Racemization assessment in alkali treated dietary proteins using high-performance liquid chromatography

Abstract

Racemization of amino acids was studied in alkali treated β-casein, oat protein (Avena sativa) and rapeseed protein (Brasica napus). Amino acid hydrolyzates containing enantiomeric amino acids, L-Xxx and D-Xxx, were converted into mixtures of N-ethoxycarbonyl-(Eoc-) derivatized diastereomeric dipeptide pairs, Eoc-Phe-L-Xxx, Eoc-Phe-D-Xxx and Eoc-Val-L-Xxx, Eoc-Val-D-Xxx upon the reaction with ethoxycarbonylphenylalanine N-hydroxysuccinimide ester (Eoc-Phe-ONSU) and ethoxycarbonylvaline N-hydroxysuccinimide ester (Eoc-Val-ONSU), respectively. The epimeric products were separated and determined by reversed phase high-performance liquid chromatography and the content of D-isomers was calculated. Racemization of β-casein using this method was in agreement with gas chromatography-mass spectrometry techniques described in the literature. In oat and rapeseed proteins exposed to alkaline conditions for 3 and 24 hours in 0.1 N NaOH at 65°C, the extend of racemization followed the sequence Ser>Asp>Phe>Glu>Val in most of the samples. In preliminar experiments, alkali-treated sunflower seed protein (Helian-thus annuus) samples were examined for the content of D-Phe. The reported HPLC technique represents a simple method for a practical assessment of racemization in dietary proteins.