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Green Rooibos Nutraceutical: Optimisation of Hot Water Extraction and Spray-Drying by Quality-By-Design Methodology

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Green Rooibos Nutraceutical: Optimisation of Hot Water Extraction and Spray-Drying by Quality-By-Design Methodology GREEN ROOIBOS NUTRACEUTICAL: OPTIMISATION OF HOT WATER EXTRACTION AND SPRAY-DRYING BY QUALITY-BY-DESIGN METHODOLOGY Neil Miller Thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Food Science Department of Food Science Faculty of AgriSciences Stellenbosch University Supervisor: Prof. E. Joubert Co-supervisor: Prof. D. de Beer December 2016 Stellenbosch University https://scholar.sun.ac.za DECLARATION By submitting this thesis/dissertation electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. Date: December 2016 Copyright © 2016 Stellenbosch University All rights reserved i Stellenbosch University https://scholar.sun.ac.za ABSTRACT Unfermented Aspalathus linearis, otherwise known as green rooibos (GR), contains high levels of aspalathin, a potent C-glucosyl dihydrochalcone antioxidant with antidiabetic bioactivity, unique to rooibos. Inherent variation in the phenolic composition of rooibos is likely to cause significant variability in the aspalathin content of different GR production batches and thus also the batch-to-batch quality of a nutraceutical green rooibos extract (GRE). The aim of this study was to optimise hot water extraction and spray-drying for the production of a shelf-stable GRE. A quality-by-design (QbD) approach was applied, entailing a preliminary risk assessment step, one-factor-at-a-time analysis, and analyses according to a central composite design (CCD) to determine the effects of process parameters on responses. Response surface methodology (RSM) was applied to identify suitable control spaces, i.e. ranges of process input factors in which optimal responses, i.e. product quality, could be expected. Significant variation in aspalathin content of different GR production batches (n = 47; 2.5–4.5%) was demonstrated. The CCD for extraction included three independent variables: extraction time (10–40 min), extraction temperature (41–93 °C) and water-to-plant material ratio (6.6:1–23.4:1; v.m-1). Prediction models and response surfaces for extract yield (EY; g.100 g-1 plant material), aspalathin extraction efficiency (Asp_EE; g.100g-1 in plant material) and aspalathin content (g.100 g-1 soluble solids) were generated. Verification of the prediction models showed good predictive ability for EY and Asp_EE. Multi-response optimisation was applied to identify levels of the independent variables which would maximise EY and Asp_EE. Optimal conditions were identified based on these results, along with considerations of cost-efficiency and practicality: extraction time, 29–31 min; extraction temperature, 90–95 °C and water-to-plant material ratio, 9:1–11:1 (v.m-1). Validation of the optimal extraction conditions using the 47 commercial GR production batches, (aspalathin content >2.5%) showed that at least 15% EY and 8% aspalathin content in the extract could be achieved. Standardisation of the maximum particle size by sieving out of large particles could potentially improve the overall process efficiency. The CCD for spray-drying included three independent variables: inlet air temperature (150–220 °C), feed concentration (5–35%) and feed flow rate (0.12–0.64 L.h-1). Powder yield (g powder recovered per 100 g solids in feed) was the only response for which a statistically significant prediction model was generated. Optimal spray-drying conditions (inlet air temperature of 210–230 °C, feed concentration of 34–36% and feed flow rate of 0.62–0.67 L.h-1) were identified and applied in the spray-drying of a pure GRE as well as GRE in a 1:1 mass ratio blend with the carriers, inulin and maltodextrin, respectively. Amorphous powders with low moisture content (<2.2%) and water activity (<0.13) and >89% retention of aspalathin were obtained. The hygroscopic character of the powders was confirmed by moisture sorption analysis, and storage conditions of <25 °C and <40% relative humidity are therefore recommended in order to maintain optimal quality. Inulin improved the flowability and wettability of the powder as compared with maltodextrin. An inulin-GRE formulation was identified as a good candidate for further development as a high-value antidiabetic nutraceutical. ii Stellenbosch University https://scholar.sun.ac.za UITTREKSEL Ongefermenteerde Aspalathus linearis, andersins bekend as groen rooibos (GR), bevat hoë vlakke van aspalatien, ‘n kragtige C-glukosiel dihidrochalkoon antioksidant met anti-diabetiese bioaktiwiteit, uniek aan rooibos. Inherente variasie in die fenoliese samestelling van rooibos sal waarskynlik variasie in die aspalatieninhoud van verskillende produksielotte van GR tot gevolg hê, en dus ook in die kwaliteit van ‘n nutraseutiese groen rooibos ekstrak (GRE). Die hoofdoelstelling van hierdie studie was die optimisering van warm water-ekstraksie en sproeidroging in die produksieproses vir ‘n rakstabiele GRE. ‘n Kwaliteit-deur- ontwerp (KdO) benadering is toegepas, insluitende ‘n aanvanklike risiko assessering, een-faktor-op-‘n-slag analise, en analises volgens ‘n sentrale saamgestelde ontwerp (SSO) om die effek van veranderlikes op die proses uitkomstes te bepaal. Respons-oppervak metodiek (ROM) is toegepas vir die identifisering van toepaslike kontroleruimtes, d.w.s. reikwydtes van die veranderlikes waarbinne optimale uitkomstes, en dus produk kwaliteit, verwag sou kon word. Beduidende variasie in die aspalatieninhoud van verskillende GR produksielotte (n = 47; 2.5–4.5%) is gedemonstreer. Die SSO vir die ekstraksie het drie onafhanklike veranderlikes ingesluit: ekstraksietyd (10–40 min), ekstraksietemperatuur (41–93 °C) en water-tot- plantmateriaal verhouding (6.6:1–23.4:1; v.m-1). Voorspellingsmodelle en respons-oppervlaktes vir die ekstrakopbrengs (EO; g.100 g-1 plantmateriaal), aspalatien ekstraksiedoeltreffendheid (Asp_ED; g.100 g-1 in plantmateriaal) en aspalatieninhoud (g.100 g-1 ekstrak), is gegenereer. Verifikasie van die voorspellingsmodelle het goeie voorspellingsvermoë aangedui vir EO en Asp_ED. Multi-respons optimisering is dus toegepas om die optimale waardes van die onafhanklike veranderlikes, waarby maksimum EO en Asp_ED bewerkstellig kan word, te vind. Optimale proses parameters is geselekteer op grond van hierdie resultate, sowel as praktiese oorwegings en koste-doeltreffendheid: ekstraksietyd, 29–31 min; ekstraksietemperatuur, 90–95 °C en water- tot-plantmateriaal verhouding, 9:1–11:1 (v.m-1). Validering van die optimale ekstraksiekondisies met die 47 kommersiële GR produksielotte (aspalatieninhoud >2.5%) het aangedui dat ten minste 15% ekstrakopbrengs en 8% aspalatieninhoud in die ekstrak bereikbaar is. Standardisering van die maksimum partikelgroottes deur die uitsifting van groot partikels kan moontlik die algehele ekstraksiedoeltreffendheid verbeter. Die SSO vir sproeidroging het drie onafhanklike veranderlikes ingesluit: inlaat lugtemperatuur (150–220 °C), toevoer konsentrasie (5–35%) en toevoer vloeitempo (0.12–0.64 L.h-1). Slegs vir poeier-opbrengs (g poeier herwin per 100 g vastestowwe in toevoer) is ‘n statisties beduidende voorspellingsmodel gegenereer. Optimale sproeidrogingskondisies (inlaat lugtemperatuur 210–230 °C), toevoer konsentrasie (34–36%) en toevoer vloeitempo (0.62–0.67 L.h-1) is geïdentifiseer en toegepas in die sproeidroging van ‘n suiwer GRE sowel as GRE in ‘n 1:1 massaverhouding met die draers, maltodekstrien en inulien, onderskeidelik. Die sproeidroging het amorfe poeiers met lae voginhoud (<2.2%) en wateraktiwiteit (<0.13) gelewer, en >89% behoud van aspalatien is bewerkstellig. Die higroskopiese karakter van die poeiers is bevestig deur vogsorpsie-analise. Opbergingstoestande van <25 °C en <40% relatiewe humiditeit word aanbeveel ten einde optimale kwaliteit te handhaaf. Inulien het die vloeikarakter en benatting van die poeier verbeter in vergelyking met maltodekstrien. ‘n Inulien-GRE formulasie is geïdentifiseer as ‘n goeie kandidaat vir verdere ontwikkeling as ‘n hoëwaarde anti- diabetiese nutraseutiese aanvuller. iii Stellenbosch University https://scholar.sun.ac.za ACKNOWLEDGEMENTS Prof. Elizabeth Joubert & Prof. Dalene de Beer of ARC Infruitec-Nietvoorbij (Stellenbosch) — thank you for your expert supervision and significant contributions to this project. The quality of your collective body of work speaks for itself. Ms Nina Muller — thank you for assisting with administration and for playing a significant supportive role in my application for these postgraduate studies back in 2014. Nico Walters, Dr Christie Malherbe, Carin de Wet, Chantelle Human, Louise Robertson, George Dico, Neliswa Matrose, Claire Pauck and Lara Alexander — colleagues at ARC Infruitec-Nietvoorbij — thank you for your assistance and advice in and around the laboratory and office. Chantelle Human — thank you for kindly carrying out the SEM imaging at the electron microbeam unit at the Central Analytical Facility of Stellenbosch University. Prof. Marique Aucamp of Pharmacen, North West University (Potchefstroom, South Africa) — for carrying out isothermal microcalorimetry, differential thermal and thermogravimetric analyses, moisture sorption studies, contact angle measurements and X-ray powder diffraction. Thank you for assisting in the interpretation of the data. Prof. Martin Kidd of the Centre
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