Dynamics of cork mycobiota throughout stopper manufacturing process: from diversity to metabolite

Dissertation presented to obtain the Ph.D degree in BiologyCork, the continuous layer of outer bark of the Quercus suber L. tree, has physical and chemical properties that are unique. Portugal possesses 33 % of the world’s cork oak forests and accounts for approximately half of total global cork production. The manufacture of cork discs (or stoppers) comprises several stages, including two boiling stages, during which slabs of cork are steeped in boiling water. In days following the boiling the humidity of the slabs decreases and they become completely covered in a white mycelium of Chrysonilia sitophila until the cork achieves a certain water activity level (ca 0.9 aw). Below this level other fungal species (e.g. Penicillium, Aspergillus or Trichoderma) can germinate and shift the fungal colonization of the cork slabs. The two main objectives of the research described in the presented PhD thesis are (1) a taxonomic identification of the mycobiota present in cork slabs throughout the manufacture of cork discs, and (2) an investigation into the chemical compounds, which can give unfavourable properties to the cork, produced by these fungi.(...)FCT Fundação para a Ciência e Tecnologia with the grant BD/19264/ 2004

Cork taint of wines: the formation, analysis, and control of 2,4,6- trichloroanisole

Cork taint has devastating effects on the aroma and quality of the wine, which can cause an annual loss of may be up to more than one billion dollars. There are many causes of cork taint, but 2,4,6-trichloroanisole (2,4,6-TCA) is a major contributor, giving the wine a wet-moldy smell. This study provided a comprehensive overview of the occurrence, detection, and control/remediation of 2,4,6-TCA. The occurrence and formation mechanisms of 2,4,6-TCA mainly include microbial O-methylation of chlorophenols and chlorination of anisole. The source of 2,4,6-TCA in wine is the cork or other woodworks, but it is also possible to contaminate wine from the environment. Due to the extremely low odor threshold concentration of 2,4,6-TCA, the effective sample pre-enrichment for instrument identification and quantification is more important. The control/remediation strategies of 2,4,6-TCA mainly include eliminating 2,4,6-TCA in cork and removing 2,4,6-TCA from wine by adsorption. Finally, the challenges and possible future research directions in this research field were discussed and proposed

Métodos de determinación de TCA (tricloroanisoles)

Uno de los principales defectos organolépticos más importantes en el vino, es el olor al corcho asociado principalmente a un aroma a moho que posee un umbral de detección extremadamente bajo. Este defecto es debido principalmente a la presencia de compuestos aromáticos que se encuentran en concentraciones del rango de ng/L. El compuesto principal asociado a este defecto es el 2,4,6-Tricloroanisol cuyo principal origen se haya en la O-metilación de triclorofenoles presentes en el corcho por parte de hongos filamentosos. Para su determinación y cuantificación se han empleado diversos métodos analíticos, destacando principalmente la cromatografía de gases, ya que ha resultado muy eficaz en su determinación. En este Trabajo de Fin de Máster se ha resumido los desarrollos analíticos más importantes en técnicas instrumentales de determinación del compuesto incluyendo técnicas de extracción tanto en matrices de vino como en matrices de corcho, constituyendo una de las etapas más importantes en el análisis de este compuestoDepartamento de Ingeniería Agrícola y ForestalMáster en Calidad, Desarrollo e Innovación de Alimento

Identifizierung und Quantifizierung von Korkfehlaromen in Naturkorken mittels multidimensionaler gaschromatographischer Methoden

The typical cork taint primarily caused by 2,4,6-trichloroanisole (TCA) in cork stoppers is considered today to be less important since its major origin, the utilization of hypochlorite as bleaching agent, during the manufacturing process is avoided and rigorous quality management is applied. Still, TCA and other haloanisoles in wine can originate from a contamination in cellars due to the usage of wood preservatives or flame retardants. Therefore, it is still important to monitor these compounds in the cork and wine industry. Particularly, the trace level analysis of such potent aroma compounds in the complex wine matrix is often hindered due to co-elutions using one-dimensional gas chromatographic (GC) analysis. Thus, a robust analytical method based on headspace solid phase microextraction (HS-SPME), heart-cut multidimensional gas chromatography (H/C MDGC) and halogen-sensitive electron capture detection (ECD) was established for routine application in wine and cork soaks that allowed a reliable quantification in the complex wine matrix below the compounds’ odor thresholds at sub-ng/l level that may be crucial in customer conflict situations. With regard to the atypical cork taint, a clear correlation of tainted wines with this off-flavor was hitherto difficult as this sensory alteration lacked any substantial information. In a study comprising H/C MDGC-olfactometry the responsible off-flavor compounds were identified by analyzing natural cork stoppers with off-odors deviant from the typical cork taint. Here, the identification of trace level aroma compounds benefitted from the additional application of heart-cut and comprehensive multidimensional GC in combination with mass spectrometric detection (H/C MDGC-MS-MS, GC×GC-MS). Basically, well-known off-flavor substances like geosmin, 2-methylisoborneol, and 3-isopropyl-2-methoxypyrazine were detected as well as chlorinated substances. Besides TCA, another potent aroma compound, 3,5-dimethyl-2-methoxypyrazine (MDMP), was present in each sub-group of the off-odorous cork stoppers, obviously playing an important role concerning the atypical cork taint. The unequivocal identification of MDMP was critical since a constitutional isomer, originally associated with another off-flavor in wine, showed similar mass spectrometric data and gas chromatographic behavior on common stationary phases. The GC separation of the isomers that was essential for the unambiguous assignment could be finally achieved on a cyclodextrin-based stationary phase. Targeted trace level analyses of the most important cork off-flavor compounds was achieved in cork soaks and wines below their odor thresholds using an analytical approach based on HS-SPME-H/C MDGC with tandem mass spectrometric detection (MS-MS). Analysis of individual off-odorous cork stoppers revealed elevated concentrations of the targeted compounds correlating with the corresponding sensory description of the stopper. The migration of off-flavor compounds (especially alkyl methoxypyrazines) from cork stoppers into wine and an associated sensory alteration of the wine could be observed in wines sealed with affected cork stoppers after an appropriate storage period. In particular, MDMP became apparent to be important for the atypical cork taint and should thus be monitored in routine quality control. However, the contribution of MDMP to the characteristic sensory alteration of wine related with the atypical cork taint e.g. reduced fruitiness has to be investigated in more detail. Furthermore, its origin has to be fully elucidated to be able to apply preventive procedures in the cork production process.Der typische Korkgeschmack, der hauptsächlich durch die Verbindung 2,4,6-Trichloranisol (TCA) in Korken verursacht wird, spielt heute nur noch eine untergeordnete Rolle, da die Hauptursache - die Bleichung der Korken mit Hypochlorit - bei deren Herstellung nicht mehr angewendet wird und strenge Qualitätskontrollen durchgeführt werden. Allerdings können TCA und andere Haloanisole aufgrund der Verwendung von Holzschutzmitteln oder Flammschutzmitteln auch im Kellerumfeld gebildet werden, so dass diese Verbindungen regelmäßig in der Kork- und Weinindustrie kontrolliert werden müssen. Die Spurenanalytik solcher potenter Aromastoffe erweist sich in der komplexen Weinmatrix oft als schwierig, da bei der Anwendung von nur eindimensionaler Gaschromatographie (GC) oft Co-elutionen beobachtet werden. Zur Routineanalytik von Wein- und Korkproben wurde daher eine robuste Methode etabliert, in der die Dampfraum-Festphasenmikroextraktion (HS-SPME), heart-cutting multidimensionale GC (H/C MDGC) und der halogenempfindliche Elektroneneinfangdetektor (ECD) eingesetzt wurden. Dadurch konnte in der komplexen Weinmatrix eine verlässliche Quantifizierung im Konzentrationsbereich unter den Geruchsschwellenwerten (sub-ng/l) erreicht werden, das gerade in kritischen Fällen von Verbraucherbeschwerden entscheidend sein kann. Beim Auftreten des untypischen Korkgeschmacks war es bisher schwierig die sensorische Veränderung eines Weines diesem Fehlaroma zuzuordnen, da das Wissen über die verantwortlichen Substanzen fehlte. Diese Verbindungen wurden mittels H/C MDGC in Kombination mit olfaktometrischer Detektion identifiziert, indem Naturkorken mit einem Fehlaroma, das sich vom typischen Korkgeschmack unterscheidet, untersucht wurden. Hierbei wurde die Identifizierung von Aromastoffen im Spurenbereich durch die Anwendung von multidimensionalen GC Methoden mit massenspektrometrischer Detektion (H/C MDGC-MS-MS, GC×GC-MS) begünstigt. Prinzipiell wurden bereits bekannte Fehlaromen wie Geosmin, 2-Methylisoborneol und 3-Isopropyl-2-methoxypyrazin sowie chlorierte Verbindungen nachgewiesen. Jedoch wurde neben TCA ein anderer potenter Aromastoff, das 3,5-Dimethyl-2-methoxypyrazin (MDMP), in allen Untergruppen der sensorisch beeinträchtigten Korken nachgewiesen und scheint daher eine wichtige Rolle im Zusammenhang mit dem untypischen Korkgeschmack zu spielen. Die eindeutige Identifizierung von MDMP war zunächst kritisch, da ein Konstitutionsisomer, das ursprünglich mit einem anderen Weinfehlaroma in Verbindung gebracht wurde, ein ähnliches Massenspektrum und ähnliches gaschromatographisches Verhalten auf üblichen stationären Phasen aufwies. Die eindeutige Zuordnung war nur möglich aufgrund der gaschromatographischen Trennung der beiden Isomere, welche schließlich mittels einer stationären Phase auf Basis eines Cylclodextrin-Derivats erreicht wurde. Die Quantifizierung der wichtigsten Korkfehlaromen in Korkextrakten und Weinen unter deren Geruchsschwelle im unteren ng/l-Bereich wurde erreicht durch den Einsatz von HS-SPME-H/C MDGC mit Tandemmassenspektrometrie (MS-MS) zur Detektion. Bei der Untersuchung von einzelnen sensorisch auffälligen Korken wurden erhöhte Konzentrationen der untersuchten Verbindungen beobachtet, die mit den entsprechenden sensorischen Beschreibungen der Korken korrelierten. Durch das Verschließen von Weinen mit sensorisch auffälligen Korken wurde nach einer entsprechenden Lagerungszeit die Migration von Fehlaromen, vor allem von Alkylmethoxypyrazinen, aus den Korken in den Wein sowie eine sensorische Beeinflussung des Weines beobachtet. Besonders MDMP ist offensichtlich von großer Bedeutung für den untypischen Korkgeschmack und sollte deshalb in die routinemäßige Qualitätskontrolle mit aufgenommen werden. Allerdings ist die Bedeutung von MDMP in der charakteristischen Wahrnehmung des untypischen Korkgeschmacks (z.B. die reduzierte Frucht des Weines) in ergänzenden sensorischen Studien zu untersuchen. Weiterhin sollten die Ursachen von MDMP auf bzw. in Korken geklärt werden, um präventive Maßnahmen im Produktionsprozess einleiten zu können

Aplicação de planejamento fatorial fracionário e box-behnken para otimização de extração com microgota para determinação de tricloroanisol e tribromoanisol de amostras de vinho por cromatografia a gás

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas, Programa de Pós-Graduação em Quimica, Florianópolis, 2007.Neste trabalho foi desenvolvida uma metodologia analítica para determinação de 2,4,6-tricloroanisol e 2,4,6-tribromoanisol, os quais proporcionam cheiro de mofo ao vinho. A metodologia desenvolvida baseou-se na extração com microgota de solvente com separação e detecção por cromatografia a gás e detector por captura de elétrons. O solvente extrator utilizado foi 1-octanol, suspenso na ponta de uma microseringa convencional para injeção de líquido. Os parâmetros que afetam a eficiência de extração dos analitos pelo solvente foram otimizados através de ferramentas multivariadas, tais como planejamento fatorial fracionário e planejamento Box-Behnken. As condições ótimas obtidas foram: 25 minutos de extração a 48ºC, 20 mL de amostra em frasco de 40 mL ajustado a pH 5,5 e contendo 2,5 mol L-1 de NaCl. Através de um estudo de efeito de matriz, concluiu-se que a amostra sintética não representava convenientemente as amostras de vinho tinto. Um estudo de diluição foi realizado, e satisfatórias recuperações entre 70 e 120% foram obtidas para a diluição 14 mL de amostra e 6 mL de água. Para as amostras de vinho branco nenhuma diluição foi necessária. Excelentes limites de detecção de 8 e 6 ng L-1, respectivamente para TCA e TBA, foram obtidos. Precisão satisfatória (RSD<16,7%, n=5) foi obtida a um nível de concentração de 40 ng L 1

At-line testing of chlorophenol and chloroanisole contaminants in commercial wine production

The research described in this thesis concerns the development of at-line test procedures for the detection of trace levels of chlorophenols and chloroanisoles in wine and related materials. Competitive ELISA assays were developed and optimised for pentachlorophenol and pentachloroanisole to enable the detection of chlorophenols and chloroanisoles in the range 0.1 to 100ng/ml in purified sample extracts, using antibodies supplied by the French consortium partner, Diaclone, together with synthesised conjugate materials, based on the enzyme horseradish peroxidase. The cross reactivity of the assay towards chlorophenol and chloroanisole congeners in wine was investigated and the pentachlorophenol assay was selected as the most efficient antibody to detect and quantify both chlorophenol and chloroanisole congeners. The use of synthetic receptors based on molecularly imprinting technology was also investigated for pentachlorophenol and pentachloroanisole, and a new assay format (Displacement Imprinted Polymer Analysis (DIPRA)) was established to measure chlorophenols in contaminated materials at a concentration range of 0.1 to 50 µg/ml, based on the displacement of a reporter molecule rebound to the synthetic receptor sites. Two alternative reporter molecules were synthesised by linking a pentachlorophenol derivative (2,3,5,6-tetrachloro-4-hydroxy phenoxy acetic acid) to the enzyme horseradish peroxidase or 7-amino-4-methylcoumarin-3-acetyl hydrazide. Alternative hyphenated sample extraction and purification methods based on solid phase extraction, steam distillation and liquid/liquid partition were evaluated to enable the ELISA and DIPRA test methods to be employed using the limited facilities of a local winery laboratory. The application of the procedures to the analysis of sample types such as wine, corks and packaging materials was undertaken to investigate the suitability of the test methods for inclusion in quality control and incident analysis protocols.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Migração de compostos voláteis através de diferentes vedantes de vinho

Estágio realizado na Amorim & Irmãos, S. A. - Investigação e Desenvolvimento e orientado pelo Doutor Miguel CabralTese de mestrado integrado. Engenharia Química. Faculdade de Engenharia. Universidade do Porto. 201

Analysis of TCA incidence and its precursors in cork and its relation with soil and climatic characteristics of origin

Mestrado em BioquímicaA cortiça é um material de excelência e dos produtos com maior destaque na economia portuguesa, quer pelo elevado nível de exportações quer pela criação de emprego associada a este setor. A aplicação dada à cortiça com maior relevância económica é a rolha natural para vedação de vinhos. Contudo, alguns compostos halogenados presentes na cortiça, ao migrarem para o vinho, podem comprometer a sua qualidade levando a perdas significativas tanto para a indústria corticeira como para os produtores de vinho. Neste sentido, é de extrema importância o desenvolvimento e a aplicação de métodos de análise que permitam a quantificação simultânea das principais moléculas identificadas como responsáveis pelos desvios organoléticos dos vinhos. O presente trabalho surge com o objetivo de validar um método para análise simultânea de 2,4,6-tricloroanisol (TCA) e clorofenóis em cortiça e a sua posterior aplicação na análise de cortiças provenientes de diferentes locais da Península Ibérica, de forma a verificar uma possível relação entre a concentração destes compostos e as características edafoclimáticas de origem da cortiça. Com o objetivo de validar o método de análise, foram realizados estudos de linearidade, limiares analíticos, precisão, exatidão e robustez. Estes estudos permitiram considerar o método analítico como validado e implementá-lo nas análises de rotina do laboratório. Ao longo deste trabalho analisaram-se 46 amostras de cortiça, das quais 32 eram provenientes de Portugal e 14 de Espanha. Os resultados obtidos permitiram concluir que a cortiça da região da Andaluzia é aquela com valores médios e máximos mais elevados tanto para o TCA como para os clorofenóis, sendo a densidade do montado uma possível explicação para esta ocorrência, uma vez que, nesta região, o montado de sobro é muito denso (elevado número de árvores, pouca luz e exposição solar e mau arejamento), possivelmente propiciando o desenvolvimento fúngico e consequente formação destes compostos.Cork is an excellence material and one of the most relevant products of the portuguese economy, either by the high level of exports and the number of jobs created by this industrial sector. The most important application of cork is as cork stopper for sealing wine bottles. However, some halogen containing compounds, by migrating to the wine, can compromise its quality causing significant financial losses for both the cork industry and winemakers. In this way, it’s extremely important the development and application of methods of analysis to allow the quantification of those molecules responsible for the sensory deviations of wine. This work arises with the aim of validating a method for the simultaneous analysis of 2,4,6-trichloroanisole (TCA) and chlorophenols in cork and it’s subsequent application in the analysis of cork samples from different locations in the Iberian Peninsula, to verify a possible relationship between the concentration of these compounds and the characteristics of the climate and the soil of the original cork tree. In order to validate the analytical method, linearity, analytical precision, accuracy and robustness studies have been performed. These studies allowed the validation of the analytical method and its implementation in the laboratory routine analysis. Throughout this thesis 46 cork samples were analyzed; 32 of them came from Portugal and 14 from Spain. The obtained results allow to conclude that the cork from Andalusian region is the one with higher values for both TCA and chlorophenols, being the density of the cork oak forest one possible explanation for this manifestation, once in this region the forest is denser (high number or trees, few light and sun exposure and bad aeration) providing, possibly, a higher fungi development and consequently the formation of these compounds