 Okay, so first of all I would like to thank the organizers for inviting me to give a talk in this fantastic forum. I'm sure we will see along the next days very interesting examples on how the scattering techniques are excellent tools to understand the structure of food. And in my case, I would like to show you some examples of the work that we have been doing on the investigation of the structure of polysaccharide gels. So first of all, a little bit of my background. I'm currently working for the Spanish Council for Scientific Research, the CSIC. It has several institutes along Spain, and I was previously working on Valencia on the extraction of polysaccharides from Mara in biomass and the development of gel-like structures. But I'm currently based on Madrid, on a different institute. But it was several years ago when I was working in Australia with Elliot Gilbert and Mike Gidley, that I had the opportunity of learning how excellent tools are the scattering techniques to understand the structure of food. So first of all, why are we interested in understanding the structure of food? Well, for scientific and for technological reasons. Because, you know, nowadays the consumers are looking for new types of foods like functional foods or dietetic foods. So we need to develop new production processes. But also the consumers are more and more concerned about the relationship between diet and health. So we want to understand also how the foods that we are consuming are affecting our health. So as you know, food has, many foods have a very complex structure. The polysaccharides proteins are, in fact, are the main macronutrients. And today I will talk to you about polysaccharides. So one example of polysaccharides I'm sure you know is cellulose we can find in the cell walls from vegetables and fruits. In general, polysaccharides are the major components in fruits and vegetables. They can account up to 90% of the right weight. And they are also used in the food industry as ingredients because they have thickening, jelly and properties. They can also be used as encapsulants and some other functionalities. And there are some examples here from the most widely known polysaccharides which can be extracted from plants, seaweed and can also be produced from microorganisms. And some particularities from polysaccharides in foods is that they are typically found in nature as highly hydrated systems. And this is an issue because typically microscopy techniques have been used to investigate the structure of these polysaccharides. But since we need to subject the samples to drying processes we are surely affecting the structure of these native materials. And then on the other hand, we can find polysaccharides having an amorphous structure such as some phycocoloids which are water soluble, but they can also be showing a crystalline structure such as cellulose. And also another important particularity of polysaccharides is that most of them or some of them are able to form gels when they are subjected to different temperature conditions. So many of you I'm sure you are familiar with these gel-like structures in gelatin, some products like that. And here I'm showing an example of based on the work that I did in Australia to investigate the structure of cellulose. And as you can see, we can find cellulose in the plant cell walls forming a hierarchical structure with different structure levels. So we need to combine different characterization techniques to get the whole picture. And in this sense, scattering techniques are very powerful because not only they allow us to cover almost the whole size range of interest, but we can also analyze the structure of these materials in their native hydrated state. So that's a very great advantage. And I would also like to mention, because you will see in my presentation that we used x-rays and neutrons. And that's because in the case of polysaccharides, x-rays are going to highlight differences between crystalline and amorphous domains, whereas when using neutrons, we can generate contrast between different components in our samples. So we need to be careful of selecting the source of radiation that we need for each type of experiment. And you will see in the results that I will show you today, some experiments that we did in Australia using the SANS and USAN instrument, but we have also done a lot of work in the sax and wax line in the Spanish synchrotron. The first example consists on agar-based hydrogels. So agar is a polysaccharide which is found on the cell walls from red seaweeds. I'm sure you are familiar with agar agar because nowadays it's commercialized and it's used in the food industry to produce gel-like products. In this case, what we wanted to explore was how to simplify the production process of the agar because it's a relatively complex process industrially, it has several steps. The main ones are an alkali per treatment where we are removing some other polysaccharides which are present in the seaweeds and other components. And then the main extraction step is a hot with water treatment. So we wanted to investigate the possibility of removing the first alkali extraction step and then also evaluate the possibility of using alternative methods such as ultrasounds to extract agar. So first thing we observe is that the sonication extraction allowed us to reduce the extraction time significantly while the extraction yields were preserved. And then, as you can see in this picture, we produced less purified agar by skipping this first alkali treatment, which of course contains some other components such as proteins and minerals. So the alkali per treatment is good when we want to produce a pure agar, but on the other hand is producing a lower molecular weight agars. And then the non-purified extracts contain agar, but some other components such as proteins, minerals and polyphenols, which gave them some bioactive properties. And therefore we wanted to study what was the gelation process in the pure agars and in the less purified agars. So the hypothesis was that when in solution agar chains are forming a random coil structure and then as we cool down, they first form the elix aggregates, which then associate to form bundles. And these bundles are responsible for the hydrogel structure. So we first started doing some rheological measurements, and here you can see the results from more purified agar versus less purified agar. Essentially, you see that the gelation temperature was very similar for both of them. So that means even if we have some impurities, they are not affecting the gel structure, the final gel structure, but they are forming softer gels. And on the other hand, when when heating down the samples back to the initial temperature, there was no real melting. So the structures that were being formed were not completely disrupted. And there was a large synergistic meaning that there was a large difference between the initial status and the final structure of the material. So now we wanted to understand what was going on on these agars and for that, the first thing we did with it was to perform sacks and stands experiments on the gels as they were formed. And in the case of using neutrons we took advantage of contrast variation so that you can see there the type of cells that we used. We put a piece of the of the gel sample and then it was soaked on different mixtures of H2O and D2O so that to generate different contrasts. So as you can see here in the scattering patterns from the sacks and stands experiments, we can differentiate three regions. A low Q region where most of the samples show the power low behavior, then an intermediate region where most of the samples showed shoulder like feature and then in current scattering. So using advantage of taking advantage of the of the contrast variation experiments and what we did was to plot the intensity values calculated at a certain Q value versus the amount of D2O in our solvent to determine the contrast match point of the samples and if you are familiar with with some techniques, you will know that if the samples are not deviating from the theoretical behavior those points would fit to a parabolic function and then we will have a minimum points, which theoretically would give us an intensity of zero and that's the contrast match point that means that the scattering density of the sample equals that of the solvent. But as you can see, in this plot, most of the samples deviated from from that behavior and that was something that was not unexpected to us, because we knew that this polysaccharide, well, it's not something particular from agar from agar we also saw that something similar in the case of cellulose, because because they have so many labile hydroxyl groups, they are exchanged when we soak the pellicles in different solvent mixtures so there will be a deviation from from the theoretical behavior and in fact, as you can see the theoretical scattering intensity of agarose was 2.16 while for most of our samples we obtain higher values. And this was especially visible in the case of the less purified samples, this could be due to a different structure for the AR but also due to the presence of other components such as the proteins. And then on the other hand, when fitting our scattering data using an empirical model, we observed that the radius of gyration, which is related to the thickness of the agar double elixus was greater in the case of the more purified agar that that made us think that probably if we have a higher concentration of agarose in those samples they were forming thicker bundles. And finally, to also understand the gelation process, we took advantage of the synchrotron beam line and perform a temperature resolved success experiments. So we use the similar program to that we previously used for for rheological measurements and the samples were heated to 95 degrees to melt the gels and then they were kept at that temperature for 30 minutes and cooled down back to 25 degrees. So we observed that there was no melting transition in the samples, but there was a disruption in the structure of the aggregates because the shoulder disappeared when we were heating the samples. That was true in the case of the less purified samples. But as you can see here for a more purified agar, even when we increase the temperature there was little change on the on the scattering intensity so that made us think that in that case the elix aggregates were much more stable. And then on the other hand when cooling down the sample back to the initial temperature we will we were not able to recover the initial structure so this process was not completely reversible. So based on this on all of these results we proposed a gelation mechanism for the purified agars and for the less purified agars. As you can see, in the case of the purified agars we have a two step gelation process. First, the agar chains combine to form double elixes and then these elixes further aggregate to form the bundles with a size around seven to 10 nanometers. And these bundles, as I said before, they are responsible for the hydro gel structure. And in the case of the less purified agars, we have proteins and other components which are not impeding the formation of the agar double elixes, which then further aggregate into bundles but the size of these bundles is smaller. So that's the reason why we observe a lower strength for the for the less purified agar hydrogels. In the case we saw that the presence of the proteins did not impair the interconnectivity of the gel network. So now another example, a different polysaccharide which is carrageenan. This polysaccharide is also found in the cell walls from red seaweed. And in this case, we have different types of carrageenan depending on the amount and the position of sulfate groups. Here you can see that we work with kappa carrageenan which has only one sulfate group per the saccharide unit, while Yota carrageenan contains two sulfate groups. So this position and amount of sulfate groups has a strong impact on the type of gels that are formed. In the case of kappa carrageenan, we typically come from very strong gels. In the case of Yota carrageenan, they are softer gels, more like a paste consistency. So in this case, we started doing a design of experiments where we were modifying the type and concentration of carrageenan. And then we also studied the incorporation of two different salts. The valent salts containing calcium are monovalent salt containing potassium. So to investigate the influence of these two salts in the type of hydrogel that was being formed. So what we saw is that the carrageenan and cell concentration had a very strong impact on the hydrogel strength. And as you can see, those were the optimal conditions for each type of carrageenan. Even with the addition of salt, in the case of the Yota carrageenan, you see that the hydrogel strength was much lower. That's because the sulfate content was much higher in this type of carrageenan. And then another interesting fact is that while potassium had a higher impact in the case of kappa carrageenan, calcium worked better for Yota carrageenan. So again, we started doing rheological measurements to see what was going on when the samples were going through the gelation process. In this case, we see only the cooling ramps and for the pure carrageenan and with the different salts. And as you can see in general, the incorporation of the salts had a positive effect in increasing the gelation temperature of the carrageenan and also in producing gels with higher strength. As I said before, while potassium worked better for the kappa carrageenan, calcium worked better for the Yota carrageenan. So the next step, we wanted to understand why and what type of structures were being formed in each case. So those are the results from SUCKS experiments and by fitting the data, we were able to determine the Gaussian parameter which is related to the mesh size in the hydrogels. And then the correlation length related to the size of the double helices. So first thing that we noticed is that more compacts and order structures were formed in the kappa carrageenan and especially with the addition of the potassium. I don't know if you can see properly but in the in the first plots, we also see two small peaks, which are appearing in the case of the kappa carrageenan, indicating the formation of very well ordered structures with the presence of the salt. In the case of the Yota carrageenan, we were able to observe a more packed structures with the addition of the calcium. And in general, the idea is that the nature of the bridges holding together the bundles was different depending on the carrageenan type and on the balance of the salt. So based on on our rheological and scattering results we proposed a model for the gelation mechanism. And so in the case of the kappa carrageenan we only have one sulfate group so the addition of the of the salts, especially the potassium is able to neutralize this sulfate groups and therefore is promoting the formation of hydrogen bonding between the polysaccharide chains. On the other hand, in the case of the Yota carrageenan we have a higher amount of sulfate groups so that the addition of salts is not able to to neutralize of these charges from the sulfate groups, but instead what what they are doing is creating cross bridges between the between the sulfate groups so that we in this case we have an ionic cross linking. So that's the main reason why the strength of these hydrogels is so different because in the case of the kappa carrageenan we have strong hydrogen bonding, whereas in the case of the Yota carrageenan, we have ionic interactions. I will show you something a little bit different. These polysaccharides are not able only to form hydrogels but they can also form a different type of structures which are emulsion gels. And in this case, what we do is that we mix a liquid oil in this case sunflower oil with a solution of the polysaccharide, which could be carrageenan or agar or any other gel in polysaccharide. We can also emulsify the material we can also incorporate surfactants and as we have this emulsion and this emulsion is cooling down the polysaccharide is gelling. So, as they form this gel structure the oil droplets are entrapped within this structure and we have something like you see in this confocal microscopy image which has gel consistency. And the polysaccharide that we use as the gelling matrix we may obtain different types of structures. Here you can see some examples from agar and from kappa carrageenan. The amount of oil that we can incorporate is also different, while in the case of agar we can go up to 50% of oil. In the case of carrageenan we can only incorporate up to 40% of oil, but this can be a little bit increased when we incorporate surfactants into the formulations. So these images are confocal microscopy images from the emulsion gels and as you see they have a more or less homogeneous distribution of the oil droplets. In the case of the agar because it has high emulsifying properties, these droplets are much smaller and homogeneous. Whereas in the case of kappa carrageenan, because this polysaccharide starts gelling at a very high temperature, it was more difficult to disperse the oil droplets so that we had a larger oil droplets and a more heterogeneous distribution. And then also interestingly the presence of surfactants was not very positive in the case of agar and we felt that this may be due to a competition between the agar and the surfactant to cover the oil droplets surface so that in that case there was some coalescence. While in the case of the kappa carrageenan, since there was a decrease in the viscosity of the emulsions, it was easier to disperse the oil and the presence of the surfactant had a positive effect. So again, we wanted to investigate the structures of these materials by small angle scattering and here you can see some examples of sacks patterns. In the case of the agar, we basically observed that we had a very similar structure to that we had previously observed in the hydrogels. So the agar double helices had more or less the same size and we had a branch network structure which is typical from hydrogels. And so the oil did not have a very strong impact at this size range. On the other hand, in the case of the kappa carrageenan, we had the formation of less compact network structures as compared to the agar. And then the incorporation of the oil gave rise to the formation of less packed bundles, although the double helices had a similar size as those observed in the hydrogels. And then when we increase the amount of oil in the formulations, we observed some particular structures which could be related to the formation of inverse emulsions. So as we have done with all the polysaccharide based samples, we also performed rheological experiments because we think they can add a lot when combined with scattering. And as you can see here in the case of agar, we had a two step gelation process, a first step, which we thought was related to the gelation of the agar present in the accuse phase and a second step which may be related to the gelation of the domains located at the surface of the oil droplets. And this was not the case for the kappa carrageenan, in that case there was only one gelation step, but then interestingly with the addition of the surfactant, we have one step gelation process in the case of agar. So that supports the hypothesis that in that case the surfactant is located at the interface of the oil droplets so that it's disrupting the emulsifying capacity of the agar. And then we also observed here with marked with the arrows the formation of metastable structures in all the samples when cooling down. We couldn't find any reference on the literature to explain these structures and we perform several variations on the rheological measurements to see if this was an artifact or something due to the type of probes that we were using, but it always appeared in our samples. So then we decided to perform some temperature result experiments on the Spanish synchrotron and in fact we observed the formation of some metastable structures in the temperature range close to the same that we observed in the rheological experiments. So we think this is due to some kind of chain reorganization, but we have to do more experiments to try to understand what what is going on on on this process but we thought it was very interesting. You have three minutes. Okay, I will be very fast. So this this last slides are to show you the final application of this type of structures in the food industry. So we have also done some some experiments on evaluating how these hydrogels are being affected when subjected to some simulated digestion conditions. So that means changing the pH and also putting the samples in contact with different bile salts and other components. And in this case, these results are from alginates, but essentially what we saw that was very interesting is that depending on the structure of the alginate on the manuronic and guluronic ratio, these structures were more resistant to the in vitro digestion conditions, or in the case of the manuronic rich alginate, they lost their structural integrity when subjected to pH values higher than two. So what is next? Well, we are currently working on the encapsulation of protein in this type of polysaccharide hydrogel structures. So what we would like to do now is to do some that in situ digestion experiments to see how the structure of the polysaccharides is being affected, and also how the structure of the encapsulated protein is being affected during the the vitro digestion So that's the next step in this type of studies. And yeah, well, these were some conclusions, but basically, I would say that as you can see from these examples, both x-ray and neutron scattering techniques are very powerful tools to understand the structure of these polysaccharide materials And they offer us the possibility of doing some very interesting work such as in situ digestion experiments. So with that, I would like to thank my colleagues from the CSIC, Salmers, Ansto and the Spanish Synchrotron. Thank you so much Marta. There is one question if you can give a quick answer to that. And that's on Krasimir, he asked what applications to consider for the emulsion yield? What kind of applications? Well, in the food industry, we were thinking of fat replacement. So, for instance, for meat products where, you know, they are looking to replace all the animal fats for alternative type of fats. But also, we have also investigated the application of these materials for biomedical applications for fat phantoms in particular. So, yeah, they could have several applications. Oh, thank you so much. Thank you for a fascinating talk.