Analysis and stability of fatty acid esterified xanthophylls from microalgae

Fatty acid esterified xanthophylls (e.g. astaxanthin) produced by microalgae are regarded as a natural alternative for food colourants, but little is known on the stability of these compounds in foods. The aims of this research were (i) to develop protocols to analyze esterified xanthophylls, and their degradation products, from microalgae by UHPLC-PDA-ESI-MS and MALDI-TOF-MS, and (ii) to assess whether esterification influences xanthophyll stability. Both analytical methods were successfully tested on complex palmitoyl astaxanthin mixtures of Haematococcus pluvialis. MALDI-TOF-MS analysis showed that sodiated [M+Na]+ palmitoyl astaxanthin parents had higher ionization efficiency compared to the radical/protonated parents [M]+•/[M+H]+. Furthermore, TOF/TOF fragmentation of [M+Na]+ parents yielded more diagnostic daughter ions than for [M]+•/[M+H]+ parents. UHPLC-PDA-ESI-MS was applied to investigate xanthophyll biosynthesis in Chlorella zofingiensis. Upon nitrogen depletion, C. zofingiensis produced, amongst other pigments, ketolutein and its fatty acid ester. Ketolutein production was independent of the production of secondary β,β-carotene derivatives. Furthermore, UHPLC-PDA-ESI-MS was used to assess the oxidation products of palmitoyl astaxanthins and the influence of the fatty acid ester on colour stability. Mild light-accelerated autoxidation led to formation of free apo-astaxanthins and a new class of palmitoyl apo-astaxanthins. Harsh hypochlorite bleaching revealed (palmitoyl) epoxide-apo-9- and 13-astaxanthinones. Similarly, the methine bridges in the tetrapyrrole biliverdin were found to be susceptible to autoxidation. An increase in palmitate esterification enhanced stability of all-trans-astaxanthin upon light-accelerated autoxidation in hexane. Esterification appeared to modulate the cis-trans equilibrium of astaxanthin, which seems to play a key role in its stability.