INFORM February 2025 Volume 36 (2)

16 • inform February 2025, Vol. 36 (2)

Compositions of the oil phases and emulsifiers in the 16 emulsions studied. Source: Tiago C. Pinto, University of Helsinki

formulation required over four hours of simulated digestion, and X-ray characterization of the samples had to occur at pre cise points in time during the digestion process. To maximize their beamtime, the team had two digestion simulations run ning simultaneously at all times, which required several scien tists to manage. Because lipids are primarily digested during the intesti nal phase, the researchers focused on tracking changes during that stage, as opposed to the oral and gastric phases of diges tion. For each sample, they characterized their structures just before the intestinal phase began, and then 2, 5, 10, 15, 60 and 120 minutes later. “We saw from our preliminary experiments that it was during the initial stage of the intestinal phase that the diges tion rate changed the fastest, so we thought that time period might yield the most interesting data,” Pinto said. SIMULATING DIGESTION OF OLEOGELS Clinical trials are considered the gold standard for nutrition studies, but they can have limited ability to provide detailed mechanistic insights compared to in-vitro methods. For this study, the researchers used a modified version of the INFOGEST protocol, an in-vitro method originally designed to simulate the digestion of five grams of food. “Usually, lipids are a small part of an entire food sample. In our case, we are analyzing oleogels by themselves,” Pinto said. In 2022, the Food Materials Science Research Group at the University of Helsinki published a paper addressing the lim itations of the traditional INFOGEST protocol as it applies to oleogels ( https://doi.org/10.1016/j.foodres.2022.111633 ). The paper also provided recommendations for how to alter the protocol for greater bio-relevance and better standardization of results from different studies. Pinto and team used this revised protocol to simulate digestion in a room next to the beamline (see page 17). During the intestinal phase, triglycerides are broken down into mono

glycerides and free fatty acids (FFAs). The release of FFAs nat urally brings a sample’s pH level down. By adding sodium hydroxide as needed, the researchers carefully maintained each sample’s pH at a constant level of seven using a method called pH-stat. Based on the volume of sodium hydroxide added, they were able to track the release of FFAs and deter mine how much of a sample had been digested at various points in time. In the meantime, the researchers periodically took sam ples from the digestion beakers and added a lipase inhibitor to stop digestion. They then incubated the samples at 37 °C to maintain the same temperature as in the digestion vessel and avoid any further modification of the lipid crystals. In this way, the samples represented snapshots during digestion, which were then brought to the beamline for structural analysis. CRYSTAL STRUCTURE FORMATION DURING DIGESTION Fats are polymorphic, which means they can display more than one crystal structure at once. It is believed that the different polymorphs present in fat can impact macroscopic character istics, such as melting temperature, rheological properties and sensory attributes. The researchers expected to see changes in polymorphic forms during digestion. Analysis is revealing the existence of polymorphs, including the alpha, beta prime and beta forms (listed in order of increasing stability). By looking for common peak positions identified in previous literature for different lip ids, the team tracked the formation evolution of the different polymorphs at the nanoscale in their XRD data and identified whether they strengthened or weakened over time for differ ent samples. A major takeaway from the study was that polymorphic changes depended more on the composition of the oil phase in the digested oleogel sample, as opposed to the emulsi fier. The researchers found that samples structured with