Lysine blockage of milk proteins in infant formula impairs overall protein digestibility and peptide release

During heat processing of milk and dairy products, for example infant formula, the Maillard reaction occurs. In vitro and animal studies suggest that Maillard reaction induced lysine blockage impairs protein digestibility. Most studies that investigate the effect of glycation on protein digestion use a mixture of isolated milk protein with reducing sugars. In this study, infant formulas with 6.5%, 8.4%, 11.2%, 14.8%, 20.8%, and 44.5% of blocked lysine (BL) were digested in an in vitro infant digestion model and tested for protein hydrolysis and peptide release. OPA (o-phthalaldehyde) assay was used to assess the degree of protein hydrolysis. SDS-PAGE was conducted to monitor the hydrolysis of specific proteins. Peptides formed after gastric and intestinal digestion were identified by LC/MS. Protein hydrolysis of the 6.5% BL sample was significantly higher after 10 minutes of intestinal digestion compared to all other samples. Most differences were observed after intestinal digestion. A significant change in peptide patterns was observed for the 45% BL sample resulting in a relatively higher number of peptides with more than 14 amino acids. Mainly casein-derived peptides were affected. Overall, the average peptide length was significantly increased for the 44.5% BL glycated product (on average 10.2 amino acids for 6-21% BL vs. 11.4 amino acids for 45% BL; p < 0.001). In conclusion, glycation of milk proteins in an infant formula product can impair overall protein digestibility. These findings emphasize the importance of mild processing and having low BL levels in infant formula to ensure optimal digestion of proteins.</p

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&rsquo;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 &beta;-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

No glycation required : Interference of casein in age receptor binding tests

For the determination of the binding of heated cow’s milk whey proteins such as β-lactoglobulin to the receptors expressed on immune cells, inhibition ELISA with the soluble form of the receptor for advanced glycation end products (sRAGE) and scavenger receptor class B (CD36) has been successfully used in the past. However, binding to heated and glycated caseins in this read-out system has not been tested. In this study, inhibition ELISA was applied to measure the binding of cow’s milk casein alone, as well as all milk proteins together, which underwent differential heat treatment, to sRAGE and CD36, and we compared those results to a dot blot read out. Moreover, binding to sRAGE and CD36 of differentially heated milk protein was measured before and after in vitro digestion. Casein showed binding to sRAGE and CD36, independent from the heat treatment, in ELISA, while the dot blot showed only binding to high-temperature-heated milk protein, indicating that the binding is not related to processing but to the physicochemical characteristics of the casein. This binding decreased after passage of casein through the intestinal phase

The effect of low vs. high temperature dry heating on solubility and digestibility of cow's milk protein

Dry heating of cow's milk protein in the presence of the milk sugar lactose leads to a loss of solubility and digestibility. Most studies that investigated the loss of solubility in milk protein powders suggested that, besides structural changes, hydrophobic interaction, hydrogen bonds, disulphide bonds, and Maillard reaction-induced crosslinking are responsible for this. However, little is known about the direct contribution of these inter- and intramolecular interactions on loss of solubility and protein digestibility. Low temperature (60 °C) and high temperature (130 °C) dry heating of cow's milk protein in the presence of lactose was applied after which both the soluble and insoluble fractions were analysed with SDS-PAGE and LC-MS/MS. The Maillard reaction was monitored by quantification of Nε-carboxymethyllysine, Nε-carboxyethyllysine, and pentosidine with LC-MS/MS. Loss of solubility was analysed with solvent solubility tests. Protein hydrolysis after simulated infant in vitro digestion, and after hydrolysis with single enzymes, was monitored using SDS-PAGE and the o-phthaldialdehyde assay. The results indicated that caseins are the main proteins that become insoluble upon dry heating, independent of the heating temperature. The decreased solubility of low temperature dry heated cow's milk protein is induced by hydrogen bonds and hydrophobic interactions and did not impair protein hydrolysis. At the same time, covalent protein crosslinking is an important determinant in protein solubility and digestibility of high temperature dry heated cow's milk protein.</p

Lysine blockage of milk proteins in infant formula impairs overall protein digestibility and peptide release

During heat processing of milk and dairy products, for example infant formula, the Maillard reaction occurs. In vitro and animal studies suggest that Maillard reaction induced lysine blockage impairs protein digestibility. Most studies that investigate the effect of glycation on protein digestion use a mixture of isolated milk protein with reducing sugars. In this study, infant formulas with 6.5%, 8.4%, 11.2%, 14.8%, 20.8%, and 44.5% of blocked lysine (BL) were digested in an in vitro infant digestion model and tested for protein hydrolysis and peptide release. OPA (o-phthalaldehyde) assay was used to assess the degree of protein hydrolysis. SDS-PAGE was conducted to monitor the hydrolysis of specific proteins. Peptides formed after gastric and intestinal digestion were identified by LC/MS. Protein hydrolysis of the 6.5% BL sample was significantly higher after 10 minutes of intestinal digestion compared to all other samples. Most differences were observed after intestinal digestion. A significant change in peptide patterns was observed for the 45% BL sample resulting in a relatively higher number of peptides with more than 14 amino acids. Mainly casein-derived peptides were affected. Overall, the average peptide length was significantly increased for the 44.5% BL glycated product (on average 10.2 amino acids for 6-21% BL vs. 11.4 amino acids for 45% BL; p < 0.001). In conclusion, glycation of milk proteins in an infant formula product can impair overall protein digestibility. These findings emphasize the importance of mild processing and having low BL levels in infant formula to ensure optimal digestion of proteins.</p

Peptide release after simulated infant in vitro digestion of dry heated cow’s milk protein and transport of potentially immunoreactive peptides across the caco-2 cell monolayer

Dry heating of cow’s milk protein, as applied in the production of “baked milk”, facilitates the resolution of cow’s milk allergy symptoms upon digestion. The heating and glycation-induced changes of the protein structure can affect both digestibility and immunoreactivity. The immunological consequences may be due to changes in the peptide profile of the digested dry heated milk protein. Therefore, cow’s milk protein powder was heated at low temperature (60 °C) and high temperature (130 °C) and applied to simulated infant in vitro digestion. Digestion-derived peptides after 10 min and 60 min in the intestinal phase were measured using LC-MS/MS. Moreover, digests after 10 min intestinal digestion were applied to a Caco-2 cell monolayer. T-cell epitopes were analysed using prediction software, while specific immunoglobin E (sIgE) binding epitopes were identified based on the existing literature. The largest number of sIgE binding epitopes was found in unheated samples, while T-cell epitopes were equally represented in all samples. Transport of glycated peptide indicated a preference for glucosyl lysine and lactosyl-lysine-modified peptides, while transport of peptides containing epitope structures was limited. This showed that the release of immunoreactive peptides can be affected by the applied heating conditions; however, availability of peptides containing epitopes might be limited

Receptor Mediated Effects of Advanced Glycation End Products (AGEs) on Innate and Adaptative Immunity : Relevance for Food Allergy

As of late, evidence has been emerging that the Maillard reaction (MR, also referred to as glycation) affects the structure and function of food proteins. MR induces the conformational and chemical modification of food proteins, not only on the level of IgG/IgE recognition, but also by increasing the interaction and recognition of these modified proteins by antigen-presenting cells (APCs). This affects their biological properties, including digestibility, bioavailability, immunogenicity, and ultimately their allergenicity. APCs possess various receptors that recognize glycation structures, which include receptor for advanced glycation end products (RAGE), scavenger receptors (SRs), galectin-3 and CD36. Through these receptors, glycation structures may influence the recognition, uptake and antigen-processing of food allergens by dendritic cells (DCs) and monocytes. This may lead to enhanced cytokine production and maturation of DCs, and may also induce adaptive immune responses to the antigens/allergens as a result of antigen uptake, processing and presentation to T cells. Here, we aim to review the current literature on the immunogenicity of AGEs originating from food (exogenous or dietary AGEs) in relation to AGEs that are formed within the body (endogenous AGEs), their interactions with receptors present on immune cells, and their effects on the activation of the innate as well as the adaptive immune system. Finally, we review the clinical relevance of AGEs in food allergies

Binding of CML-Modified as Well as Heat-Glycated β-lactoglobulin to Receptors for AGEs Is Determined by Charge and Hydrophobicity

Intake of dietary advanced glycation end products (AGEs) is associated with inflammation-related health problems. Nε-carboxymethyl lysine (CML) is one of the best characterised AGEs in processed food. AGEs have been described as ligands for receptors present on antigen presenting cells. However, changes in protein secondary and tertiary structure also induce binding to AGE receptors. We aimed to discriminate the role of different protein modifications in binding to AGE receptors. Therefore, β-lactoglobulin was chemically modified with glyoxylic acid to produce CML and compared to β-lactoglobulin glycated with lactose. Secondary structure was monitored with circular dichroism, while hydrophobicity and formation of β-sheet structures was measured with ANS-assay and ThT-assay, respectively. Aggregation was monitored using native-PAGE. Binding to sRAGE, CD36, and galectin-3 was measured using inhibition ELISA. Even though no changes in secondary structure were observed in all tested samples, binding to AGE receptors increased with CML concentration of CML-modified β-lactoglobulin. The negative charge of CML was a crucial determinant for the binding of protein bound CML, while binding of glycated BLG was determined by increasing hydrophobicity. This shows that sRAGE, galectin-3, and CD36 bind to protein bound CML and points out the role of negatively charged AGEs in binding to AGE receptors.</p

Amino Acid Modifications during the Production (Shearing, Sterilization) of Plant-Based Meat Analogues : An Explorative Study Using Pet Food Production as an Example

Shear cell technology is a promising method for the production of meat analogues. Meat analogues are also studied as alternative proteins for dogs and cats, which require high-quality protein. This study monitored the effect of shearing, using shear cell technology, and sterilization (26 min at 125.5 °C) on selected amino acids, advanced glycation end products (AGEs), lysinoalanine, o-phthalaldehyde-reactive lysine, as well as oxidation markers, free thiols, and dityrosine, in soy protein- and pea protein-based meat analogues. These are compared with animal-based pet foods. Processing resulted in modified amino acids, especially cysteine. Reductions in amino acid levels were higher in the soy-based meat analogue, but markers indicated more pronounced oxidation in the pea-based meat analogue. AGEs and lysinoalanine were not formed on shearing, only during sterilization. Despite extensive thermal treatment, the effects of processing on the protein quality of plant-based products were comparable or less than those in animal-based pet food