Periodic Reporting for period 4 - BINDING FIBRES (Soluble dietary fibre: unraveling how weak bonds have a strong impact on function)
Okres sprawozdawczy: 2020-10-01 do 2022-03-31
Dietary fibres are recognized for their health promoting properties; nevertheless, many of the physicochemical mechanisms behind these effects remain poorly understood. While it is understood that dietary fibres can associate with small molecules influencing, both positively or negatively their absorption, the molecular mechanism, by which these associations take place, needed further elucidatations. We proposed a study of the binding in soluble dietary fibres at a molecular level to establish binding constants for various fibres and nutritionally relevant ligands. The interactions between fibres and target compounds in general may be quite weak, but still have a major impact on the bioavailability. To gain insight to the binding mechanisms at a level of detail that has not earlier been achieved, we applied novel combinations of analytical techniques (MS, NMR, EPR) and both natural as well as synthetic probes to elucidate the associations in these complexes from macromolecular to atomic level. Glucans, xyloglucan, arabinoxylan and galactomannans served as model soluble fibres, representative of real food systems, allowing us to determine their binding constants with nutritionally relevant micronutrients, such as monosaccharides, bile acids, food dyes, and metals. Furthermore, we examined supramolecular interactions between fibre strands to evaluate contributions of several fibre strands to the micronutrient associations. At the atomic level, we applied complementary spectroscopies to identify the functional groups and atoms involved in the bonds between fibres and the ligands. The project executed a unique approach to quantify binding of small molecules by dietary fibres, which can further in the future be translated to polysaccharide interactions with ligands in a broad range of biological systems and disciplines. The findings originating from this study will further facilitate us to predictably utilize fibres in functional foods, which can have far-reaching consequences in human nutrition, and thereby also public health.
The project was focused on the method development to study biomolecular interactions between dietary fibres and small molecules. We successfully synthesised different kinds of probes (namely dietary fibre tethered nano particles as well as spin labelled dietary fibres), which were used to study the existence and strength of interactions, and to provide complementary information about the possible binding mechanisms. This included creation of a well-defined fibre substrates with molecular structures that were characterised in detail with complementary analytical techniques (MS, SEC, DLS). The methods for molecular interactions were applied to measure the interactions between fibres and ligands, and the results show that highest sensitivity and resolution is obtained with the most labor intensive methods based on Electron Paramagnetic Resonance, but that meaningful results for stronger interactions (which are still very weak interactions on the overall scale) can also be obtained with faster methods such as nanoparticle probing and isothermal calorimetric titration. Of the ligands, strongest interactions were obtained with partially charged molecules (such as food dyes) and weak acids. Further application of the established methods will allow for collecting a database of interactions and use the data for predictions of the effects in food systems.
The project utilised previously unexplored analytical techniques for the study of molecular interactions between fibres and ligands. Analytical tools like direct mass spectrometry, electron paramagnetic resonance, and other methods for biomolecular interactions are commonly used for proteins, but seldom for carbohydrate polymers like dietary fibres, which was successfully achieved in this project. The major advantage of stretching the methods out to utilize them for polysaccharides lies in the fact that these methods provide much more detailed information about the nature of the interaction and identify the functional groups involved, rather than merely confirming the existence of an interaction.