Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Aspalathus linearis is a leguminous shrub native to the Cedarberg Mountains in the Western Cape, of which the leaves and stems are used for the preparation of rooibos tea. Over the past few decades, rooibos tea and other related products have gained popularity due to their health promoting properties. These beneficial properties can partly be ascribed to the phenolic constituents that are trapped within the cellulolytic plant material of the tea leaves as glycoconjugated aroma and phenolic compounds. Although many fungal species are known for their efficient hydrolysis of plant material, fungal enzymes have not been evaluated for the bioprocessing of rooibos tea to improve its commercial value. It was the objective of this study to identify a specific cocktail of microbial enzymes to enhance the maceration of the rooibos plant material, while retaining the antioxidant content. During this study, 11 fungal species known for the production of hydrolytic enzymes, as well as 12 species isolated from rooibos tea products, were screened for their potential to improve aroma development and/or increased extraction of soluble matter and/or antioxidants from rooibos tea material. After culturing in Potato Dextrose medium, the crude enzyme extracts of the 23 isolates were evaluated on spent rooibos tea for enhanced extraction of soluble solids (SS) and/or total polyphenols (TP). Nine strains increased the yield in SS (improvement varying from 3% to 42%), while 14 strains yielded higher levels of TP (increase varying from 1% to 36%). Little improvement in colour development from green (unfermented) rooibos tea was observed, but the enzyme extracts from Pleurotus ostreatus var. florida, Lentinula edodes, Aspergillus oryzae, Aspergillus tubingensis, Paecilomyces variotti and Trichoderma reesei improved the aroma development from green tea to some extent. Ten-fold concentrated enzyme extracts from four of these isolates were able to release at least an additional 10% in SS from the green tea. The crude enzyme extracts prepared from three food-grade strains, i.e. Aspergillus oryzae, Lentinula edodes and Pleurotus ostreatus var.florida, contained relatively high levels of endoglucanase, xylanase and pectinase activities. Eight different culture media were evaluated for optimal hydrolase and laecase production by these food-grade fungi. MYPG proved to be the best growth medium, while 1% spent grain, 1% wheat straw and 1% pineapple peel gave the best induction of xylanase, cellulase, pectinase and laecase activities for L. edodes. When cultured in the Yeast Peptone (YP) medium + 1% wheat straw, the L. edodes enzyme cocktail showed the best improvement in both the aroma and colour development of green tea and may be considered for shortening of the fermentation time required for green tea processing. Traditional open-air fermentation of rooibos tea can take up to -1-6hours, which results in a significant loss in antioxidants and therefore also in its pharmaceutical and nutraceutical value. The Rhizopus oryzae cocktail prepared in YP + 1% wheat straw showed potential for the development of a quick-draw fermented tea made by infusion, where there is improved colour release and more than 20% improved extraction of soluble solids without a loss in the TP content. When cultured in Potato Dextrose medium, the L. edodes cocktail can be used for aroma and colour development from green tea, while the R. oryzae cocktail can be used for increasing the antioxidant content in rooibos extracts from green or fermented tea. This was confirmed with small-scale industrial treatments of fermented tea where the L. edodes YP + wheat straw cocktail improved the release in SS by more than 10% and the R. oryzae yP + wheat straw cocktail increased the yield in SS by more than 30% and the TP by more than 20%. / AFRIKAANSE OPSOMMING: Aspalathus linearis is 'n fynbosplant inheems aan die Sederberge in die Wes-Kaap, waarvan die blare en stingels vir die voorbereiding van rooibostee gebruik word. Die afgelope paar dekades het die gewildheid van rooibostee en verwante produkte aansienlik toegeneem weens die gesondheidsvoordele wat dit inhou. Hierdie voordelige eienskappe kan toegeskryf word aan die fenoliese komponente wat binne die sellulolitiese plantweefsel van die teeblare as gekonjugeerde geur- en fenoliese verbindings vasgevang is. Alhoewel verskeie swamspesies vir hul doeltreffende degradering van plantmateriaal bekend is, is fungale ensieme nog nie geëvalueer vir die prosessering van rooibostee om die kommersiële waarde daarvan te verbeter nie. Die doelwit van hierdie studie was om 'n spesifieke kombinasie van mikrobiese hidrolitiese ensieme te identifiseer wat die maserasie van rooibos plantmateriaal sal verhoog met behoud van die anti-oksidant inhoud. Tydens hierdie studie is 11 swamspesies wat bekend is vir die produksie van hidrolitiese ensieme, asook 12 swamspecies wat vanaf rooibostee produkte geïsoleer is, geëvalueer vir hul potensiaalom geurontwikkeling en/of ekstraksie van oplosbare stowwe en/of anti-oksidante vanuit rooibostee materiaal te verbeter. Die kru ensiemekstrakte van die 23 isolate, wat ID Aartappel-Dextrose medium opgegroei is, is op oorskot rooibostee geëvalueer vir verhoogde ekstraksie van oplosbare vastestowwe (SS) en/of totale polifenole (TP). Nege rasse het die opbrengs van oplosbare vastestowwe verhoog (verbetering tussen 3% en 42%), terwyl 14 rasse die totale polifenoliese vlakke laat toeneem het (tot so hoog as 36%). Baie min verbetering in kleurontwikkeling van groen (ongefermenteerde) rooibostee is waargeneem, maar ensiemekstrakte van Pleurotus ostreatus var. florida, Lentinula edodes, Aspergillus oryzae, Aspergillus tubingensis, Paecilomyces variotti en Trichoderma reesei, het wel die aroma ontwikkeling vanaf groen tee tot 'n mate verbeter. Tienvoudig gekonsentereerde ekstrakte van vier van hierdie isolate het 'n verbetering van meer as 10% in die ekstraksie van opgeloste vastestowwe uit groen tee tot gevolg gehad. Die ensiemekstrakte van drie swarnme bekend vir hul gebruik in die voedselindustrie, nl. A. oryzae, L. edodes and P. ostreatus var. florida, het relatief hoë vlakke van endoglukanase, xylanase en pektinase aktiwiteit getoon. Agt verskillende kultuur-media is vir die optimale produksie van hidrolitiese and lakkase ensieme vanaf hierdie voedsel-graad swarnme geëvalueer. MYPG was die beste groeimedium vir L. edodes, -terwyl 1% koringstrooi, 1% oorskot graan en 1% pynappelskil die beste induksie van xylanase, pektinase, endoglukanase en lakkase aktiwiteite vir hierdie organisme getoon het. Lentinula edodes opgegroei in YP medium + 1% koringstrooi, het die beste verbetering in aroma en kleur getoon vanaf groen tee getoon. Hierdie ekstrak kan dus moontlik gebruik word vir die verkorting van die fermentasietyd wat vir groen tee benodig word. Ope-lug fermentasie van groen tee duur gewoonlik tot 16 uur en lei tot 'n aansienlike verlies in antioksidant-inhoud. Die R. oryzae ekstrak het die beste potensiaal vir die vervaardiging van 'n "quick-draw" tee getoon met 'n goeie kleurvrystelling sonder enige verlies in SS en TP opbrengs. Wanneer die swamme in Aartappel-Dextrose medium opggegroei word, kan die L. edodes ensiemekstrak vir aroma en kleurontwikkeling van groen tee aangewend word, terwyl die R. oryzae ensiemekstrak vir die verhoging van die antioksidant-inhoud in rooibos ekstrakte van groen tee of gefermenteerde tee gebruik kan word. Dit is bevestig met die kleinskaalse behandeling van gefermenteerde tee waar die L. edodes YP + 1% koringstrooi ensiemekstrak die vrystelling van SS met meer as 30% en die TP met meer as 20% verbeter het.

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