characteristics like the skin Young’s Modulus, which is of importance for stability.
When we look a bit more detailed into the twin cone type of interfacial rheometer or the pendant drop so we can even exchange phases. That means when a skin has formed, we may, with the double capillary, change the content of these drop or we can also pump in and out some liquid in these twin cone cell. And from this, we can modulate the conditions, like shown on the right-hand side with the elastic and viscous moduli. Shear moduli have been measured under a simulated gastric digestion of such capsule interfaces. And you can see here temperature adjustment, stomach pH adjustment very low. Then comes the pepsin as an enzyme and the hydrolysis, in principle, degrades or it dissolves the skin.
And then peptide networks can form and then a new type of layer is built up. So you can follow this versus time and have an exact mirroring of what happens during the gastric digestion. Looking a bit more detail into structure by atomic force and neutron reflectometry, it’s a very good combination to look at morphologies of such structures. Atomic force gives you a visual impression and these can then be correlated to the scattering curves received from the neutron scattering, which then allows for building models, like shown here for pure beta-lactoglobulin. Other components, like for mixed systems with nanocrystalline cellulose, means carbohydrates. This frame can be wide, and so we chose the powerfulness of this tool box.
A listing of dairy proteins and processes for encapsulation of hydrophilic, hydrophobic compounds, as well as probiotics, just to give an overview– how much is there already around and related references, if you want to go into some further detail. For the milk proteins, exemplarily also showing a bit systematics of the scheme– how you can work with. So looking at caseins and whey proteins as the two building blocks for milk, you can have the formation of some building blocks by interaction binding of these molecules to each other and entrap some functional compounds, so the stars denote the encapsulated compounds.
You can also use the casein micelles to stabilise emulsions, for example, and then entrap some of the encapsulated compounds into an oil phase of such an emulsion. This is also called the top-down approach. And you can also use whey proteins or caseins to form assemblies. Whey protein self-assembly can also entrap some active compounds. And also gelation, so gel beads or gel layers forming can also be microporous gel entrapment of the compounds you want to encapsulate. So different ways to go and nicely demonstrated here with the scheme for the milk proteins. You could go even further.
So if you say, I’m interested in these emulsion interfaces being stabilised by casein, for example, you could also go for enzymes to further cross-link at the interface if one’s placed at the interface. So this would be one of these known enzymes being efficient in that respect– it’s transglutaminase. And here we see the surface shear viscosities of an absorbed casein film– how it goes very quickly up or more slowly up, depending on the system when you do the networking by transglutaminase triggered cross-linking. So the same is shown here for a stabilised emulsion gels– are forming emulsion.
And then you can also stabilise the gel by further cross-linking, and you also can see the different increases if this type of cross-linking is done on top. Having a gel system finalised, there is certainly also special gel testing and here we can see, for mechanical testing, the breakage strength versus the deformation. And one can also easily get from these percentages of gelatin, in this case, there is gelatin gels– how the strength develops as a function of the deformation.
This can also be modelled and, from these models, you can derive– from the molecular and the concentration information– you can get some molecular junction association constant for the forming of these gel junctions, as well as the shear modulus that has been derived from David Oakenfull in the paper which is cited here. Finally, and very interesting, the mixing of, in this case, gelatin and some other components from the carbohydrate space, which has been treated before in the first part of the lecture course to the encapsulation materials– but uncommon, so a protein and polysaccharide mixture is shown here. They can either have attractive or repulsive forces interacting.
And accordingly, you form different structures where, for the attractive interaction, we will find biopolymers in the same phase because the materials attract each other, or in the different phases for the segregation phenomena or phase separation phenomena when repulsion is acting. And so you have the chance to build different structures, like droplets, deformed droplets, or some types of networks or a disintegrated type of filament structures. The examples on the right-hand side show you such practise mixtures of gelatin carrageenan, so it means always protein and carbohydrates, gelatin, sodium alginate, as well as gelatin/sodium CMC, which is successfully treated here to generate these beads.
Last but not least, it’s of importance again to do the mechanical testing for the proteins, as we have seen before for the carbohydrates. We go for mechanical testing of tensile strength, tear strength, puncture strength, and elongation at break, with mechanical testing machines, accordingly. The permeability with respect to oxygen is very important to avoid oxidative effects, if encapsulation should protect against these shown here for 4% of difference of the proteins. So we have here the sodium caseinate, then followed by the whey protein isolates, and then followed by the gelatin, as shown here– gelatin being, obviously, very efficient in oxygen permeability reduction.
Water resistance is important when you have once encapsulated something– moisture in the air during storage or, also then, contacting watery liquids with acids and alkaline properties. In addition, oil permeability, so you can see that oil permeability is very good with respect to whey protein layers, but not so good, as we see, for the gelatin, and even worse for the sodium caseinate.
Optical appearance is important for some of the encapsulation tasks. And we can see, again, here turbidity and some colour changes, as well as microscopic pictures of the morphology. We can see here porosity of layers, which may also give an idea about the permeability for the different gels formed from 4% sodium caseinate, 8% sodium caseinate, 8% and 12% whey protein isolates, and 4% and 8% of gelatin. Finally, a ranking of all these proteins, so the blue boxes are just for proteins. The other ones in the same table had been shown before, in the course part number one for the carbohydrates.
So for the proteins, which we have here taken into account– the sodium caseinate, whey protein isolate, and gelatin– you see the ranking of the characteristics which have been mentioned before. At the end of the day, it needs that these layers have to guarantee for a controlled release. And I’ll just show you here one example of such a cell, a shear cell where, under well-defined shear, you can investigate the release of components out of the capsules through a membrane. The membrane can simulate the intestinal mucosa, for example, mixed conditions in the other hemisphere.
And so, under well-defined shear, you can study the mass transfer or you get the mass transfer coefficient here expressed in a dimensionless manner with a Sherwood number versus Reynolds and Schmidt, as you would do for preparing this data for upscaling. With this, I will just briefly summarise. We have seen quite a narrow range of different proteins, but, with growing potential on the plant protein side, it’s available for encapsulation layering and gel formation. Edible film coding and these microporous protein gels are the two formats of relevance.
The major difference between proteins and carbohydrates is the protein reactivity, amphiphilicty, and the related surface activity, which relates to emulsion and foam stabilisation, as well as the temperature sensitivity, which is relevant for denaturation under higher temperatures. A powerful tool box which I showed you for bulk and interfacial elongation and shear rheometry, X-ray neutron scattering, which is quite powerful in order to get quantitative information on the process-structure-property relationship. In addition, certainly, microscopic techniques. The functional differentiation of proteins for encapsulation can then, again, be done by mechanical testing, permeability testing with respect to water, oil, and oxygen.
And one can just sum up that there is a large potential in combining proteins and carbohydrates to achieve enhanced techno-functional properties combining the advantages of both of these systems. There is already a wide range of applications in combining the carbohydrates and the proteins. With this, I would like to thank you for your attention.
Proteins as raw material for encapsulation. Focus on protein structuring in film-/gel bead format
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