Joseph James Kehoe

Tryptophan-Mediated Denaturation of β-Lactoglobulin A by UV Irradiation

beta-Lactoglobulin A, a genetic variant of one of the main whey proteins, was irradiated at 295 nm for 24 h. After irradiation, 18% of the protein was denatured (determined by reverse-phase chromatography). The fluorescence spectrum of the irradiated protein was red-shifted compared to that of the native protein, indicating a change in protein folding. Sulfhydryl groups, which are buried in native beta-lactoglobulin, were exposed following irradiation and became available for quantification using the Ellman assay. The quantity of exposed sulfhydryls increased, but the number of total sulfhydryl groups decreased. Gel permeation chromatography showed that some protein aggregation occurred during irradiation. Fourier transform infrared (FTIR) spectroscopy of irradiated beta-lactoglobulin revealed changes in the secondary structure, comparable to that of early events during heat-induced denaturation. There was evidence for some photo-oxidation of tryptophan.

Heat-Induced Denaturation, Aggregation and Gelation of Whey Proteins

Whey proteins, a group of acid-soluble proteins, represent approximately 20 % of the total protein in bovine milk. The two main proteins, β-lactoglobulin (18.3 kDa) and α-lactalbumin (14.2 kDa) have been the subject of numerous studies. The purpose has mostly been to elucidate and exploit potential structure/function relationships. Both proteins provide high nutritional value and are utilised in the production of nutritional beverages such as infant formula and energy drinks. In addition, β-lactoglobulin offers a range of useful techno-functional properties, such as thickening, emulsification, gelation or foaming. Exposure of whey proteins to heat is a common industrial processing step that causes structural changes in proteins and that can lead to increases in viscosity and/or formation of potentially extensive gel networks above a critical protein concentration. This chapter describes such heat-induced unfolding, denaturation and aggregation processes, and their kinetics, as well as cases of ordered protein structures such as fibrils or nanoparticles. Heat-induced and cold gelation of whey proteins is also described. Gel formation is brought about by the assembly of soluble aggregates formed during the initial stages of heating. Such gel networks develop through electrostatic, hydrophobic and covalent interactions between denatured whey proteins. The micro- and macro-structure of whey protein gels vary widely and are dependent on the nature of the aggregation processes involved. The influence of protein type and concentration, salt type and ionic strength, pH and heating conditions on the above processes are reviewed.

Gastric digestion of α-lactalbumin in adult human subjects using capsule endoscopy and nasogastric tube sampling

In the present study, structural changes in the milk protein α-lactalbumin (α-LA) and its proteolysis were investigated for the potential formation of protein-fatty acid complexes during in vivo gastric digestion. Capsule endoscopy allowed visualisation of the digestion of the test drinks, with nasogastric tubes allowing sampling of the gastric contents. A total of ten healthy volunteers had nasogastric tubes inserted into the stomach and ingested test drinks containing 50 g/l of sucrose and 25 g/l of α-LA with and without 4 g/l of oleic acid (OA). The samples of gastric contents were collected for analysis at 3 min intervals. The results revealed a rapid decrease in the pH of the stomach of the subjects. The fasting pH of 2·31 (SD 1·19) increased to a pH maxima of pH 6·54 (SD 0·29) after ingestion, with a subsequent decrease to pH 2·22 (SD 1·91) after 21 min (n 8). Fluorescence spectroscopy and Fourier transform IR spectroscopy revealed partial protein unfolding, coinciding with the decrease in pH below the isoelectric point of α-LA. The activity of pepsin in the fasting state was found to be 39 (SD 12) units/ml of gastric juice. Rapid digestion of the protein occurred: after 15 min, no native protein was detected using SDS-PAGE; HPLC revealed the presence of small amounts of native protein after 24 min of gastric digestion. Mirocam® capsule endoscopy imaging and video clips (see the online supplementary material) revealed that gastric peristalsis resulted in a heterogeneous mixture during gastric digestion. Unfolding of α-LA was observed during gastric transit; however, there was no evidence of a cytotoxic complex being formed between α-LA and OA.