Rooibos fermentation - characterising phenolic changes using chemometric analysis and kinetic modelling

[摘要] ENGLISH ABSTRACT: Rooibos is a popular herbal tea produced from Aspalathus linearis, a South African endemic fynbos plant species. Health-promoting properties associated with rooibos are closely linked to its phenolic composition. 'Fermentation' (oxidation) is essential to the development of characteristic sensory attributes, including colour, of traditional fermented rooibos, however oxidation reactions are detrimental to the quality of green rooibos. In this study, advanced chemometric methods were used to uncover the phenolic changes occurring in rooibos during fermentation in an unbiased manner. Chromatographic fingerprints of green, semi-fermented and fermented rooibos extracts were constructed using a recently improved high-performance liquid chromatography with diode-array detection (HPLC-DAD) method. Pre-processing techniques were applied to eliminate unwanted variation that may be related to the nature of the data or the analytical instrument used, i.e. signal enhancement (baseline elimination and signal denoising), peak alignment, peak detection and integration, normalisation, centering and transformation. The 'size effect', an artefact related to the overall differences in concentration between samples, was eliminated by application of a new normalisation method called robust pair-wise log ratios (rPLR). rPLR also allowed for easy identification of phenolic compounds showing significant (p<0.01) changes between treatments. Results showed that all 56 peaks detected were significantly (p<0.01) changing between green, semi-fermented and fermented rooibos. A 30% change in peak area threshold was used to ensure that the selected markers showed reasonable degradation, which reduced their number to 34. Given that this is still a relatively large number of compounds, the number of phenolics examined in targeted analysis of rooibos fermentation is small in comparison to the number of compounds significantly changing. To gain insight into the reaction kinetics of rooibos phenolics during elevated temperatures and humid conditions, rooibos plant material was subjected to a 6 h simulated fermentation under highly controlled conditions, at four different temperatures between 37 and 50°C. Preceding the determination of the temperature dependence, two preliminary experiments were performed. Firstly, an extraction optimisation experiment showed that heating is not necessary for extraction and that a 5 min sonication period was sufficient. It was also necessary to investigate the role of endogenous rooibos enzymes since no direct evidence of their involvement in fermentation was available. For the second experiment, a dry heat treatment (170°C for 30 min) prior to simulated fermentation was postulated to partially inactivate the enzymes based on aspalathin and nothofagin content changes and differences in reaction rate constants between the control and heat-treated plant material. The fractional conversion reaction kinetics model, based on first-order kinetics, best described the degradation of aspalathin and nothofagin, as well as the formation of four eriodictyol-glucoside isomers, during simulated fermentation. The kinetic parameters estimated from the model (i.e. reaction rate constant and equilibrium concentration) suggested that the reactions proceeded faster, but, achieved a lower extent of degradation/formation at higher temperatures. Based on these results, it was postulated that enzymes were partially inactivated at higher fermentation temperatures or that there was a limiting factor during the simulated fermentation, e.g. oxygen depletion.

[发布机构] Stellenbosch University