Bovine α-lactalbumin (BLA) is a calcium ion binding protein with high nutritional value and can be widely found in dairy products. As an immediate response mediated by immunoglobulin E (IgE), the allergic response signs to BLA occur within minutes of exposure. It is estimated that 15% of the population suffer from these allergic reactions. Different methods including heating, phosphorylation, and lactic acid fermentation have been used to reduce the allergenicity of BLA. BLA is digested into peptides in digestive system and gastrointestinal tract is the main route of exposure to food allergens. Therefore, it is important to study the effects of gastrointestinal digestion on the allergenicity of BLA. It was previously reported that phosphorylation can reduce the allergenicity of BLA, which was explained by the masking of the allergic epitopes from negatively charged phosphate group. All of these explains the importance of studying the effect of phosphorylation on allergenicity of BLA.

To this end, the digestive products of BLA were isolated, separated with LC-MS and the phosphorylation sites were identified using PEAKS.

The results showed that phosphorylation before gastrointestinal digestion can reduce the allergic reaction of BLA significantly. It is suggested that this significant reduction is resulted from the combination of change in both molecular weight and conformational structure.

How was PEAKS used?

The peptides and modified sites initially separated using Nano-LC-ESI-Q-Orbitrap-MS/MS. Positive ion mode, mass range of m/z 250–1250, fragmentation mode of HCD, and fragmentation energy of 27% were chosen for this study and the top 20 peptides were selected for fragmentation according to the signal intensity of MS. PEAKS DB function module software was used to process the MS/MS data and analyse peptides.

Chen, Wen-mei, et al. “Simulated in vitro digestion of α-lactalbumin modified by phosphorylation: Detection of digestive products and allergenicity.” Food Chemistry 372 (2022): 131308. https://doi.org/10.1016/j.foodchem.2021.131308

Abstract

The effects of phosphorylation on the allergenicity of bovine α-lactalbumin (BLA) and digestive products were studied in vitro digestion. Two components with different molecular weight and conformation were obtained from natural and phosphorylated BLA. In vivo and in vitro assessment of allergenicity showed that phosphorylation prior to digestion significantly decreased the IgE/IgG binding capacity and allergic response in KU812 cells, and reduced the levels of IgG, IgE, IL-4 and histamine, with an increase in IFN-γ levels in mouse serum, depending on the changes in BLA structures, producing numerous small peptides. There were four phosphorylated sites (S22, T29, S47 and S70) in the high molecular weight components of phosphorylated BLA after digestion. These phosphorylated sites could mask the linear epitopes of digestive products, resulting in reduced allergic activity. Phosphorylation prior to digestion of dairy products can reduce the risk of anaphylaxis in patients with milk allergy to some extent.