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

Tobin, Jade (2018-03)

Thesis (MSc Food Sc)--Stellenbosch University, 2018.

Thesis

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.

AFRIKAANSE OPSOMMING: Rooibos is ‘n gewilde kruie-tee wat van Aspalathus linearis, ‘n endemiese Suid-Afrikaanse fynbos plantspesie, geproduseer word. Die gesondheidsvoordele van rooibos is geassosieer met sy fenoliese samestelling. “Fermentasie” (oksidasie) van die plantmateriaal is van kardinale belang vir die ontwikkeling van tradisionele rooibos se karakteristieke sensoriese eienskappe, inlsuitend kleur, maar oksidasiereaksies is nadelig vir die kwaliteit van groen rooibos. Chemometriese metodes is in hierdie studie gebruik om die fenoliese veranderinge wat tydens fermentasie plaasvind op ‘n onsydige manier te onthul. Chromatografiese vingerafdrukke van groen, semi-fermenteerde en gefermenteerde rooibos ekstrakte is saamgestel deur van ‘n onlangs verbeterde hoë druk vloeistof chromatografiese metode met ultraviolet-diode deteksie (HPLC-DAD) gebruik te maak. Data prosesseringstegnieke is toegepas on ongewensde variasie verwant aan die inherente eienskappe van die data of die analitiese instrument wat gebruik is uit te skakel. Dit het seinversterking (verwydering vab agtergrond en seingeraas), piek belyning, piek deteksie en integrasie, normalisering, sentrering en transformasie behels. Die “grootte effek”, wat ‘n gevolg is van oorhoofse verskille in konsentrasie tussen monsters, is ge-elimineer deur gebruik te maak van ‘n nuwe normaliseringsmetode bekend as robuuste paargewyse logaritmiese verhoudings (rPLR). Hierdie tegniek het ook die identifisering van fenoliese verbindings wat beduidend (p<0.01) tussen behandelings verskil het vergemaklik. Die resultate het getoon dat al 56 pieke wat detect is beduidend (p<0.01) verander het tussen groen, semi-fermenteerde en gefermenteerde rooibos. ‘n Verandering van ten minste 30% is geselekteer om te verseker dat die merkers ‘n redelike degradasie ondergaan het, wat die aantal na 34 verminder het. Gegewe dat dit steeds ‘n groot aantal is, is die aantal fenole wat in doelgerigte analise van rooibos fermentasie ondersoek is redelik min. Rooibos plantmateriaal is onderworpe aan ‘n 6 uur gesimuleerde fermentasie onder streng beheerde toestande, by vir temperature tussen 37 en 50°C, om insig te verkry oor die reaksie kinetika van rooibos fenole inderworpe aan hoë temperatuur en vogtigheid. Eerstens het ‘n ekstraksie optimeringseksperiment getoon dat ‘n 5 min sonikeringstydperk voldoende was. Dit was ook nodig om die rol van endogene rooibos ensieme te ondersoek, aangesien direkte bewys van hul betrokkenheid nie beskikbaar was nie. Vir die tweede eksperiment is ‘n droë hitte behandeling (170°C vir 30 min) voor gesimuleerde fermentasie gepostuleer om die ensieme gedeeltelik te inaktiveer, gebaseer op die verandering in aspalatien en nothofagin inhoud en die verskille in reaksietempo konstantes tussen die kontrole en hitte-behandelde plantmateriaal. Die gedeeltelike omskakeling reaksie kinetika model, wat baseer is op eerste orde reaksie kinetika, het die degradasie van aspalatien en nothofagin, sowel as die vorming van vier eriodictyol-glukosied isomere, tydens gesimuleerde fermentasie die beste beskryf. Die kinetiese parameters wat deur die model beraam is (maw reaksie tempo konstante en ekwilibrium konsentrasie) het getoon dat die reaksies vinniger was, maar ‘n verlaagde degradasie-/vormingsomvang bereik het by hoër temperature. Hierdie waarnemings het gelei to die postulasie dat ensieme gedeeltelik ge-inaktiveer word by hoër temperature of dat ‘n beperkende faktor ;n rol speel tydens gesimuleer fermentasie, bv. opgebruik van suurstof.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/103461
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