Evaluation of commercial enzymes for the bioprocessing of Rooibos tea

Coetzee, Gerhardt (2005-04)

Thesis (MSc) -- University of Stellenbosch, 2005.

Thesis

ENGLISH ABSTRACT: The Rooibos tea plant (Aspalathus linearis) is indigenous to South Africa and occurs only in the Western Cape's Cedarberg region. Rooibos tea is produced from the leaves and fine stems of the plant. The tea is normally prepared by brewing the leaves and consuming the liquor. However, the Rooibos plant is not only used to prepare tea; the plant extracts are also used in various neutraceutical and pharmaceutical products, including health drinks, iced tea, soaps and moisturising creams. Although the tea plant contains native enzymes responsible for the colour and aroma development of Rooibos tea, the disruption and maceration of the plant material during processing is insufficient to allow these enzymes proper access to the substrates responsible for Rooibos tea's characteristics. The current processing of Rooibos tea is also time consuming and is done under uncontrolled conditions, leading to unnecessary loss in aroma and antioxidant content. The addition of enzymes could improve the maceration of the plant material, shorten the processing time and improve the extraction of aroma, colour and antioxidant components. During this study, 16 commercially available microbial enzymes were evaluated on three different Rooibos substrates for the improvement of aroma and colour development, as well as the extraction of soluble solids (SS) and total polyphenols (TP). Thirteen enzymes were evaluated on spent tea for the enhanced extraction of soluble solids and to determine the best candidates for further evaluation on fermented and green Rooibos tea. Seven of the enzymes improved the yield in SS from spent tea. Up to 232% improvement was obtained, depending on the type of enzyme and dosage applied. The best six enzyme preparations were further evaluated on fermented Rooibos tea. For Depol™ 670L at 20 ul/g tea, the laboratory treatment increased the yield in SS by 44%, while small-scale industrial simulations increased the SS by 26%. However, an increase in the yield in SS was usually accompanied by a decrease in the %TP/SS ratio, indicating that mainly inactive compounds were extracted. Based on the results with the commercial enzymes, twelve "synthetic" enzyme cocktails, consisting of different combinations of commercial enzymes were designed, of which three cocktails released increased amounts of SS without decreasing the %TP/SS ratio significantly. Thirteen enzymes were evaluated on dried and freshly cut green Rooibos tea, with three enzymes (Depol™ 670L, Pectinex Ultra SP-L and Depol™ 692L) increasing the yield in SS between 21% and 66%, and the TP content between 11% and 47%. Laccase was the best candidate in improving colour development from green tea, with the improvement being slightly better at 50°C than at 40°C. All the "synthetic" cocktails containing laccase improved the colour extract of all three substrates evaluated, but also significantly decreased the TP and antioxidant content. However, lower dosages of laccase resulted in colour development with little loss in the antioxidant content. Due to the promising results obtained with the treatments of Rooibos tea with laccases, it was decided to clone and express the laccase gene (lacA) of Pleurotus ostreatus into Aspergillus niger. The gene was successfully transformed into A. niger, but the expression of the recombinant gene was not effective.

AFRIKAANSE OPSOMMING: Die Rooibostee plant (Aspalathus linearisi is inheems tot Suid-Afrika en kom slegs in die Sederberg-omgewing in die Wes-Kaap voor. Rooibostee word van die blare en fyn stingels van die plant geproduseer. Die tee word normaalweg voorberei deur die blare in kookwater te laat trek en dan die aftreksel te drink. Die Rooibos plant word nie net gebruik om tee te maak nie; die tee ekstrak word ook gebruik vir verskeie neutraseutiese en farmaseutiese produkte, insluitende gesondheidsdrankies, ystee, seep en bevogtigingsrome. Ten spyte daarvan dat die teeplant sy eie ensieme vir die kleur en aroma ontwikkeling van Rooibostee bevat, is die verbreking en maserasie van die plantmateriaal tydens prosessering onvoldoende om die ensieme genoeg toe gang tot die substrate verantwoordelik vir die kenmerkende eienskappe van Rooibostee te gee. Die huidige prosessering van Rooibostee is ook tydrowend en geskied onder onbeheerde toestande, wat tot 'n onnodige verlies in aroma en antioksidante lei. Die toevoeging van ensieme kan die afbraak van die plantmateriaal verbeter, die behandelingsproses verkort en die aroma, kleur en antioksidant inhoud van ekstrakte verbeter. Tydens hierdie studie is 16 kommersieel-beskikbare mikrobiese ensieme op drie verskillende Rooibos substrate vir die verbetering van aroma, kleur en ekstraksie van oplosbare vastestowwe (SS) en totale polifenole (TP) getoets. Dertien ensieme is op oorskot tee vir die verbeterde ekstraksie van oplosbare vastestowwe geevalueer, waama die beste kandidate vir evaluering op gefermenteerde en ongefermenteeede Rooibostee gekies is. Sewe ensieme het die SS vanaf oorskot tee verhoog. Tot 232% verhoging is waargeneem, afhangende van die tipe ensiem en die dosis wat gebruik is. Die beste ensiern preparate IS verder op gefermenteerde Rooibostee geevalueer, Labarotoriurn behandelings met Depol™ 670L teen 20 ul/g tee het die SS inhoud met 44% verhoog, terwyl die kleinskaalse industriele simulasie die SS inhoud met 26% verhoog het. 'n Verhoging in SS het egter gewoonlik met 'n afname in die %TP/SS verhouding gepaard gegaan, wat aandui dat hoofsaaklik onaktiewe stowwe vrygestel IS. Na aanleiding van die resultate met die kommersiele ensieme, is twaalf "sintetiese" ensiemmengsels met verskillende ensiemkombinasies getoets, waarvan drie mengsels ook meer SS vrygestel het met byna geen verlaging in die %TP/SS verhouding nie. Dertien ensieme was op gedroogde en vars gekerfde groen Rooibostee getoets met drie ensieme (Depol™ 670L, Pectinex Ultra SP-L en Depol™ 692L) wat die SS met tussen 21% en 66%, en die TP inhoud met tussen 11% en 47% verhoog het. Lakkase was die beste kandidaat vir die verbetering van kleur ontwikkeling by groen Rooibostee met die verbetering effens beter by 50°C as by 40°C. Al die "sintetiese" ensiem mengsels wat lakkase bevat het, het die kleur by al die verskillende substrate verbeter, maar het ook die TP en antioksidant inhoud aansienlik verlaag. Laer lakkase dosisse het goeie kleurontwikkeling tot gevolg gehad met minimale verlies in die antioksidant inhoud. Vanwee die goeie resultate wat met die lakkase behandelings verkry is, is daar besluit om die lakkase geen (lacA) van Pleurotus ostreatus te kloneer en in Aspergillus niger uit te druk. Die geen is suksesvol in A. niger getransformeer, maar die uitdrukking daarvan was nie effektief nie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/50343
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