Volatile Metabolite Profiles Research Articles

Chemical Variation in Essential Oil Profiles Detected Using Headspace Solid-Phase Microextraction Gas Chromatography Spectrometry in Response to Potassium, Nitrogen, and Water Available to Micropropagated Plants of Salvia stenophylla (Burch. ex Benth.)

The effects of nitrogen, potassium, water stress, and phytohormones were studied using a Salvia stenophylla (Burch. ex Benth.) microplant system. In vitro regeneration was monitored and then followed with volatile secondary metabolite profiling through headspace solid-phase microextraction gas chromatography. Plantlet growth was most prolific on half-strength Murashige and Skoog medium without phytohormones. An increase in macronutrients supplied to the microplants enhanced accumulation of the commercially important (−)-α-bisabolol, while no significant changes to the relative abundance of β-bisabolene, α-muurolene, α-patchoulene, and D-limonene (among others) became apparent. Water-stressed plants, treated with sorbitol and polyethylene glycol, had a lowered rooting capacity in vitro. Overall, as a plant production system, micropropagation did not have deleterious effects on the biochemistry of S. stenophylla as no significant differences in metabolic profiles existed between conventional garden plants and in vitro propagules, regardless of phytohormone treatment. We also show that nutrient manipulation can be used efficiently as a strategy for positively altering secondary metabolism. This will ultimately benefit the domestication of this commercially important medicinal herb.

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Metabolic engineering of geranic acid in maize to achieve fungal resistance is compromised by novel glycosylation patterns

Many terpenoids are known to have antifungal properties and overexpression of these compounds in crops is a potential tool in disease control. In this study, 15 different mono- and sesquiterpenoids were tested in vitro against two major pathogenic fungi of maize ( Zea mays), Colletotrichum graminicola and Fusarium graminearum. Among all tested terpenoids, geranic acid showed very strong inhibitory activity against both fungi (MIC<46 μM). To evaluate the possibility of enhancing fungal resistance in maize by overexpressing geranic acid, we generated transgenic plants with the geraniol synthase gene cloned from Lippia dulcis under the control of a ubiquitin promoter. The volatile and non-volatile metabolite profiles of leaves from transgenic and control lines were compared. The headspaces collected from intact seedlings of transgenic and control plants were not significantly different, although detached leaves of transgenic plants emitted 5-fold more geranyl acetate compared to control plants. Non-targeted LC–MS profiling and LC–MS–MS identification of extracts from maize leaves revealed that the major significantly different non-volatile compounds were 2 geranic acid derivatives, a geraniol dihexose and 4 different types of hydroxyl-geranic acid-hexoses. A geranic acid glycoside was the most abundant, and identified by NMR as geranoyl-6- O-malonyl-β- d-glucopyranoside with an average concentration of 45 μM. Fungal bioassays with C. graminicola and F. graminearum did not reveal an effect of these changes in secondary metabolite composition on plant resistance to either fungus. The results demonstrate that metabolic engineering of geraniol into geranic acid can rely on the existing default pathway, but branching glycosylation pathways must be controlled to achieve accumulation of the aglycones.

Impact of the synbiotic combination of Lactobacillus casei shirota and oligofructose‐enriched inulin on the fecal volatile metabolite profile in healthy subjects

Hypothesis-driven approaches have mainly focused on the quantification of SCFAs as mediators of beneficial effects of synbiotics. However, the emergence of metabolite profiling strategies allows to evaluate the colonic metabolism from a top-down approach. In the present study, we evaluated the impact of a synbiotic combination on fecal metabolite profiles. A synbiotic combination (Lactobacillus casei Shirota cells+oligofructose-enriched inulin) was evaluated in nine healthy volunteers. Before the start, during and after 4-wk treatment, fecal samples were obtained. GC-MS technology was applied to analyze the volatile metabolites. Application of a Type III test revealed that the metabolite profiles from the three conditions were significantly different. We identified three volatile organic compounds, acetate, dimethyl trisulfide and ethyl benzene, which were significantly affected. The acetate levels increased, whereas the dimethyl trisulfide levels decreased during and after the intervention. For ethyl benzene only an effect during the synbiotic intervention period was observed. We report a detailed analysis of the influence of L. casei Shirota combined with oligofructose-enriched inulin on fermentation metabolites. Our results indicated a stimulation of saccharolytic fermentation and, importantly, a reduction of potentially toxic protein fermentation metabolites dimethyl trisulfide and ethyl benzene attended these effects.

Effectiveness of different solid-phase microextraction fibres for differentiation of selected Madeira island fruits based on their volatile metabolite profile—Identification of novel compounds

A headspace solid-phase microextraction (HS-SPME) procedure based on five commercialised fibres (85 μm polyacrylate – PA, 100 μm polydimethylsiloxane – PDMS, 65 μm polydimethylsiloxane/divinylbenzene – PDMS/DVB, 70 μm carbowax/divinylbenzene – CW/DVB and 85 μm carboxen/polydimethylsiloxane – CAR/PDMS) is presented for the characterization of the volatile metabolite profile of four selected Madeira island fruit species, lemon ( Citrus limon), kiwi ( Actinidia deliciosa), papaya ( Carica papaya L.) and Chickasaw plum ( Prunus angustifolia). The isolation of metabolites was followed by thermal desorption gas chromatography–quadrupole mass spectrometry (GC–qMS) methodology. The performance of the target fibres was evaluated and compared. The SPME fibre coated with CW/DVB afforded the highest extraction efficiency in kiwi and papaya pulps, while in lemon and plum the same was achieved with PMDS/DVB fibre. This procedure allowed for the identification of 80 compounds, 41 in kiwi, 24 in plums, 23 in papaya and 20 in lemon. Considering the best extraction conditions, the most abundant volatiles identified in kiwi were the intense aldehydes and ethyl esters such as (E)-2-hexenal and ethyl butyrate, while in Chicasaw plum predominate 2-hexenal, 2-methyl-4-pentenal, hexanal, (Z)-3-hexenol and cyclohexylene oxide. The major compounds identified in the papaya pulp were benzyl isothiocyanate, linalool oxide, furfural, hydroxypropanone, linalool and acetic acid. Finally, lemon was shown to be the most divergent of the four fruits, being its aroma profile composed almost exclusively by terpens, namely limonene, γ-terpinene, o-cymene and α-terpinolene. Thirty two volatiles were identified for the first time in the fruit or close related species analysed and 14 volatiles are reported as novel volatile metabolites in fruits. This includes 5 new compounds in kiwi (2-cyclohexene-1,4-dione, furyl hydroxymethyl ketone, 4-hydroxydihydro-2(3H)-furanone, 5-acetoxymethyl-2-furaldehyde and ethanedioic acid), 4 in plum (4-hydroxydihydro-2(3H)-furanone, 5-methyl-2-pyrazinylmethanol, cyclohexylene oxide and 1-methylcyclohexene), 4 in papaya (octaethyleneglycol, 1,2-cyclopentanedione, 3-methyl-1,2-cyclopentanedione and 2-furyl methyl ketone) and 2 in lemon (geranyl farnesate and safranal). It is noteworthy that among the 15 volatile metabolites identified in papaya, 3-methyl-1,2-cyclopentanedione was previously described as a novel PPARγ (peroxisome proliferator-activated receptor γ) agonist, having a potential to minimize inflammation.

Phytochemical variability of populations of Aloysia citriodora from Argentina

Aloysia citriodora Palau (Verbenaceae) is one of the most widely used herbs in the Argentine traditional medicine as antispasmodic, sedative and for the treatment of stomachache. Its worldwide use has promoted its cultivation not only in our country but also in several Latin American countries, Europe and Asia. There are official monographs in Argentine (FA) and European Pharmacopoeias (PhEU, as „lemon verbena) for the pharmaceutical quality control. Our working group is devoted to update the respective monograph of the FA, taking in account the detected phytochemical variability of local populations. Parameters for a quality profile of volatile metabolites were previously established [1,2]. At this time we have evaluated the phytochemical variability of several populations using the technique by the European Pharmacopoeia (HPLC), in order to identify the best materials for crops affordable for the pharmaceutical industry. We analyzed 22 populations from 12 regions from Argentina, both in culture and wild materials. Our results show significant differences in the contents of verbascoside: from 0.5 to 4.8%, being 2.5% the minimum accepted value in the PhEu. Other peaks were detected in the HPLC profiles, some of them with similar UV spectra as verbascoside, meanwhile others with polyphenolic UV pattern. Therefore, in vitro assays will be undertaken to determine if the quantity of verbascoside itself affects proportionally the pharmacological activity.

Target list building for volatile metabolite profiling of fruit

Fruit flavour is the combination of numerous biochemicals: sugars for sweetness, acids for sourness and volatile metabolites for aroma. The objective of this study was to establish a method to develop a target list of statistically relevant compounds for the characterization of melon from non-targeted data, while preserving the profile information. Five different varieties were sampled (sampling 12 biological replicates from 12 plants) using dynamic headspace extraction, then analysed by gas chromatography–mass spectrometry in full scan mode. Using Metalign and SIMCA-P software the raw data was spectrally aligned and then subjected to principal component analysis (PCA). The principal component analysis plot showed good separation of the five varieties based on their full scan GC–MS profile. Mass spectral data points responsible for the differences between varieties were highlighted by further statistical analysis. The mass spectra were then reconstructed and the corresponding chemicals identified using library search or reference standards were available to create a new target component list. To validate the new target list, the initial data set was re-processed using the targeted approach and the results subjected again to principal component analysis. The two representations showed excellent agreement on the separation of the five varieties. The new target list obtained from this study can be applied to differentiate and characterize the volatile profile of melon varieties using a list of statistically significant compounds.

Identification and profiling of volatile metabolites of the biocontrol fungus Trichoderma atroviride by HS-SPME-GC-MS

In the present study we describe a method, which is based on solid phase microextraction (SPME) coupled to gas chromatography–mass spectrometry (GC-MS) and which can be used for the profiling of microbial volatile organic compounds (MVOCs) in the headspace (HS) of cultures of filamentous fungi. The method comprises the following successive steps: 1. growth of the fungus on a solid culture medium directly in headspace vials, 2. measurement of volatiles by HS-SPME-GC-MS, 3. deconvolution of mass spectra, 4. identification of volatiles by comparison of measured, deconvoluted mass spectra and linear temperature programmed retention indices (LTPRI) on two stationary GC phases with database entries and LTPRI published in the literature, and 5. profiling of the identified MVOCs. The developed method was successfully applied to cultures of the biocontrol fungus Trichoderma atroviride. An in-house library consisting of mass spectra and LTPRI values of fungal VOCs was established and used to study the profiles of MVOCs of this fungus. In total, 25 different MVOCs were identified by applying strict criteria (spectral match factor at least 90% and a maximum relative deviation of LTPRI of ± 2% from literature values). The MVOCs were assigned to the compound classes of alcohols, ketones, alkanes, furanes, pyrones (mainly the bioactive 6-pentyl-alpha-pyrone), mono- and sesquiterpenes, 13 of which have never been reported to be produced by Trichoderma spp. before. Eleven of these volatiles have been additionally confirmed using authentic standards. Finally, time course experiments and cultivation of T. atroviride in the presence of the mycotoxin fusaric acid demonstrated the potential of the method to study the dynamics of MVOC profiles as well as the effect of different environmental/biological conditions on the expression of MVOCs of filamentous fungi.

Misidentification of Tansy, Tanacetum Macrophyllum, as Yarrow, Achillea Grandifolia: A Health Risk or Benefit?

Tansy, Tanacetum macrophyllum (Waldst. & Kit.) Sch. Bip., is often misidentified by herb collectors as yarrow, Achillea grandifolia Friv. With the former, cases of poisoning induced by its ingestion are well documented, but the latter is widely used for ethnopharmacological purposes. The aim of this study was to estimate, based on the volatile metabolite profiles of the two species, the potential health risk connected with their misidentification. GC and GC-MS analysis of the essential oils hydrodistilled using a Clevenger-type apparatus from A. grandifolia, T. macrophyllum, and two plant samples (reputedly of A. grandifolia, but in fact mixtures of A. grandifolia and T. macrophyllum) obtained from a local market, resulted in the identification of 215 different compounds. The main constituents of A. grandifolia oil were ascaridole (15.5%), alpha-thujone (7.5%), camphor (15.6%), borneol (5.2%) and (Z)-jasmone (6.4%), and of T. macrophyllum oil, 1,8-cineole (8.6%), camphor (6.4%), borneol (9.1%), isobornyl acetate (9.5%), copaborneol (4.2%) and gamma-eudesmol (6.2%). The compositions of the oils extracted from the samples obtained from the market were intermediate to those of A. grandifolia and T. macrophyllum. Significant differences in the corresponding volatile profiles and the literature data concerning the known activities of the pure constituents of the oils, suggested that the pharmacological action of the investigated species (or their unintentional mixtures) would be notably different. It seems, however, that misidentification of T. macrophyllum as A. grandifolia does not represent a health risk and that the absence of the toxic alpha-thujone from T. macrophyllum oil may in fact be regarded as a benefit.

Effect of tomato pleiotropic ripening mutations on flavour volatile biosynthesis

Ripening is a tightly controlled and developmentally regulated process involving networks of genes, and metabolites that result in dramatic changes in fruit colour, texture and flavour. Molecular and genetic analysis in tomato has revealed a series of regulatory genes involved in fruit development and ripening, including MADS box and SPB box transcription factors and genes involved in ethylene synthesis, signalling and response. Volatile metabolites represent a significant part of the plant metabolome, playing an important role in plant signalling, defence strategies and probably in regulatory mechanisms. They also play an important role in fruit quality. In order to acquire a better insight into the biochemical and genetic control of flavour compound generation and links between these metabolites and the central regulators of ripening, five pleiotropic mutant tomato lines were subjected to volatile metabolite profiling in comparison with wild-type Ailsa Craig. One hundred and seventeen volatile compounds were identified and quantified using SPME (Solid Phase Microextraction) headspace extraction followed by Gas Chromatography–Mass Spectrometry (GC–MS) and the data were subjected to multivariate comparative analysis. We find that the different mutants each produce distinct volatile profiles during ripening. Through principal component analysis the volatiles most dramatically affected are those derived from fatty-acids. The results are consistent with the suggestion that specific isoforms of lipoxygenase located in the plastids and the enzymes that provide precursors and downstream metabolites play a key role in determining volatile composition.

Headspace sorptive extraction and GC-TOFMS for the identification of volatile fungal metabolites

Volatiles from fungi cultivated in Petri dishes were collected by a simple headspace polydimethylsiloxane (PDMS) sorptive extraction technique (HSSE), thermally desorbed into a gas chromatographic capillary column and detected and identified by gas chromatography–time-of-flight mass spectrometry (GC-TOFMS). The method was used to compare metabolite profiles of seven species of fungi grown on two types of sterile agars — potato dextrose and Sabouraud dextrose. Three species from the genus Penicillium ( P. italicum, P. camemberti, and P. roqueforti) and four outgroups, each from a different phylum ( Saprolegnia sp.; Sordaria fimicola, wild-type; Coprinus cinereus; and Rhizopus stolonifer) were grown on the two types of agars and analyzed. Multivariate analysis (PCA) was used to determine whether separate classes of fungi can be distinguished from one another based on their metabolite profiles. PCA showed clear class separation between the three Penicillium samples and the outgroups. Slight differences were observed in metabolite profiles as a function of growth medium. HSSE/GC-TOFMS appears to be a relatively simple and accurate technique for classification of fungi based on their volatile metabolite profiles. The volatiles sampling technique reported here is non-destructive, so it can be applied with traditional methods for studying fungal growth and metabolism.

Differentiation of Closely Related Fungi by Electronic Nose Analysis

In this work the potential of electronic nose analysis for differentiation of closely related fungi has been described. A total of 20 isolates of the cheese-associated species Geotrichum candidum, Penicillium camemberti, P. nordicum, and P. roqueforti and its closely related species P. paneum, P. carneum as well as the noncheese-associated P. expansum have been investigated by electronic nose, GC-MS, and LC-MS analysis. The isolates were inoculated on yeast extract sucrose agar in 20-mL headspace flasks and electronic nose analysis was performed daily for a 7-d period. To assess which volatile metabolites the electronic nose potentially responded to, volatile metabolites were collected by diffusive sampling overnight onto tubes containing Tenax TA, between the 7th and 8th day of incubation. Volatiles were analyzed by gas chromatography coupled to mass spectrometry and the results indicated that mainly alcohols (ethanol, 2-methyl-1-propanol, and 3-methyl-1-butanol) and ketones (acetone, 2-butanone, and 2-pentanone) were produced at this stage. The volatile metabolite profile proved to be species specific. Nonvolatile metabolites were collected on the 8th day of incubation and mycotoxin analysis was performed by high pressure liquid chromatography coupled to a diode array detector and a time of flight mass spectrometer. Several mycotoxins were detected in samples from the species P. nordicum, P. roqueforti, P. paneum, P. carneum, and P. expansum. Differentiation of closely related mycotoxin producing fungi incubated on yeast extract sucrose agar has been achieved, indicating that there is a potential for predicting production of mycotoxins on food and feedstuffs by electronic nose analysis.

Prediction of Penicillium expansum Spoilage and Patulin Concentration in Apples Used for Apple Juice Production by Electronic Nose Analysis

Classification models for Penicillium expansum spoilage of apples and prediction models for patulin concentration in apples usable for apple juice production were made on the basis of electronic nose (e-nose) analysis correlated to HPLC quantification of patulin. A total of 15 Golden Delicious and 4 Jonagold apples were surface sterilized and divided into three groups per variety. The Golden Delicious group consisted of five apples each. Group 1 was untreated control, group 2 was surface inoculated with P. expansum, and group 3 was inoculated in the core with P. expansum. The apples were incubated at 25 degrees C for 10 days. E-nose analysis was performed daily. At day 10 the Golden Delicious apples were individually processed for apple juice production. During apple juice production the mash and juice were analyzed by e-nose, and samples were taken for patulin analysis by HPLC. The volatile metabolite profile was obtained by collection of volatile metabolites, on tubes containing Tenax TA, overnight between the 9th and 10th days of incubation and subsequent analysis of the collected compounds by GC-MS. Prediction models using partial least-squares, with high correlation, for prediction of patulin concentration in shredded apples as well as apple juice were successfully created. It was also shown that it is possible to classify P. expansum spoilage in apples correctly on the basis of soft independent modeling of class analogy classification of e-nose analysis data. To the authors' knowledge this is the first report of a regression model between e-nose data and mycotoxin content in which actual concentrations are reported. This implies that it is possible to predict mycotoxin production and concentration by e-nose analysis.

Detection and discrimination of two fungal diseases of mango (cv. Keitt) fruits based on volatile metabolite profiles using GC/MS

Volatile organic compounds collected from the headspace of mango cv. Keitt inoculated with Lasiodiplodia theobromae (stem-end rot), Colletotrichum gloeosporioides (anthracnose), mock (as the first control) and non-wounded-non-inoculated mango (as the second control) were analyzed using GC/MS to investigate the feasibility of automatic detection and diagnosis of diseases of mango in stores. A total of 37 metabolites, relatively consistent in 8 replicates, were identified based on mass spectral match using NIST library. Several of these were specific to a disease/inoculation or when common to all treatments, they varied in their abundances. 1-Pentanol was specific to Lasiodiplodia-inoculated mangoes while thujol was detected only in Colletotrichum-inoculated mangoes. Discriminant analysis models based on normalized abundances of 35 consistent metabolites and normalized abundances of 150 mass ions correctly classified 67 and 75% of the observations, respectively, based on cross-validation. The study has shown for the first time that it is possible to detect and differentiate between anthracnose and stem-end rot diseases of mangoes (cv. Keitt) based on their volatile production patterns using GC/MS. The methods developed here have the potential applications to mango industry to detect and diagnose diseases of mango fruits, at relatively early stages of the disease progress, after validation under commercial conditions.

Volatile metabolite profiling to detect and discriminate stem‐end rot and anthracnose diseases of mango fruits

The volatile metabolites from the headspace gas of containerised mango (Mangifera indica) cv. Tommy Atkins fruits, surface wounded and inoculated with the two fungal anamorphic pathogens Colletotrichum gloeosporioides and Lasiodiplodia theobromae, or non‐inoculated (controls), were profiled using a portable gas chromatograph/mass spectrometer to discriminate diseases of mango. Thirty‐four compounds were detected relatively consistently among replicates. Several of these were disease/inoculation‐discriminatory and were classified into three groups: (i) compounds unique to only one treatment; (ii) compounds common to two or more treatments, but not to all; and (iii) compounds common to all treatments, but varying in their abundance. Two compounds, 1‐pentanol and ethyl boronate, were detected in L. theobromae‐inoculated mangoes alone, while thujol was observed only in C. gloeosporioides‐inoculated mangoes. Discriminant analysis models based on the abundance of significant mass ions and consistent compounds correctly classified diseases/inoculations in up to 100% of cases. The disease‐discriminatory compounds and discriminant analysis models developed here have the potential to be used in the early detection of postharvest diseases of mango fruits after validation under commercial conditions.

Annual Meeting, Edmonton, Alberta, 2005

Annual Meeting, Edmonton, Alberta, 2005

Differentiation of Species from the Penicillium roqueforti Group by Volatile Metabolite Profiling

Species from the Penicillium roqueforti group were differentiated by volatile metabolite profiling primarily of sesquiterpenes. A total of 24 isolates from species P. roqueforti, Penicillium carneum, and the recently described species Penicillium paneum were inoculated on yeast extract sucrose agar. Volatile metabolites were collected by diffusive sampling onto tubes containing Tenax TA, overnight between the fifth and sixth days of incubation. Volatiles were thermally desorbed and analyzed by gas chromatography coupled to mass spectrometry. The sesquiterpene area of the chromatogram was investigated, and potential sesquiterpenes were tabulated by comparison of their Kovats retention index and mass spectrum. In general, P. carneum isolates produced the lowest number of sesquiterpenes, all of which were unique for P. carneum within the P. roqueforti group. P. roqueforti and P. paneum produced a larger variety of volatile metabolites, some of which they have in common and some of which are unique for the two species. (+)-Aristolochene was found in samples from P. paneum and P. roqueforti. Other Penicillium species in which (+)-aristolochene was also detected were P. commune, P. glandicola, and P. solitum.

Volatile metabolite profiling to discriminate diseases of McIntosh apple inoculated with fungal pathogens

AbstractGas chromatography–mass spectrometry (GC/MS) technology was used to profile volatile metabolites from the headspace gas of McIntosh apple (Malus domestica Borkh.), which were non‐inoculated or inoculated with four different fungi, Botrytis cinerea Pers, Penicillium expansum Link, Mucor piriformis Fischer and Monilinia sp. The study yielded a total of 498 different volatile metabolites. Among them only 35 occurred relatively consistently in six replicates over three incubation periods. Of the consistent metabolites, 20 were specific to one or more inoculation agents/diseases, including seven that were unique to apples inoculated with different pathogens. Fluoroethene and 3,4‐dimethyl‐1‐hexene were specific for Penicillium, while butanoic acid butyl ester, 4‐methyl‐1‐hexene and 2‐methyltetrazole were specific for Mucor. Similarly, acetic acid methyl ester and fluoroethane were specific to Botrytis and Monilinia, respectively. The method developed in this study can be used by storage managers to detect apple diseases at an early stage of disease progress and use this to manage apple diseases in storage, after further validation under commercial conditions. Copyright © 2004 Society of Chemical Industry

The Canadian Phytopathological Society Annual Meeting, Montréal, Quebec, 2003

The Canadian Phytopathological Society Annual Meeting, Montréal, Quebec, 2003

Comparison of different methods for collection of volatile chemical markers from fungi

Volatile metabolites from Penicillium vulpinum, cultured on Czapek yeast autolysate agar and broth, were collected by three different methods: (1) diffusive sampling from headspace, (2) purging and trapping of headspace gases, and (3) steam distillation extraction (SDE) of biomass and liquid medium. The qualitative nature and semiquantitative amounts of mainly unsaturated compounds like mono- and sesquiterpenes collected by diffusive sampling were comparable to volatiles collected by purging and trapping. However, the quantitative profiles of volatiles changed towards the lower boiling components with low purge flows, probably due to accumulation of carbon dioxide, demonstrating that the actual purge flow to a great extent determines the quantitative profile of volatile metabolites produced. SDE gave rise to a very different profile compared to the two other methods. None of the major volatiles liberated to the surroundings during growth were dominant in SDE extracts. Instead, lipid degradation products like 1-octene-3-ol and 3-octanone were dominating. The latter method is therefore not recommended as a screening method for volatile chemical markers of active fungal growth.