essential oils and plant extracts - Completed Projects

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RIRDC Completed Projects in 1998-1999 & Research in Progress as at June 1999
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SUB-PROGRAM 2.4 - ESSENTIAL OILS AND PLANT EXTRACTS

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COMPLETED PROJECTS
Project No.	Project Title	Researcher	Phone	Organisation
ANU-24A	Establishment of a genetic and biodiversity resource of Eucalyptus polybractea	Dr Michael U. Slee	02 6286 3224	Department of Forestry Australian National University
DAV-101A	Feasibility of Native and Scotch Spearmint Oil production in Tas. & Vic	Fred Bienvenu	03 5731 1222	Agriculture Victoria,
UM-16A	Control of mint rust (Puccinia menthae) on peppermint - epidemiology and chemical control	J. Edwards	03 9344 6458	Institute of Land and Food Resources, The University of Melbourne,
UNC-4A	Generation of high quality echinacea products	Professor Ron Wills	02 4348 4140	Centre for Food Industry Research and Development
UT-10A	Yield and quality of concrete from Boronia megastigma (Nees)	Professor Bob Menary, Dr Hazel MacTavish	03 6226 2650	University of Tasmania
UT-11A	A New Commercial Flavour Product from Tasmannia lanceolata	Professor Robert Menary	03 6226 2723	University of Tasmania,
UT-13A	Determination of pesticide minimum residue limits in essential oils	Professor Robert Menary	03 6226 2723	University of Tasmania,
UT-15A	Enantiomeric composition of essential oils	Professor Robert Menary	03 6226 2723	University of Tasmania,
UT-16A	Best Practice in Peppermint	Professor Robert Menary	03 6226 2723	University of Tasmania,
UT-21A	Improved Quality in Floral Products From Brown Boronia	Prof RC Menary; Dr HS MacTavish	03 6226 2650	University of Tasmania

Completed Projects

Project Title	Establishment of a genetic and biodiversity resource of Eucalyptus polybractea
RIRDC Project No.:	ANU-24A
Researcher:	Dr Michael U. Slee
Organisation:	Department of Forestry Australian National University CANBERRA ACT 0200
Phone:	02 6286 3224
Fax:	02 6249 0746
Email:	mike.slee@anu.edu.au
Objectives	The project aimed to establish a permanent, readily accessible and protected genetic resource of Eucalyptus polybractea. The trees included would represent a cross section of the species and be a permanent reservoir of its genetic variation. The resource would be managed for conservation, for research and for general use involving production of seed, cuttings or other material.
Background	Oil producing plantations based on mallee species have agroforestry and land rehabilitation potential in areas subject to drought and salinity. In Western Australia, major land conservation programs are in place based on the use of mallees. Similar developments in other low rainfall areas are possible. Breeding programs have been established to support the development. The resource is a fundamental support to these programs
Research	The resource has been established at Tallimbalong, West Wyalong NSW on the G.R.Davis property. The coverage is comprehensive. There are 1800 trees representing seven NSW and four Victorian locations. Within each location the seed from individual seed trees has been kept separate and this parent tree distinction has been carried through into the field. There are 47 individual parents represented.
Outcomes	Initial assessments show wide variation in growth, oil production, and in leaf size. For example, oil yields, as a proportion of leaf dry weight, varied between 1% and 16%, and mean leaf size between 0.4 and 7.5 sq cm. This variation is typical of the species and indicates the success of the project
Implications	The establishment of this resource with 11 different seed sources and 47 individual progenies in a uniform environment presents a unique opportunity to examine the cause of the extreme variation exhibited in almost every feature in this mallee species of eucalypt. Such a study could yield unexpected and probably invaluable information on the species, its breeding habits and possibly its history. It may produce guidelines for ecological management of trees in semi arid regions.

Project Title	Feasibility of Native and Scotch Spearmint Oil production in Tas. & Vic
RIRDC Project No.:	DAV-101A
Researcher:	Fred Bienvenu
Organisation:	Agriculture Victoria, Ovens Research Station, PO.Box 235, MYRTLEFORD VIC 3737
Phone:	03 5731 1222
Fax:	03 5731 1223
Objectives	To investigate the potential of Native and Scotch spearmint for high quality oil in Victoria and Tasmania by determining the effects of major production variables on yield and composition of oil produced. To assess the effect of oil composition on the marketability of spearmint oils. To estimate cost of production and potential returns.
Background	Spearmint oil crops are mainly grown in the USA with some recent development in other regions. The world market for spearmint oils is approximately 1500 tonne/yr and increasing and is one of the larger essential oil commodities. The biosynthesis of chemical constituents in the spearmint plant does not have the strict geographic or climatic constraints that peppermint oil production involves.
Research	The fungicide bitertanol (Baycor) gave the most effective control of mint rust and resulted in the highest oil yields of the six fungicides evaluated on Scotch spearmint at the ORS, Vic. and in Tas. Tebuconazole (Folicur) was also effective, its use resulting in high levels of disease control and good oil yields. Victorian peppermint growers rely on a single registered fungicide, propiconazole (Tilt) for rust control. Several readily available are more effective at controlling mint rust on Scotch spearmint than propiconazole, with a corresponding increase in oil yield. Tasmanian peppermint growers use tebuconazole in addition to propiconazole. Large scale harvesting and distillation were used to obtain commercial yield figures likely in a full-scale spearmint oil production enterprise. The spearmint oil yield from semi-commercial plots in 1998 was higher than for peppermint (113kg/ha c/f: 105kg/ha for peppermint. Native spearmint oil production was some 20-30 % less than Scotch spearmint oil and with lower prices paid for Native spearmint oils there is little reason to produce the Native variety.
Outcomes	The two main producers of peppermint in Victoria have indicated that they are keen to put in trial blocks of Scotch spearmint using the capital advantages in having multiple uses for their expensive harvest distillation equipment and diversification of their production base. The oil produced in south eastern South Australian was of acceptable quality. The results obtained so far are encouraging. Samples of Scotch spearmint oil were evaluated for their commercial acceptance by the flavour and fragrance industry. The results were very encouraging and indicated that Scotch spearmint trialed in this study are commercially acceptable. Gross margin analysis has shown production of Scotch spearmint oil to be equally profitable to peppermint in the areas studied.
Implications	Sufficient information has been established to steer potential producers towards the establishment of a second mint oil industry. Initially it is expected that the existing peppermint industry should act on these findings to augment their peppermint enterprises and make more use of the extensive capital infrastructure already in place. It is recommended that growers and governments pursue minor-use registration of bitertanol and tebuconazole which would allow these fungicides to be alternated. The lack of a negative oil component in spearmint allows for potential production sites from a wider area. The most limiting resource will be the availability of water in some regions. Further work may be required to study spearmint oil production in the wider geographic context.

Project Title	Control of mint rust (Puccinia menthae) on peppermint - epidemiology and chemical control
RIRDC Project No.:	UM-16A
Researcher:	J. Edwards
Organisation:	Institute of Land and Food Resources, The University of Melbourne, Parkville, VICTORIA 3052.
Phone:	03 9344 6458
Fax:	03 9344 5570
Email:	j.edwards@landfood.unimelb.edu.au
Objectives	To determine and demonstrate an effective strategy for the control of peppermint rust based on minimal use of chemical sprays. To identify fungicides which will effectively control Puccinia menthae without contamination of distilled oil. To conduct detailed studies of the epidemiology of mint rust eg. life cycle and the influence of temperature and humidity on its capacity for growth and reproduction.
Background	Peppermint oil production is an emerging new industry in the river valleys of north east Victoria. The long term viability of the industry depends upon production of an oil of consistent quality and reliable yield. The disease mint rust, caused by the fungus Puccinia menthae, has caused major problems for peppermint growers, reducing oil yields by more than 50% and lowering oil quality. Control measures have been based on those effective in the USA, yet little is known about the behaviour of the disease under local conditions.
Research	The research examined the epidemiology of the disease in north east Victoria and the influence of the environment on disease development. An action threshold for timely initiation of chemical control was developed, and several fungicides were screened for their effectiveness. The uses of flaming and the herbicide, paraquat, for mint rust control were investigated. Variation within the pathogen population was examined, using both host range differences and the molecular technique known as RAPD analysis.
Outcomes	The full life cycle of the rust does not occur on peppermint growing in this region. No spermogonia or aecia were ever seen on peppermint, whereas the summer spores, the urediniospores, persisted all year round. A study of the infection process of urediniospores on peppermint found that, under artificial conditions, the minimum, optimum and maximum temperatures for infection were <5, 20 and 27°C respectively, and the minimum leaf wetness duration was 6 hours. Under field conditions, daily maximum temperatures above 35°C caused disease levels to fall. An action threshold of 60% infected leaves per shoot was proposed for the initiation of chemical control of the disease. This would usually be in October or November. Mint rust significantly increased leaf loss, and significantly reduced leaf area, leaf fresh weight, oil content, stem and root dry weights, and the numbers of stolons per plant. These effects were seen to have implications for the long-term health of the crop, and a plot of peppermint where mint rust was allowed to develop without control died out after three and a half years. Autumn flaming of peppermint resulted in increased oil yields, but spring flaming did not. Autumn flaming cannot currently be recommended due to legal constraints, but could be substituted by use of the herbicide, paraquat. Six currently available fungicides were tested for mint rust control on M. x gracilis cv. Scotch spearmint over two years. Bitertanol and tebuconazole were identified as significantly more efficient than the other fungicides in controlling the disease, which resulted in higher oil yields. Variation within the pathogen population was examined by comparing host ranges, teliospore morphology and RAPD-PCR profiles. It became evident that there are two types of Puccinia menthae found in Victoria which differ in morphology, host range and disease cycles, and they appear to be genetically isolated from each other. One type infects peppermint and related hosts, and the other type infects spearmint and its relatives. These results raise doubts about the current taxonomic classification of this rust as a single species.
Implications	Mint rust can be controlled if managed carefully. Disease progress in the crop should be monitored and chemical control initiated early, when less than 60% of leaves are infected. Three currently available fungicides are effective against the disease: bitertanol, tebuconazole and propiconazole.

Project Title	Generation of high quality echinacea products
RIRDC Project No.:	UNC-4A
Researcher:	Professor Ron Wills
Organisation:	Centre for Food Industry Research and Development University of Newcastle P.O. Box 127, OURIMBAH NSW 2258
Phone:	02 4348 4140
Fax:	02 4348 4145
Email:	ftrbhw@cc.newcastle.edu.au
Objectives	To assist the industry to become and international supplier of high quality echinacea crops and manufactured end products.
Background	Echinacea is one of the leading medicinal herbs that is expanding rapidly in many countries due to consumer demand for natural or alternate therapies. Australia has a growing vibrant echinacea industry in terms of production and processing. To be fully internationally competitive in the long term, the Australian industry needs to become a reliable supplier of high quality echinacea products; high quality being products with high levels of active constituents that confer a health benefit. To achieve this end, industry needs to understand the quality of harvested crops and to maintain this quality throughout the many operations in the marketing chain.
Research	There were four major components to the research project. First stage was to develop reliable and efficient analytical methods for the analysis of the active constituents in echinacea. The second stage was to determine the levels of active constituents in crops during growth and in dried, traded plant material. The third stage was to investigate various stages in postharvest handling and drying to determine the potential sources of loss of active constituents. The fourth stage was to examine each step in the processing of dried material to a manufactured end product to determine where losses of active constituents may occur then to determine the level of active constituents in manufactured products available on the retail market.
Outcomes	A commercial analytical service is now available to the industry to enable certificates of quality to be obtained for marketing purposes or for quality management of in-house processes. The level of active constituents was found to vary during plant development and between plant parts. For alkylamides, the root was the major source while for chicoric acid, the root and aerial parts are of equal commercial value. It seems that the concentration of actives remains reasonably constant and hence efforts to increase crop yield will be beneficial in retaining quality while increasing production tonnage. A national survey of traded dried echinacea found a reasonable consistency in quality. The industry should consider setting quality standards for high grade and acceptable product to assist in raising the quality profile of the Australian industry. It is believed that the industry has the capability of greatly improving the quality of traded raw materials as the levels of active constituents were well below that achieved in controlled laboratory trials. Causes of loss in the handling and drying system were identified and introduction of a quality management program could substantially increase the quality of the final product. A surprising finding was that natural air drying rather than mechanical drying was a possibility and may even give a superior quality product. Examination of the processing parameters in extracting the active constituents revealed substantial losses will occur unless optimum conditions are used. The survey of quality in manufactured end products reflected the many stages where losses can occur with a substantial number of products having near zero levels of active constituents ranging up to very high quality products. The need for better labelling was considered desirable to retain consumer confidence.
Implications	Australia has real potential to become a high quality supplier of echinacea products provided it adopts quality standards for marketed products and industry sectors adopt appropriate quality management strategies to eliminate those handling and processing aspects which lead to losses of active constituents.

Project Title	*Yield and quality of concrete from Boronia megastigma* (Nees)**
RIRDC Project No.:	UT-10A
Researcher:	Professor Bob Menary, Dr Hazel MacTavish
Organisation:	University of Tasmania GPO Box 252-54 HOBART TAS 7001
Phone:	03 6226 2650
Fax:	03 6226 7450
Objectives	To produce boronia concrete at higher yields and with improved quality by developing pre-harvest, harvesting, and post-harvest management practices and by value-adding the existing product.
Background	Previous research illustrated key areas with the potential to induce significant increases in the concentration of floral extract, including volatiles such as ?-ionone via management practices. These include harvest timing, post-harvest incubation of flowers and the extraction protocol used; the potential of the first two practices being modified by clonal differences. The possibility that biosynthesis of floral volatiles or hydrolysis of precursors in boronia flowers continued after harvest was novel and required extensive verification. The opportunity to extract an increased yield of floral volatiles from a standard quantity of flower material has economic significance, and implications for in vitro production.
Research	Research presented in this report has illustrated that not only is post-harvest production of floral volatiles reproducible, it has considerable potential in some clones under specific environmental conditions. Our research has also shed some light on the biological processes behind, and the limitations to, post-harvest changes. In addition, specific information regarding accumulation of extract throughout flowering has been obtained for each of the commercial clones, allowing optimisation of harvest timing; plus optimal extraction methods.
Outcomes	The opportunity for production of floral extracts with new, and perhaps improved, organoleptic properties is demonstrated via several means: harvest timing, post-harvest incubation, solvent extraction protocol and pre-extraction flower treatment. Thus the objectives of the project have in the main been achieved.
Implications	The most appropriate point for harvest in terms of % open flowers has been established for each clone. Post-harvest activity has the potential to produce 20-25% more extract and 30-300% more volatiles from present flower material, depending on clone and harvest timing. Thermal and atmospheric control of incubations is imperative. Manipulation of extraction conditions including pre-extraction flower treatment, extraction time and solvent volume can produce 75-100% more extract with new organoleptic properties. We are now aware of the rapid response of floral volatiles to external changes in the light environment. The importance of genetic control of phenology, extract concentration and quality and potential for post-harvest production of volatiles has been demonstrated.

Project Title	A New Commercial Flavour Product from Tasmannia lanceolata
RIRDC Project No.:	UT-11A
Researcher:	Professor Robert Menary
Organisation:	University of Tasmania, GPO Box 252C, HOBART TAS 7001
Phone:	03 6226 2723
Fax:	03 6226 7609
Email:	r.menary@utas.edu.au
Objectives	The project is aimed towards the specification of the solvent extract as a new flavour product. This involves the survey, propagation, assessment and documentation of collected samples from around the state. Client preferences can be gauged through the evaluation of a range of extracts and fractions. The monitoring of safrole levels and the further chemical elucidation of the extract are important steps towards the registration of the product with an international body.
Background	There has been international interest in the leaf extract of T. lanceolata since the late 1980’s. A commercial extraction protocol has been developed and semi-commercial quantities of extract have been provided to a large Japanese flavour and fragrance company. To date, most of the leaf material used in the commercial scale extractions are collected from natural stands. The two areas of concern that have been identified are the ability to supply increasing quantities of product in the short to medium term, and the apparent product variability. In addition, there has been some difficulty with specifying market preferences. Part way through this project, in July ’97, discussions with growers highlighted the fact that further market penetration was being hindered through the lack of registration of the extract with any internationally recognised regulatory body. Consequently, field trial work was relegated to ‘observation-only’ status, and a greater emphasis was placed on chemical characterisation and safrole level investigations.
Research	A widespread search of the state produced over 100 clones for assessment in terms of essential oil composition, organoleptic impact, yield and physical properties. The most promising clones have been propagated and a clone bank has been established to house these plants on a long term basis. An integrated database has been set up, which gives ready access to valuable information concerning the clones that have been collected. A time trial to monitor the level of safrole over the course of the season was initiated to investigate concerns over safrole accumulation patterns. In order to further refine our target standard extract, fractions of the extract, a distilled essential oil and an absolute sample were sent to Japan for assessment. The components of the extract have been largely identified through mass spectrometry, high performance liquid chromatography and gas chromatography, after initial fractionation by column chromatography.
Outcomes and Implications	The research findings showed that the levels of safrole in the extract do not vary significantly over the course of the growing season. The implication is that a low safrole content clone will not accumulate sufficient safrole to cause concern, irrespective of when samples are taken. A clone bank which holds the most promising clones in terms of yield, organoleptic assessment and vigour now exists at the Horticultural Research Centre. The properties of these clones, and others, which were examined, are stored in a Filemaker Pro database. The on-going work of registration of Tasmannia lanceolata extract with FEMA on the GRAS list is an involved one, and has to date been addressed through the intensive investigation of the chemical components of the extract, resulting in the identification of 98% of components. Other considerations such as toxicological studies are being addressed through liaisons with other institutions that specialise in this area, and will be addressed fully in future work.

Project Title	Determination of pesticide minimum residue limits in essential oils
RIRDC Project No.:	UT-13A
Researcher:	Professor Robert Menary
Organisation:	University of Tasmania, GPO Box 252C, HOBART TAS 7001
Phone:	03 6226 2723
Fax:	03 6226 7609
Email:	r.menary@utas.edu.au
Objectives	Develop a research base with facilities and validated methods for analyses of pesticides in essential oil crops Conduct field trials in accordance with the NRA Provide industry with data supporting assurances of quality for all exported products
Background	International markets often require that essential oils are presented with certification detailing the analyses of pesticide residue contamination. Temporary permits issued by the NRA for the use of specific pesticides require that research continues towards achieving pesticide registration.
Research	The use of these supercritical fluids to extract and pre concentrate residues of pesticides in the matrices of essential oils, was investigated. Preliminary field trials and analytical method development for a large range of herbicides were undertaken. Extensive field trials of the fungicides Folicur and Tilt were established in commercial boronia crops. The effect of storage conditions on residue concentration in leaves and oil was assessed for Tilt, Folicur and Nuvacron.
Outcomes	Extraction of residues of Folicur and Tilt from boronia leaves using supercritical fluids was successful. The technique was not successful in the extraction of residues of Nuvacron. Analytical methods were developed for the analyses of samples from preliminary field trials for residues of Sinbar (terbacil), Goal (oxyflurofen), Krovar (bromacil and diuron) Allicide (chloropham), Solicam (norflurazon), Stomp (pendimethalin) Lorsban (chlorpyriphos), Friontier (dimethenamid), Sertin (sethoxydim), Simazine Garlon (trichlopyr) Lontrel (clopyralid), Dicamba, MCPA, and Verdict (haloxyfop). The large number of herbicides investigated limited the extent of method validation undertaken for each such that results can only be used as an indicator as to which pesticides warranted further investigation in terms of weed control and contamination of oils with residues. The dissipation rate of the ai of Folicur, tebuconazole, and Tilt, propiconazole, in peppermint and boronia crops was established. Storage experiments indicated that pesticide residues do not degrade under the conditions trialed.
Implications	The results for the field trials highlight the potential for product contamination when introducing new herbicides in boronia crops. The high levels of residue detected in some of the essential oils monitored through the two years of this study emphasise the need for continued application of the protocols developed. The achievement of best agricultural practices, which is critical to continued success of the marketing of Australian produce, is reliant on the continued close liaison between research and industry
Publications	Dissipation of propiconazole and tebuconazole in Peppermint Crops (Mentha piperita (Labiatae)) and their Residues in Distilled Oils. J. Agric. and Food Chem. Vol 47, (1) 1999

Project Title	Enantiomeric composition of essential oils
RIRDC Project No.:	UT-15A
Researcher:	Professor Robert Menary
Organisation:	University of Tasmania, GPO Box 252C, HOBART TAS 7001
Phone:	03 6226 2723
Fax:	03 6226 7609
Email:	r.menary@utas.edu.au
Objectives	To investigate the application of chiral columns in gas chromatography (GC) to the elucidation of the enantiomeric composition of essential oils
Background	The adulteration of oils with synthetic derivatives and the bulking of extracts with oils from cheaper varieties, are methods that may be used to undercut the price of natural extracts. Oils may be authenticated through elucidation of the structural detail of the chemicals components. The orientation of atoms within a molecule is very specific. Molecules with the same molecular formula, but with one atom orientated differently about a carbon atom, can have very different properties. These molecules are said to be chiral and each form is called an enantiomer. The relative abundances of two enantiomeric forms are specific to the species and geographical origin of an essential oil. Correlating enantiomeric excesses of structurally related terpenes can elucidate biosynthetic pathways. New GC technology now facilitates the separation of enantiomers.
Research	Separation of enantiomeric pairs using chiral GC has been undertaken. The feasibility of differentiating the geographical or clonal origin of essential oils using this technique was investigated
Outcomes	Several limitations in the use of enantiomeric ratios in essential oil research were identified. Only volatiles of high optical purity and small ranges of enantiomeric excesses can be used to validated origin of oil components, or detect the blending of natural and synthetic flavour compounds. The mass spectra of enantiomers are identical and there is a paucity of data on the relative retention times and Kovat indices of chemicals on chiral columns. Peak identity is reliant on the availability of standards. Nevertheless, enantiomeric ratios have been shown to be a valuable criterion for differentiation of natural flavour compound from those of synthetic origin. The application of ?-cyclodextrin chiral columns to the separation of racemic mixes of enantiomer standards was tested. Baseline separation was achieved for a great number of enantiomers investigated. There was no obvious structural feature unique to chemicals which could not be resolved. Separation of enantiomers within the matrix of essential oils was achieved. Oils from a range of geographical and clonal origin were analysed. The technique was effective in establishing the profiles of the chiral components of oils. Preliminary results indicated that some boronia clones might be distinguished by the distribution of the enantiomers of some of the terpenes containing a chiral centre.
Implications	Single dimension GC methods proved to be robust and reproducible. The obvious direction for further investigation within this field is the application of multi-dimensional GC.

Project Title	Best Practice in Peppermint
RIRDC Project No.:	UT-16A
Researcher:	Professor Robert Menary
Organisation:	University of Tasmania, GPO Box 252C, HOBART TAS 7001
Phone:	03 6226 2723
Fax:	03 6226 7609
Email:	r.menary@utas.edu.au
Objectives	To understand why this was so, and to provide a management framework designed to lift yields from historical averages of less than 60kg/ha to more than 75-80kg/ha, at which level mint enterprises could compete effectively for market share on the one hand, and the available land on the other.
Background	Essential oil of peppermint is used extensively in confectionary and toothpaste preparations throughout the world, and in dollar terms is one of the most important of the very many essential oils used in the international flavour and fragrance trade. The Australian peppermint oil industry, based in Tasmania and northeastern Victoria, has been established for almost 20 years, but growers are still struggling to obtain yields which will enable them to compete effectively with the northeastern states of the US, where most of the world's mint oil is produced.
Research	An examination of current practices revealed: that planting material was often of poor quality, no practical benchmarks were being used to regulate irrigation and nitrogen fertiliser, the two most important inputs during the growing season, in many fields and for many growers, a lack of uniformity of inputs, and resulting variation in yield of herb and oil wase the main contributor to the poor overall yields, that many plantings lapsed after relatively few years of production, usually due to poor weed control, general loss of vigour or change of enterprise.
Outcomes	A working 'manual' was devised which focusses the attention of the mint manager on a limited number of key points during the production cycle, provides basic management guidelines and a documentation system to allow comparisons between sites, growers, districts and seasons. An important objective is to refine the guidelines each year, reset the benchmarks for management and yield, and to share experience and succesful strategies among Australian producers.
Implications	Several practical demonstrations of nitrogen and soil water monitoring were conducted on commercial crops during the project, and the results of these were used to establish the initial recommendations for production. The manual will be provided to current and future producers on the understanding that they meet all the recording and information exchange requirements of the plan, and participate in a redrafting of the plan at regular intervals.

Project Title	Improved Quality in Floral Products From Brown Boronia
RIRDC Project No.:	UT-21A
Researcher:	Prof RC Menary	Dr HS MacTavish
Organisation:	University of Tasmania GPO Box 252-54 HOBART Tasmania Australia 7001	University of Tasmania GPO Box 252-54 HOBART Tasmania Australia 7001
Phone:	03 6226 2723	03 6226 2650
Fax:	03 6226 7609	03 6226 7609
Email:	R.Menary@utas.edu.au	Hazel.MacTavish@utas.edu.au
Objectives	To increase the market share held by floral products from brown boronia by improving the range and quality of products through improved post-harvest handling and value-adding, and to quantify changes in bound volatiles and hydrolytic enzymes in boronia flowers throughout post-harvest incubations.
Background	As a result of previous work supported by RIRDC, several regulatory factors affecting the production of floral extract including volatiles such as ??ionone by brown boronia flowers during post-harvest incubation were identified. The need for commercial scale verification of the potential for adoption of post-harvest incubation practices on-farm was identified. Identifying the biosynthetic processes that produce this increase in volatiles after harvest is of paramount importance.
Research	Flower samples (18 kg) were incubated in shade cloth bags for up to 33 h after harvest under ambient conditions; flowers were extracted on a pilot-scale, absolutes were prepared, and the effect of post-harvest incubation on the 8 major volatiles was detailed. The effect of manually mixing flowers to replenish oxygen and avoid accumulation of excess thermal load under these conditions was studied by comparison with not mixing. Thirty six volatiles bound as glucosides (to sugars) or bound in other forms were quantified and the activity of boronia enzymes capable of releasing free volatiles from these bound forms were compared in flowers incubated at 12 and 30°C for up to 48 h after harvest.
Outcomes	Mixing flowers during pilot-scale post-harvest incubation did not affect temperature profiles, but did slow the rate of production of volatiles, probably due to reduced availability of oxygen. Increases in floral extract (20%) and volatiles (65%) occurred after 8-16 h with mixing, and after 24 h without mixing. Bound forms of volatiles were hydrolysed during the first 24 h of incubation as a result of increased activity of glycosidases and ‘volatile-forming enzymes’. Glucosides of volatiles formed after prolonged incubations, suggesting a role in catabolism.
Implications	The potential for technology transfer of laboratory scale results from post-harvest incubations to pilot-scale, on-farm incubations without regulated temperature chambers or novel, expensive equipment has been demonstrated. The products from on-farm post-harvest incubations have the potential to be marketed as new, since organoleptic properties are markedly different. We can now verify that hydrolysis of bound forms of volatiles produces the increase in free volatiles after harvest. Limitation of activity of transferases that produce glucosidically bound volatiles as a first step in catabolism of volatiles may represent a means to increase the magnitude or duration of post-harvest production of volatiles in boronia flowers. There is proven potential, at least on a laboratory scale, for re-extraction of pigment-free boronia volatiles after incubation of previously solvent-extracted flowers with either boronia enzymes or ??glucosidase.
Publications	MacTavish HS and Menary, RC (1999). Production of volatiles in brown boronia flowers after harvest: pilot-scale research. Annals of Botany, submitted MacTavish HS, Davies NW and Menary, RC (1999). Post-harvest incubation of boronia flowers: Changes in bound volatiles and hydrolytic enzymes. Journal of Horticultural Science and Biotechnology, submitted.

Last updated: 6 September 1999
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