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Abstract
Whey proteins and alginate are vastly available biomolecules, coming from dairy production and purified from brown algae, respectively. The utilization of both whey protein and alginate in food and pharma products, is a move towards a more sustainable world, where waste is minimized and CO2 neutral biomaterials are essential. Both biomolecules are already being applied, especially in the food, but also to some extent in the pharma industry, they are however rarely used in the same products. The few studies that already exist on the interaction between alginate and whey proteins have established some basic knowledge. The complexation of these molecules results in phase separation due to charge neutralization, thus the basis of the interaction is opposite charge attraction. The interaction is influenced by pH, ionic strength of the solvent, as well as the composition and molecular size of the alginate. Thus the available information of whey protein alginate interaction on a molecular level is rather limited with regard to alginate structure. This thesis focuses on the understanding of how the above mentioned factors influence the interaction and whether other forces, such as hydrophobicity play a role or can be neglected. Knowledge on the mechanisms of alginate and whey protein interaction can serve for food product related purposes.
This thesis is written in two sections that both have alginate and whey proteins as the main topic, but while the first section concerns the fundamental science of the interaction, the second section has a more applied focus. Many of the findings in the first section also play a role in the second, illustrating the importance of understanding the basic chemistry in the more applied research field.
Section 1 comprises 1 paper and 2 manuscripts. Paper 1 focuses on describing how varying mannuronate and guluronate content (M/G ratio) in alginate affects the complexation with the model protein β-Lactoglobulin (β-Lg). Through isothermal titration calorimetry (ITC), dynamic light scattering (DLS) and turbidimetry we found that increased G content leads to less β-Lg binding, higher Kd and larger complexes with alginate. These effects were found at both pH 4.00 and pH 2.65, but were most prominent at pH 2.65. Especially the deprotonation of alginate with high G content hampered binding of β-Lg. This is due to alginate self-association in the initial acid gel formation phase, which is more prevalent for G blocks than MG or M blocks. Thorough analysis of ITC results, further made it possible to introduce a new stoichiometric term of the number of uronic acid residues bound to one molecule of β-Lg. This stoichiometric interpretation revealed that one molecule of β-Lg binds 16-20 uronic acid residues at pH 4.00.
Manuscript 1 is a molecular level interaction study, aimed to reveal where on β-Lg alginate oligosaccharides (AOSs) binds. Here AOSs of varying composition and length are used to form complexes with 15N labeled β-Lg that stay in solution, making it possible to determine which amino acid residues are affected when an AOS binds, by 1H,15N hetero singular quantum coherence NMR. The NMR data were supported by ITC and molecular modelling, resulting in a detailed description of the fuzzy binding of AOSs in recognized binding sites on β-Lg and with distinct lowest energy poses in the binding sites.
Manuscript 2 describes the difference in interaction of β-Lg and alginate between pH 4.00 and 2.65, as well as the influence of increased solvent ionic strength and heat treatment. It is found that complexation at pH 4.00 results in an uptake of protons from the solvent, as opposed to at pH 2.65 where it resulted in a release of protons to the solvent. At pH 3.00 there was little to no change in the proton concentration of the solvent. Thus variation in protonation state of alginate and the acidic side chains of β-Lg controls very different binding mechanisms. The change in proton concentration of the solvent during complex formation was quenched at high NaCl concentrations, however, this also reduced the turbidity thus indicating less complex formation. Temperature studies showed macromolecular changes in the complexes, resulting in formation of larger complexes at higher temperature. The interaction also affects protein stability and Tm decreased by 10°C in the presence of alginate. All reflecting a hydrophobic effect, where alginate stabilizes the unfolded β-Lg state.
Section 2 includes 2 manuscripts. Manuscript 3 describes the enzymatic cross-linking effect of whey protein or β-Lg on the interaction with alginate. Manuscript 4 addresses the effect of alginate on the in vitro digestion of native and cross-linked whey protein.
The enzymatic cross-linking in manuscript 3, leads to loss of tertiary structure of β-Lg, resulting in covalently linked poly-β-Lg with no defined tertiary structure as determined by fluorescence spectroscopic techniques. The loss of tertiary structure was linked to the degree of polymerization, as β-Lg oligomers with lesser degree of polymerization retained some tertiary structure. The interaction between alginate and the cross-linked product resulted in completely different enthalpy, Kd and stoichiometry than previously shown for the native protein. We further investigated this by using ITC at different temperatures and found that the interaction between cross-linked protein and alginate was driven by hydrophobic forces, instead of the conventional charge-charge interaction. DLS analysis of the alginate protein complexes revealed that cross-linking of protein prior to complexation resulted in smaller particles than for the non-cross-linked protein. Thus the same network was not achieved with enzymatically cross-linked protein as with native protein.
This was also found in manuscript 4, where complexes made with alginate of varying M/G ratio with both cross-linked and native whey protein were subjected to simulated in vitro gastro-intestinal digestion. Here the complex sizes depended both on the M/G ratio and whether protein was cross-linked or not, as also observed in paper 1 and manuscript 3. We used the gold standard protocol INFOGEST, to study in vitro protein digestion and the effect of alginate. The alginate was found to prevent protein degradation in the gastric phase (pH 3.0), where the degree of protection depends on the concentrations of alginate and protein. In the intestinal phase (pH 7.0) we did not observe protection of protein, agreeing with the observation that alginate and whey proteins do not form complexes under these conditions. Alginate moreover in the gastric phase (pH 3.0) protected crosslinked whey protein less than native whey protein, indicating that the change in binding and morphology makes the protein more accessible for the protease. Finally, we looked into the effect of adding an alginate lyase (that degrades alginate) in the intestinal phase, which showed a slight increase in protein degradation in the sample containing alginate lyase as also visualized by C-PAGE supporting alginate degradation.
This thesis brought together elements from the basic interaction mechanism between alginate and whey proteins, and put it in relation to eventual application in food production and gastro-intestinal digestion.
This thesis is written in two sections that both have alginate and whey proteins as the main topic, but while the first section concerns the fundamental science of the interaction, the second section has a more applied focus. Many of the findings in the first section also play a role in the second, illustrating the importance of understanding the basic chemistry in the more applied research field.
Section 1 comprises 1 paper and 2 manuscripts. Paper 1 focuses on describing how varying mannuronate and guluronate content (M/G ratio) in alginate affects the complexation with the model protein β-Lactoglobulin (β-Lg). Through isothermal titration calorimetry (ITC), dynamic light scattering (DLS) and turbidimetry we found that increased G content leads to less β-Lg binding, higher Kd and larger complexes with alginate. These effects were found at both pH 4.00 and pH 2.65, but were most prominent at pH 2.65. Especially the deprotonation of alginate with high G content hampered binding of β-Lg. This is due to alginate self-association in the initial acid gel formation phase, which is more prevalent for G blocks than MG or M blocks. Thorough analysis of ITC results, further made it possible to introduce a new stoichiometric term of the number of uronic acid residues bound to one molecule of β-Lg. This stoichiometric interpretation revealed that one molecule of β-Lg binds 16-20 uronic acid residues at pH 4.00.
Manuscript 1 is a molecular level interaction study, aimed to reveal where on β-Lg alginate oligosaccharides (AOSs) binds. Here AOSs of varying composition and length are used to form complexes with 15N labeled β-Lg that stay in solution, making it possible to determine which amino acid residues are affected when an AOS binds, by 1H,15N hetero singular quantum coherence NMR. The NMR data were supported by ITC and molecular modelling, resulting in a detailed description of the fuzzy binding of AOSs in recognized binding sites on β-Lg and with distinct lowest energy poses in the binding sites.
Manuscript 2 describes the difference in interaction of β-Lg and alginate between pH 4.00 and 2.65, as well as the influence of increased solvent ionic strength and heat treatment. It is found that complexation at pH 4.00 results in an uptake of protons from the solvent, as opposed to at pH 2.65 where it resulted in a release of protons to the solvent. At pH 3.00 there was little to no change in the proton concentration of the solvent. Thus variation in protonation state of alginate and the acidic side chains of β-Lg controls very different binding mechanisms. The change in proton concentration of the solvent during complex formation was quenched at high NaCl concentrations, however, this also reduced the turbidity thus indicating less complex formation. Temperature studies showed macromolecular changes in the complexes, resulting in formation of larger complexes at higher temperature. The interaction also affects protein stability and Tm decreased by 10°C in the presence of alginate. All reflecting a hydrophobic effect, where alginate stabilizes the unfolded β-Lg state.
Section 2 includes 2 manuscripts. Manuscript 3 describes the enzymatic cross-linking effect of whey protein or β-Lg on the interaction with alginate. Manuscript 4 addresses the effect of alginate on the in vitro digestion of native and cross-linked whey protein.
The enzymatic cross-linking in manuscript 3, leads to loss of tertiary structure of β-Lg, resulting in covalently linked poly-β-Lg with no defined tertiary structure as determined by fluorescence spectroscopic techniques. The loss of tertiary structure was linked to the degree of polymerization, as β-Lg oligomers with lesser degree of polymerization retained some tertiary structure. The interaction between alginate and the cross-linked product resulted in completely different enthalpy, Kd and stoichiometry than previously shown for the native protein. We further investigated this by using ITC at different temperatures and found that the interaction between cross-linked protein and alginate was driven by hydrophobic forces, instead of the conventional charge-charge interaction. DLS analysis of the alginate protein complexes revealed that cross-linking of protein prior to complexation resulted in smaller particles than for the non-cross-linked protein. Thus the same network was not achieved with enzymatically cross-linked protein as with native protein.
This was also found in manuscript 4, where complexes made with alginate of varying M/G ratio with both cross-linked and native whey protein were subjected to simulated in vitro gastro-intestinal digestion. Here the complex sizes depended both on the M/G ratio and whether protein was cross-linked or not, as also observed in paper 1 and manuscript 3. We used the gold standard protocol INFOGEST, to study in vitro protein digestion and the effect of alginate. The alginate was found to prevent protein degradation in the gastric phase (pH 3.0), where the degree of protection depends on the concentrations of alginate and protein. In the intestinal phase (pH 7.0) we did not observe protection of protein, agreeing with the observation that alginate and whey proteins do not form complexes under these conditions. Alginate moreover in the gastric phase (pH 3.0) protected crosslinked whey protein less than native whey protein, indicating that the change in binding and morphology makes the protein more accessible for the protease. Finally, we looked into the effect of adding an alginate lyase (that degrades alginate) in the intestinal phase, which showed a slight increase in protein degradation in the sample containing alginate lyase as also visualized by C-PAGE supporting alginate degradation.
This thesis brought together elements from the basic interaction mechanism between alginate and whey proteins, and put it in relation to eventual application in food production and gastro-intestinal digestion.
Original language | English |
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Place of Publication | Kgs. Lyngby, Denmark |
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Publisher | DTU Bioengineering |
Number of pages | 241 |
Publication status | Published - 2021 |
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Dive into the research topics of 'Whey Protein Alginate Complexation: A Multifaceted Interaction, Tuneable for Application'. Together they form a unique fingerprint.Projects
- 1 Finished
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Structure and Design of Whey Protein Alginate Complexes
Madsen, M. (PhD Student), Imberty, A. (Examiner), Nicolai, T. (Examiner), Svensson, B. (Main Supervisor), Kragelund, B. B. (Supervisor), Aachmann, F. L. (Supervisor) & Büll, A. (Examiner)
01/05/2018 → 08/11/2021
Project: PhD