Lipid oxidation induced protein scission in an oleogel as a model food.

Lipid oxidation induced protein scission was investigated in oleogel using beta-lactoglobulin (whey protein isolate) as gelator. Extracted cleaved peptides were measured using high resolution mass spectrometry (FT-ICR-MS), which was provided by an automatically generated annotation list approach to identify relevant masses and sum formula using the isotopic pattern. The identified oxidized peptides were then further evaluated using partial least squares regression to relevant lipid hydroperoxide formation data, which provide the significance and importance of the peptides toward lipid induced scission. Thereby, the most important peptides are located at the surface of the protein in random coil segments and especially at the ends of the protein sequence. The most important amino acids were cysteine and aliphatic amino acids, which undergo scission mostly by the α-amidation pathway. The findings compare well with studies investigating depletion of amino acids initiated by lipid oxidation in systems containing bovine albumin or gamma-globulin.

Changes in Protein Fluorescence in a Lipid-Protein Co-oxidizing Oleogel.

High and low levels of lipid-induced protein oxidation (tuned by the addition of 0%-8.4% water) were investigated in oleogels, using excitation-emission matrix (EEM) fluorescence spectroscopy, coupled with a partial least-squares (PLS) regression and lipid hydroperoxide data. In high-level oxidation models, the intrinsic tryptophan fluorescence decreased and the emission maxima increased from 352.5 to 356.0 nm indicating the presence of protein modifications, which was further supported by size-exclusion chromatography. PLS recognized 3 latent components, with several excitation-emission points of interest. These apparent compounds include a region associated with radical mediated protein modifications (approximately 325 and 410 nm), lipid oxidation product adducts (approximately 350 nm and 420-425 nm), and malondialdehyde adducts (approximately 375 and 425 nm). The separate evaluation of these apparent compounds, at a 420 nm emission, indicated that lipid oxidation promotes protein lipid adduct fluorescence at high water levels, rather than radical mediated protein fluorescence.

Cooxidation of proteins and lipids in whey protein oleogels with different water amounts.

Protein- and lipid oxidation were investigated in whey protein based oleogels with varying water addition. Lipid oxidation was low (~30 mmol O2/kg lipid hydroperoxides after 6 weeks) in gels with < 0.23% water and a high (>1,000 mmol O2/kg lipid hydroperoxides after 4 weeks) in gels with > 2.4% water addition. In systems with > 2.4% water addition fluorescence (excitation 325 nm / emission 410 nm) as indicator of tyrosine oxidation and carbonyl content significantly increased and remained at low levels in oleogels with < 0.23% water addition. Primary amines as indicator for protein backbone breakage increased in early stages of oxidation in high water oleogels and decreased after 28 days. Degradation has been suggested to occur through interactions with reactive secondary lipid oxidation products and was confirmed by spiking experiments using respective compounds. The results suggest that secondary lipid oxidation markers are masked dependent on water addition in the presence of proteins.

Protein oxidation during temperature-induced amyloid aggregation of beta-lactoglobulin.

Although the connection between protein oxidation, amyloid aggregation and diseases such as Alzheimer's is well known there is no information on such effects during preparation of beta-lactoglobulin fibrils. Different morphologies of amyloid aggregates of beta-lactoglobulin were prepared by incubation at pH 2 or pH 3.5 for up to 72 h. After 5 h, amyloid aggregates at pH 2 formed typical fibrils, which consisted of peptides. At pH 3.5, the amyloid aggregates were worm-like and consisted of intact protein. After 72 h, the building blocks at both pH values changed towards smaller peptides. The apparent tyrosine oxidation reached a maximum after 5 h at both pH values, whereas N-formylkynurenine and carbonyls increased continuously during 72 h. In case amyloid structures are used as edible material, the health related effects caused by protein oxidation needs to be considered.

Structure-reactivity relationship of Amadori rearrangement products compared to related ketoses.

Structure-reactivity relationships of Amadori rearrangement products compared to their related ketoses were derived from multiple NMR spectroscopic techniques. Besides structure elucidation of six Amadori rearrangement products derived from d-glucose and d-galactose with l-alanine, l-phenylalanine and l-proline, especially quantitative (13)C selective saturation transfer NMR spectroscopy was applied to deduce information on isomeric systems. It could be shown exemplarily that the Amadori compound N-(1-deoxy-d-fructos-1-yl)-l-proline exhibits much higher isomerisation rates than d-fructose, which can be explained by C-1 substituent mediated intramolecular catalysis. In combination with a reduced carbonyl activity of Amadori compounds compared to their related ketoses which results in an increased acyclic keto isomer concentration, the results on isomerisation dynamics lead to a highly significant increased reactivity of Amadori compounds. This can be clearly seen, comparing approximated carbohydrate milieu stability time constants (ACuSTiC) which is 1 s for N-(1-deoxy-d-fructos-1-yl)-l-proline and 10 s for d-fructose at pD 4.20 ± 0.05 at 350 K. In addition, first NMR spectroscopic data are provided, which prove that α-pyranose of (amino acid substituted) d-fructose adopts both, (2)C5 and (5)C2 conformation.