Towards understanding the role of heat-induced structural changes on immunoreactivity and digestibility of cow’s milk protein

This thesis focused on the physicochemical changes that occur upon heating of cow’s milk protein (MP) and connect this to their possible effects on immunoreactivity. The underlying hypothesis of this work was that 3D-modifications of the protein structure that occur upon dry heating as well as the Maillard reaction (MR), in particular the formation of advanced glycation end products (AGEs), affect the immunoreactivity and digestibility of the protein.

In chapter 2, a mixture of casein and whey protein was dry-heated in the presence of the reducing milk sugar lactose at high temperature and low temperature to assess the impact of aggregation and MR vs. minimal aggregation and MR on protein digestibility. Both heat treatments resulted in loss of solubility. Therefore, investigations on the inter- and intramolecular interactions that lead to insolubility were performed to determine their role in both the decrease of protein solubility as well as digestibility. Results indicated that mainly caseins end up in the insoluble fraction. Different inter- and intramolecular interactions were responsible for the loss of solubility after both heat treatments. Among all changes occurring, after high temperature heating, both MR-induced crosslinking and other mechanisms of crosslinking were suggested to play a crucial role in decreasing both solubility and digestibility of MP. This also resulted in the release of larger particles from the insoluble material upon gastric digestion and a slower degradation in the intestinal phase. This, as well as the high levels of AGEs, was proposed to affect immunoreactivity of MP.

To disentangle the role of aggregation and the MR on immunoreactivity, chapter 3 used a simplified model system containing only isolated β-lactoglobulin (BLG). Differential heat treatments comparing low vs. high temperature, dry vs. wet heating, as well as presence and absence of lactose were applied. By using a combination of western blot and ELISA assays, the binding of immunoglobulin E (IgE) as well as the soluble form of the receptor for AGEs (sRAGE) towards the different protein structures (high and low molecular weight) that are visible on the SDS-PAGE was determined. Results showed that protein aggregation plays an important role in the formation of IgE-binding epitopes and sRAGE binding ligands. While high individual differences between patients did not allow a clear distinction between the effect of aggregation and glycation on IgE binding, it was observed that sRAGE binding strongly increased in the presence of lactose. The promoting effect of glycation on protein aggregation as well as the formation of glycation structures were found as possible explanations for this. In contradiction to literature, sRAGE binding was not observed to all structures visible on the SDS-PAGE, that contained Nɛ-carboxymethyllysine (CML). Moreover, it is unclear whether these ligands would encounter the immune system in the gastrointestinal tract in the same form after in vitro digestion.

In chapter 4, BLG was chemically modified to create CML-BLG, to gain more insights in the role of CML in binding to three different AGE receptors: sRAGE, galectin-3 (Gal-3), and CD36. By using this approach, other 3D-modifications of the protein were minimised and allowed a better assessment of the specific role of CML. This was additionally compared to low temperature glycated BLG. Results showed that protein-bound CML was indeed a ligand for AGE receptors which was related to the negative charge of CML. At the same time, binding to low temperature glycated BLG seemed to be related to its increasing hydrophobicity, however results also suggested a possible role of other MR products which may have affected the charge state of the protein and thus its binding to AGE receptors.

The immunological mechanisms following the binding of heated and glycated BLG to AGE receptors was studied in chapter 5. BLG wet heated in the presence and absence of two reducing sugars (glucose and lactose) was incubated with THP-1-derived macrophages and monocyte-derived dendritic cells, respectively. The receptor binding was assessed using ELISA and the role of different AGE receptors in internalisation as well as cytokine production was measured. Results showed that especially aggregation led to the formation of AGE-binding ligands and internalisation via CD36, Gal-3 and scavenger receptor class A type I by both cell types. After in vitro digestion of heated and glycated BLG, binding to sRAGE and Gal-3 was higher to the digest of glycated BLG compared to other samples. This suggested that even after digestion, glycated milk protein could still trigger immunological responses that could possibly lead to T-cell activation. Moreover it was suggested that immnological responses that are induced by binding to AGE receptors occur via different pathways, with sRAGE functioning in signalling pathways and Gal-3 as a putative receptor responsible for antigen uptake.

Next to aggregates that survive in vitro digestion, also digestion-derived peptides can carry immunoreactive structures. In chapter 6, peptides generated after simulated infant in vitro digestion of dry-heated MP were monitored. sIgE binding epitopes were identified based on literature, while potential T-cell epitopes were predicted using a software tool. Similar numbers of epitopes were found in all samples, however in heated samples more of these peptides were glycated. This suggested that epitope recognition could be altered in heated samples. Overall, relatively more glycated peptides were generated in heated samples and Caco-2 cell experiments indicated a preferred transport of especially lactosyllysine and glucosyllysine- modified peptides, but also CML-modified peptides in high temperature heated MP. This suggests that relatively more AGE receptor ligands are available in heated samples. On the other hand, only a low number of peptides carrying an epitope structure were transported, suggesting that the availability of T-cell and sIgE binding epitopes following transport via normal enterocytes might be limited.

This thesis showed that several heat-induced structural modifications e.g. increased hydrophobicity, protein aggregation, and presence of AGEs, can affect immunoreactivity of MP. Moreover, it suggested that especially those immunoreactive structures that are able to survive gastrointestinal digestion could also modulate the immunological responses in vivo.