Towards commercial production of Kunzea ambigua (Sm.) Druce distilled oil : maximising the oil yield and enhancing the bioactive constituents

Essential oil, extracted from the leaves and twigs of Kunzea ambigua (Sm.) Druce, is increasingly in high demand by industry for its ‘soft, medicinal and soothing aroma’ and claimed muscle relaxant properties. Production is moving from wild-harvested to orchardised stands populated with germplasm selected from native flora based on higher yield and improved quality of oils. However, there is limited research regarding the growth and production of K. ambigua and kunzea essential oil. To produce large quantities of kunzea essential oil with a consistent, high quality profile for commercial size, agronomic and post-harvest management protocols need to be established. This study first investigated extraction technology and the potential to optimise the distillation time to maximise oil yield and produce kunzea essential oil with a targeted chemical profile. This study showed that the highest oil yield was collected between 30 and 60 mins, much of which was dominated by monoterpenes and oxygenated monoterpenes. Longer distillation times resulted in oil fractions that were relatively high in levels of sesquiterpenes and the bioactive component viridiflorol (22.45 %) which reached a maximum at 240 mins. Steam distillation requires large quantities of material to predict oil yield and quality which can be problematic when the amount of vegetation is limited, such as in the evaluation of young plants in a germplasm selection program. Experiments investigating the use of small-scale solvent extraction to extrapolate the quality of steam-distilled oil were also performed. Sample preparation (frozen or oven-dried at 40°C) and extraction solvents (petroleum ether and ethanol) were investigated. The ethanol extraction of frozen K. ambigua leaves was shown to effectively predict the oil yield and chemical constituents of steam distilled oil. This technique was used for selecting superior, high yielding cultivars which contained higher levels of viridiflorol (highly desired by the market). Kunzea is still predominantly wild-harvested in North East Tasmania, and the first step for establishing plantations is to choose superior clones. This study aimed to (i) compare the chemical constituents of ethanol extracts from wild-grown and cultivated kunzea and (ii) investigate the genetic variation in oil yield and chemical constituents of distilled oil, sourced from cultivated plants. Although the qualitative chemical composition of ethanol extracts from kunzea grown in the natural habitat was similar to that subsequently produced by their cloned progeny undergrown in ideal conditions, the results showed quantitative variability, and associated phenotypic factors. The amount of chemical components in kunzea extract was higher in cultivated plants compared to wild-harvested samples, possibly due to optimised growing conditions and plant age. The variations in oil yield and chemical profile among distilled oils from different germplasms grown under identical conditions, confirmed genetic variability within the wild populations, presenting the opportunity to selectively propagate and breed elite K. ambigua cultivars by appropriate plant selection. Intensity and season of harvest has significant impacts on the recovery of the plants and the quality and quantity of kunzea essential oil produced. The responses to the depth of cut at harvest (harvest intensity) of shallow (0.2m above ground) and deep-cut harvest (0.1m above ground), undertaken in early summer, were compared to uncut material (control). The cumulative above-ground biomass of shallow-cut was two-fold higher, relative to deep-cut treatment with a concomitant higher oil content in shallow-cut (1.84 ± 0.11% DW) compared to deep-cut treatment (1.54 ± 0.32% DW) in spring. In particular, deep-cut treatment resulted in the plants having inadequate resources for re-growth and essential oil biosynthesis. There was no seasonal variation in oil content while the kunzea essential oil extracted from autumn cropping showed enhanced levels of bioactive components such as 1-8 cineole and viridiflorol. Therefore, the recovery of biomass post-harvest is optimised by shallow-cut harvests and autumn cropping maximised oil quantity, yielding premium kunzea essential oil with enhanced levels of bioactive components. Essential oils are inherently volatile and contain thermolabile components. The retention of these chemicals is key to aroma quality. This study investigated the changes in oil colour and chemical compositions of kunzea essential oil over eight months of storage at -20°C (freezer), 4°C (refrigerator) and 20°C (room temperature), respectively. An additional influence with/without the light was also implemented at 20°C (room temperature). Colour indicated alterations in stored kunzea essential oil where poor storage conditions resulted in a loss of ‘greenness’ (a* values). This study also showed that lower levels of bicyclogermacrene and enhanced concentration of α-terpineol could be an indicator of degraded kunzea essential oil. Notably, kunzea essential oil exposed to light had significantly decreased components of germacrene D, β-caryophyllene, and α-humulene, probably due to thermal isomerisation. Overall, kunzea essential oil stored in a freezer or refrigerator (below 4°C) was the most stable and consistent in colour and chemical profile whereas there is evidence that some components may be oxidized when exposed to light with negative consequences for oil colour and quality. This thesis has provided for improved production strategies to maximise the quantity and quality of kunzea essential oil from the view of the extraction technique (distillation time), selection of superior cultivars, the productivity of crop (plant recovery and oil quantity/quality depending on harvest intensity and timing), and stability of oils. High market demand requires that more studies be undertaken, focused on increasing the oil quantity and quality through crop management with appropriate plant selection for a sustainable and profitable industry