Metabolomics in natural products research: application to in vivo bioactivity studies involving nutraceuticals

The analysis of urinary metabolite changes can provide information on the effects of food supplements or health-promoting products on healthy subjects or animal models. Specimen collection is non-invasive, long-term experiments can be easily conducted and urinary biomarkers of oxidative stress can also be measured, offering the opportunity to study the effects of a nutritional intervention and evaluate the redox status of the considered organism. In this thesis, three natural extracts were selected, namely Polygonum cuspidatum Sieb. et Zucc. (rich in resveratrol), Vaccinium macrocarpon Aiton (cranberry, rich in type A procyanidins) and green Coffea canephora Pierre ex Froehn. beans (GCBE, rich in chlorogenic acids), on the basis of their distribution on the market and on the basis of the information regarding their in vivo activity actually available in literature. In the first part of the work, the effects of P. cuspidatum dry extract were studied in healthy adult rats during a 49-days supplementation, using a combined 1H NMR and UPLC-HRMS metabolomics approach. Because of the reported antioxidant activity of resveratrol, the urinary amounts of two oxidative stress biomarkers were measured by targeted HPLC–MS/MS analyses and, due to the supposed “anti-aging” effects of resveratrol, multivariate models were designed in order to compare the aging effects between control and treated animals. Specific biomarkers were then selected and identified, and their amounts in urine were monitored throughout the experimental period. UPLC-MS metabolomics approaches were used to evaluate the mode of action of cranberry against uropathogenic Escherichia coli in two independent experiments, using an animal model and enrolling healthy adult volunteers, respectively. The experimental design was similar for the two trials, and the aim was to observe if the results obtained from the first animal experiment were reproducible in humans, being cranberry supplements claimed for treatment of UTIs in human consumers. In the first experiment, healthy Sprague−Dawley rats were orally supplemented with a standardized cranberry extract for 35 days, to mimic a prolonged consumption of cranberry by healthy subjects. 24-h urinary outputs were collected weekly during the experiment, and samples were subjected to UPLC−MS analysis using an untargeted approach. In a second experiment on the same animal model, a single dose of cranberry was administered to animals and the changes of urinary composition at 2, 4, 8, and 24 h after extract administration were monitored. Anti-adhesive properties of all the urine samples were studied. Furthermore, the markers related to cranberry intake were discovered using a multivariate data analysis approach. Finally, a specific chromatographic method was developed for the measurement of unmodified PAC-A in urine. In the experiment involving human volunteers, these consumed an oral sachet containing 360 mg of dry cranberry extract and 100 mg of quercetin. Urine samples were collected at 2, 4, 6, 8 ad 24 hours after product administration and the anti-adhesive properties of urine samples were tested using an in vitro assay on E. coli. In order to correlate possible observed bioactivity with modification of urinary composition, LC-MS-based targeted and untargeted metabolomics approaches were used. Finally, a clinical trial on a small number of healthy adult volunteers was performed to study the effects of a prolonged (30 days) supplementation with 400 mg of green coffee bean extract. The 24-h urinary samples were collected weekly, and analyzed by LC-MS. Multivariate data analysis approaches were applied and also targeted analysis were performed to measure urinary oxidative stress biomarkers, namely allantoin and 8-hydroxydeoxyguanosine (8-OHdG), in order to assess the potential antioxidant activity of GCBE in vivo

NMR Metabolomics of Foods – Investigating the Influence of Origin on Sea Buckthorn Berries, Brassica Oilseeds and Honey

The origin of foods plays an important role in their metabolome (the set of compounds present as products of metabolic events). The compositions of food plants are inevitably determined by a number of inherent and external factors – most importantly by the genotype (species, subspecies, cultivar, variety) and the prevailing conditions and weather parameters at each growth environment. The declaration of food origin can be defined and protected by law. The constantly increasing consumer awareness towards food origin, authenticity and quality has set the need for efficient tools for their verification. Metabolomics based on nuclear magnetic resonance (NMR) spectroscopy is increasingly being applied in analysing food composition and quality and in detecting food frauds and adulterations. The aim of the current work was to determine the influence of origin-related variables in food composition and quality by using 1H NMR metabolomics. The model foods – sea buckthorn (Hippophaë rhamnoides) berries, oilseeds of Brassica spp. and varietal honey – represent different foods with special sensory, nutritional, bioactive, commercial and national significance. The sea buckthorn berry metabolites were investigated in respect to the genotype (subspecies, cultivar) and geographical origin, with special emphasis on the influence of northern latitudes and related conditions. In the oilseeds, the inter-species variation and the influence of environmental and developmental stage on the seed composition was investigated. NMR profiling was applied in characterising the marker compounds for different honey types for botanical authentication. Multivariate analysis methods such as principal component (PCA) and discriminant analyses (PLS-DA, OPLS-DA) were applied in every sub-study to determine the key metabolites and origin-related factors characterising the food samples. The sea buckthorn subspecies were mainly distinguished by the relatively high content of ethyl-β-D-glucopyranoside (ssp. rhamnoides) and malic acid and vitamin C (ssp. sinensis). The northern latitude and respective conditions (the length of growth season, temperature, radiation and precipitation) was shown to alter the chemical composition of berries of the same genetic origin. In subarctic latitudes, the berries formed more ascorbic acid while the levels of ethyl glucose remained relatively low. The berries of cultivar 'Tytti' contained more ethyl glucoside while the berries of 'Terhi' contained more quinic acid in comparison. Calculated from the start of the growing season until harvest, the effective temperature sum (degree days) and the radiation sum correlated positively with ethyl glucoside that accumulated up to six-fold in overripe berries in southern Finland. The sea buckthorn berries (ssp. sinensis) grown at over 2000 m altitude contained typically more ascorbic and malic acids. The seeds of turnip rape was characterised by a relatively higher sucrose and polyunsaturated fatty acid content over oilseed rape that had a higher content of sinapine and oil in general. Growth conditions with reduced temperature added to the level of unsaturation in the oilseed lipids and delayed the seed development. The varietal honeys were classified with the aid of NMR profiling, as the typical sugar composition and other botanical markers were characterised. Also, previously unreported markers were designated for dandelion honeys. The correlations between complex food metabolomes and the origin-related variables were easily accomplished with NMR metabolomics. Especially, the effect of northern conditions on the growth place-dependent compositional flexibility (phenotypic plasticity) of the plant foods was deemed considerable. The results of this thesis can be further used to determine food quality, origin and authenticity and as an aid in plant breeding operations.Alkuperällä on suuri vaikutus elintarvikkeen metabolomiin eli aineenvaihduntatuotteiden kokonaisuuteen. Erityisesti kasviperäisten elintarvikkeiden koostumukseen vaikuttavat lukuisat sisäiset ja ulkoiset alkuperään liittyvät tekijät, kuten perimä (laji, alalaji, lajike) ja kasvupaikalle tyypilliset ympäristö- ja sääolosuhteet. Elintarvikkeen alkuperä voidaan määritellä ja suojata lainsäädännöllisin perustein. Kuluttajien kasvanut kiinnostus ja tietämys elintarvikkeiden alkuperää, aitoutta ja laatua kohtaan on lisännyt tehokkaiden ja luotettavien laadunvarmistusmenetelmien tarvetta. Varsinkin ydinmagneettista resonanssispektroskopiaan (NMR) perustuvaa metabolomiikkatutkimusta hyödynnetään yhä useammin elintarvikkeiden koostumuksen, laadun ja aitouden analysoinnissa. Tämän tutkimuksen tarkoituksena oli selvittää alkuperän vaikutusta tyrnimarjojen (Hippophaë rhamnoides), rypsin- ja rapsinsiementen (Brassica spp.) sekä lajihunajan koostumukseen 1H-NMR-metabolomiikan avulla. Nämä elintarvikkeet ovat kansallisesti ja kaupallisesti arvokkaita ja mielenkiintoisia niille tyypillisten aistittavien, ravitsemuksellisten ja bioaktiivisten ominaisuuksien ansiosta. Tyrnimarjojen koostumusta vertailtiin eri alalajien (ssp. rhamnoides ja ssp. sinensis) ja lajikkeiden ('Terhi' ja 'Tytti') sekä kasvupaikkojen (Suomi, Kiina, Kanada) välillä. Tavoitteena oli erityisesti selvittää, miten erityisesti pohjoisille leveysasteille tyypilliset olosuhteet vaikuttavat marjojen aineenvaihduntatuotteisiin. Öljysiementen kohdalla tutkittiin myös miten lajikohtainen perimä sekä kasvupaikan/-olosuhteiden ja siemenen kehittymisvaihe vaikuttavat siementen kemialliseen koostumukseen ja laatuun. Hunajien tapauksessa NMR-metabolomiikkaa hyödynnettiin kasvialkuperäkohtaisten sormenjälkiyhdisteiden tunnistamiseen ja kotimaisten lajihunajien kasvialkuperän varmentamiseen. Kaikissa osatutkimuksissa sovellettiin pääkomponentti-(PCA) ja diskriminanttianalyysiin (PLS-DA, OPLS-DA) perustuvia monimuuttujamenetelmiä tärkeimpien näyteryhmiä erottavien ja määrittävien yhdisteiden ja taustatekijöiden selvittämiseksi. Tyrnin alalajit erottuivat pääasiassa suhteellisesti korkean etyyli-β-D-glukopyranosidin (ssp. rhamnoides) sekä omenahappo- ja C-vitamiini-pitoisuuden (ssp. sinensis) perusteella. Pohjoisen leveysasteen ja sille tyypillisten olosuhteiden (kasvukauden pituus, lämpötila, säteily, sademäärä) todettiin muokanneen samaa geneettistä alkuperää olevien marjojen kemiallista koostumusta. Subarktisilla leveyksillä tyrnimarjaan muodostui enemmän askorbiinihappoa ja etyyliglukosidin määrä oli alhainen. 'Tytti'-lajikkeen marjat sisälsivät enemmän etyyliglukosidia, kun taas 'Terhi' sisälsi vastaavasti enemmän kviinihappoa. Kasvukauden tehoisa lämpösumma ja säteilysumma korreloivat positiivisesti etyyliglukosidin kanssa, jota kertyi ylikypsiin marjoihin Etelä-Suomessa jopa kuusinkertainen määrä kypsiin verrattuna. Yli 2000 metrin korkeudessa kasvaneissa tyrnimarjoissa (ssp. sinensis) oli tyypillisesti korkeampi omena- ja askorbiinihappopitoisuus. Suhteellisesti korkeampi sakkaroosipitoisuus ja monityydyttymättömien rasvahappojen osuus oli tyypillisempää rypsille, kun taas rapsi erottui rypsistä korkeamman öljy- ja sinapiinipitoisuuden perusteella. Kylmempi kasvupaikka lisäsi monityydyttymättömien rasvahappojen osuutta öljysiemenissä ja hidasti siemenen kehittymistä. NMR-profiloinnin avulla lajihunajat pystyttiin luokittelemaan kullekin hunajalle ominaisen sokerikoostumuksen ja muiden kasvialkuperästä kertovien merkkiyhdisteiden perusteella. Voikukkahunajalle tunnistettiin myös aiemmin raportoimattomia merkkiyhdisteitä. NMR-metabolomiikan avulla pystyttiin helposti selvittämään monimutkaisten aineenvaihduntatuotteiden kokonaisuuksien ja elintarvikkeen alkuperään liittyvien muuttujien välisiä yhteyksiä. Varsinkin pohjoisten kasvuolosuhteiden vaikutus kasviperäisten elintarvikkeiden koostumukselliseen vaihteluun oli huomattava. Väitöskirjan tuloksia voidaan hyödyntää elintarvikkeiden laadun, alkuperän ja aitouden varmistamisessa sekä kasvinjalostuksen apuna.Siirretty Doriast

Combined Untargeted and Targeted Fingerprinting by Comprehensive Two-Dimensional Gas Chromatography to Track Compositional Changes on Hazelnut Primary Metabolome during Roasting

This study focuses on the detectable metabolome of high-quality raw hazelnuts (Cory- lus avellana L.) and on its changes after dry-roasting. Informative fingerprinting was obtained by comprehensive two-dimensional gas chromatography with fast-scanning quadrupole mass spectrom- etry (GC×GC-qMS) combined with dedicated data processing. In particular, combined untargeted and targeted (UT) fingerprinting, based on pattern recognition by template matching, is applied to chromatograms from raw and roasted samples of Tonda Gentile Trilobata and Anakliuri hazelnuts harvested in Italy and Georgia. Lab-scale roasting was designed to develop a desirable organoleptic profile matching industrial standards. Results, based on 430 peak features, reveal that phenotype expression is markedly correlated to cultivar and pedoclimatic conditions. Discriminant components between cultivars are amino acids (valine, alanine, glycine, and proline); organic acids (citric, aspartic, malic, gluconic, threonic, and 4-aminobutanoic acids); and sugars and polyols (maltose, xylulose, xylitol, turanose, mannitol, scyllo-inositol, and pinitol). Of these, alanine, glycine, and proline have a high informational role as precursors of 2-acetyl- and 2-propionylpyrroline, two key-aroma com- pounds of roasted hazelnuts. Roasting has a decisive impact on metabolite patterns—it caused a marked decrease (−90%) of alanine, proline, leucine and valine, and aspartic and pyroglutamic acid and a −50% reduction of saccharose and galactose

Identification of markers for dietary intake and health status by GC-MS based metabolite profiling approaches

Markers are compounds that can be used as indicators of an exposure, a metabolic state, or any other effect. Metabolomics and metabolite profiling approaches for marker discovery will increasingly gain significance. In the context of food, diet, and health, these approaches allow among others the identification of dietary intake markers, which can complement and verify traditional dietary assessment methods in epidemiologic studies. Consequently, the investigation of associations between diet and health status in general, and also in particular diet-related diseases will be improved. Compared to classical biomarker studies, metabolomics enables a more comprehensive investigation of clinical markers for diagnosis, prognosis and monitoring of diseases, such as type 2 diabetes mellitus. Especially, early diagnosis in pre-disease states, where symptoms are not yet evident, are of particular interest. The aim of this thesis was to evaluate the application of GC-MS based metabolite profiling approaches for the identification of markers for dietary intake and health status. In this respect, volatile organic compounds and sugar compounds were analyzed to discover marker candidates in urine and plasma samples from a cross-sectional study with 300 participants, as well as from a human intervention study with diabetic, prediabetic and healthy participants. In the past, the search for markers of dietary intake mostly focused on non-volatile metabolites. To explore the potential of the volatilome, urine samples of a cross-sectional study were analyzed aiming to exemplary identify markers of coffee consumption using an untargeted HS-SPME-GC×GC-MS method. Six marker candidates were identified from a profile of 138 volatile organic compounds with the most robust represented by 3,4-dimethyl-2,5-furandione. Moreover, the correlation with the general dietary intake data highlighted the volatilome as a particularly interesting source for the detection of new dietary markers. The chromatographic separation of sugar compounds is often insufficient due to the high structural similarities. Therefore, in most studies common and well-known sugar compounds are analyzed in human body fluids. Within the scope of this thesis, a semitargeted GC-MS sugar profiling method was developed, which revealed a more complex sugar profile, both in urine and plasma, than described so far or expected. Rare sugar compounds such as psicose and trehalose were detected. However, the knowledge about their origin and presence in urine or plasma is limited to date. Moreover, the maltose concentration in urine was shown to be dependent on sex and menopause status (pre- and post-menopausal) a relationship with the vaginal microbiota is suggested here. In addition, the association of the urinary sugar profile with dietary intake data enabled the identification and confirmation of several new and also known marker candidates as for example, for consumption of avocado, dairy products and alcohol. The plasma sugar profiles of healthy, prediabetic and diabetic volunteers after an oral glucose tolerance test could be clearly distinguished, independent of glucose. Remarkably, a variety of sugar compounds showed marked postprandial differences dependent on health status. For example, trehalose showed a profile similar to the insulin-dependent profile of glucose. However, the origin and underlying biological mechanism for those sugar compounds remain to be elucidated. During the application of the one-dimensional GC-MS sugar profiling method to urine and plasma samples, it became evident that even more sugar compounds might be present, although in low concentrations, but were not detected due to limitations of the analytical method. Therefore, the one-dimensional method was transferred into a two-dimensional GC×GC-MS method. Improved sensitivity and separation finally enabled the detection of 84 instead of 55 sugar compounds in urine. The two-dimensional method was applied in an intervention study with apples, and revealed marker candidates for apple consumption for future validation. Overall, the results illustrate the benefit of a comprehensive analysis of sugar compounds in urine and plasma, including minor and rare sugar derivatives. The GC-MS based metabolite profiling approaches addressing the volatilome and the sugar profile, respectively, were demonstrated to be promising approaches for the identification of markers for dietary intake and health status. Future work should address the identification of unknown compounds, the adaptation of the GC×GC-MS sugar profiling method for quantitative purposes, and especially the validation of the identified marker candidates with respect to their suitability to more accurately assess dietary intake or diabetic state. High priority should also be given to the biochemical mechanisms and the origin of the compounds as well as their physiological or pathophysiological function in human metabolism.Marker sind Substanzen, die als Indikatoren für eine Exposition, einen metabolischen Zustand oder einen Effekt herangezogen werden. Metabolomics und Metabolite profiling-Ansätze gewinnen in der Markerforschung zunehmend an Bedeutung. Metabolomics ermöglicht die Identifizierung von Markern für den Lebensmittelverzehr, die in Zukunft unter anderem in epidemiologischen Studien zur Ergänzung und Überprüfung traditioneller Ernährungserhebungsmethoden Verwendung finden werden. In der Konsequenz können Zusammenhänge zwischen Ernährung und Gesundheit im Allgemeinen, sowie ernährungsabhängigen Erkrankungen im Speziellen, besser beschrieben werden. Außerdem können mittels Metabolomics auch Marker identifiziert werden, die im klinischen Rahmen eine Diagnose, Prognose oder Überwachung von Behandlungsmaßnahmen für eine Erkrankung ermöglichen, wie z.B. bei Typ 2 Diabetes mellitus. Von besonderem Interesse sind dabei Marker, die eine frühzeitige Diagnose, das heißt vor der Manifestation von Symptomen, ermöglichen. Ziel der vorliegenden Arbeit war es, die Verwendung von GC-MS basierten Metabolite profiling-Ansätzen zur Identifizierung von Markern für den Lebensmittelverzehr und den Gesundheitsstatus zu prüfen. Einen besonderen Schwerpunkt bildeten dabei volatile organische Verbindungen und Zuckerverbindungen, die in Urin- und Plasmaproben einer Querschnittsstudie mit 300 Probanden sowie einer humanen Interventionsstudie mit Diabetikern, Prädiabetikern und Gesunden analysiert wurden. Bei der bisherigen Suche nach Markern für den Lebensmittelverzehr lag das Augenmerk vor allem auf nicht-volatilen Metaboliten. Um das Potential des Volatiloms zu eruieren, wurden Urinproben einer Querschnittsstudie mithilfe einer ungerichteten HS-SPME-GC×GC-MS Methode analysiert und darin beispielhaft nach Markern für den Kaffeekonsum gesucht. Aus dem Urinprofil mit 138 volatilen Verbindungen wurden sechs plausible Kandidaten identifiziert, von denen sich 3,4-Dimethyl-2,5-furandion als der robusteste Marker erwies. Mittels einer Korrelationsanalyse anhand von Verzehrsdaten weiterer Lebensmittel wurde darüber hinaus gezeigt, dass das Volatilom eine vielversprechende Quelle neuer Marker für den Lebensmittelverzehr ist. Zucker lassen sich aufgrund ihrer strukturellen Ähnlichkeit häufig nur unzureichend chromatographisch trennen, daher werden in humanen Matrices bisher mehrheitlich nur wenige bekannte Zuckerverbindungen erfasst. Im Rahmen dieser Arbeit wurde eine semi-gerichtete GC-MS Zuckerprofiling-Methode entwickelt, mit der gezeigt werden konnte, dass das humane Zuckerprofil im Urin und im Plasma erheblich komplexer ist, als bisher beschrieben und angenommen. Verschiedene Zuckerverbindungen, wie beispielsweise Psicose oder Trehalose, über deren Herkunft und Vorhandensein im Urin oder in Plasma fast nichts bekannt ist, wurden nachgewiesen. Im Urin zeigten sich darüber hinaus Unterschiede in der Maltosekonzentration in Abhängigkeit vom Geschlecht sowie dem prä- und postmenopausalen Status, die vermutlich im Zusammenhang mit der vaginalen Mikrobiota stehen. Die Assoziation der Zuckerprofile mit dem Lebensmittelverzehr ermöglichte zudem die Identifizierung neuer und Bestätigung bekannter Marker, beispielsweise für den Verzehr von Avocado und Milchprodukten, sowie für Alkoholkonsum. Im Plasma von Gesunden, Prädiabetikern und Diabetikern wurden nach einem oralen Glucosetoleranztest deutliche Unterschiede im Zuckerprofil festgestellt. Interessanterweise zeigten eine Reihe neuer Zuckerverbindungen markante postprandiale Unterschiede abhängig vom Gesundheitszustand. Beispielsweise zeigte Trehalose ein ähnliches Profil wie die insulinabhängige Glucose. Jedoch ist weder über den Mechanismus noch zur Herkunft dieser Zucker etwas bekannt. Bereits die bisherigen Ergebnisse des Zuckerprofilings in Urin und Plasma zeigten, dass zusätzliche Zuckerverbindungen, wenn auch in sehr geringer Konzentration, vorhanden sind. Daher wurde die eindimensionale Methode zu einer zweidimensionalen GC×GC-MS-Methode mit verbesserter Sensitivität und Trennung weiterentwickelt, was nun die Erfassung von 84 statt 55 Zuckerverbindungen in Urin ermöglicht. Erste Auswertungen der Messdaten einer Interventionsstudie mit Äpfeln zeigten, dass diese Methode die Identifizierung von potentiellen Markern für den Verzehr von Äpfeln ermöglicht. Die Ergebnisse verdeutlichen, welches Potential in der umfassenden Analyse von Zuckern, einschließlich seltener Verbindungen, steckt. GC-MS basierte Metabolite profiling-Ansätze, wie hier für das Volatilom und das Zuckerprofil gezeigt, sind geeignete Methoden für die Identifizierung von Markern des Lebensmittelverzehrs und des Gesundheitsstatus. Die Identifizierung bisher unbekannter Verbindungen, die Weiterentwicklung der Zuckeranalytik zu einer quantitativen Methode und insbesondere die Validierung der identifizierten Marker bezüglich ihrer Eignung, den Lebensmittelverzehr bzw. den diabetischen Status akkurater zu erfassen, sind zukünftige Ziele. Besonders herausfordernd ist es dabei, die mechanistischen Zusammenhänge aufzuklären, insbesondere im Hinblick auf Herkunft, Vorhandensein und Funktion der detektierten Zuckerverbindungen im menschlichen Metabolismus

Incorporating standardised drift-tube ion mobility to enhance non-targeted assessment of the wine metabolome (LC×IM-MS)

Liquid chromatography with drift-tube ion mobility spectrometry-mass spectrometry (LCxIM-MS) is emerging as a powerful addition to existing LC-MS workflows for addressing a diverse range of metabolomics-related questions [1,2]. Importantly, excellent precision under repeatability and reproducibility conditions of drift-tube IM separations [3] supports the development of non-targeted approaches for complex metabolome assessment such as wine characterisation [4]. In this work, fundamentals of this new analytical metabolomics approach are introduced and application to the analysis of 90 authentic red and white wine samples originating from Macedonia is presented. Following measurements, intersample alignment of metabolites using non-targeted extraction and three-dimensional alignment of molecular features (retention time, collision cross section, and high-resolution mass spectra) provides confidence for metabolite identity confirmation. Applying a fingerprinting metabolomics workflow allows statistical assessment of the influence of geographic region, variety, and age. This approach is a state-of-the-art tool to assess wine chemodiversity and is particularly beneficial for the discovery of wine biomarkers and establishing product authenticity based on development of fingerprint libraries

Expression of multidisciplinary flavour science : proceedings of the 12th Weurman Symposium

The 12th Weurman Flavour Research Symposium contributed 177 lectures and posters to the wealth of flavor knowledge; these were presented in eight sessions: biology, retention and release, psychophysics, quality, thermal generation, bioflavors, impact molecules, and analytics. Emerging topics were discussed in three workshops dealing with flavor and health, in vivo flavor research, and flavor metabolomics. It has been an excellent forum for passionate exchange of recent results obtained in traditional and emerging fields of flavor research. The symposium allowed coverage of the broad diversity of flavor-related topics: comprising odor and taste; applying targeted and holistic approaches; using sensorial, chemical, biological, physical, and chemometric techniques; as well as considering nutrition and health aspects

Expression of multidisciplinary flavour science : proceedings of the 12th Weurman Symposium

The 12th Weurman Flavour Research Symposium contributed 177 lectures and posters to the wealth of flavor knowledge; these were presented in eight sessions: biology, retention and release, psychophysics, quality, thermal generation, bioflavors, impact molecules, and analytics. Emerging topics were discussed in three workshops dealing with flavor and health, in vivo flavor research, and flavor metabolomics. It has been an excellent forum for passionate exchange of recent results obtained in traditional and emerging fields of flavor research. The symposium allowed coverage of the broad diversity of flavor-related topics: comprising odor and taste; applying targeted and holistic approaches; using sensorial, chemical, biological, physical, and chemometric techniques; as well as considering nutrition and health aspects