Dissertations / Theses on the topic 'Flavor ester'

Gli esteri svolgono un ruolo rilevante nell'industria alimentare, sono tra i composti più importanti e versatili di aromi e fragranze naturali in alimenti, bevande e cosmetici. La loro preparazione da substrati naturali e l'utilizzo di bioprocessi (eg. fermentazione o reazioni enzimatiche) è attrattivo, perché il prodotto finale può essere etichettato e commercializzato in UE e USA come naturale. Pertanto, nuovi approcci biotecnologici per ottenere aromi sono desiderati, una volta che siano efficienti e sostenibili. Molti esteri con proprietà aromatiche possono essere ottenuti enzimaticamente usando lipasi che catalizzano reazioni di esterificazione, transesterificazione o interesterificazione. In questa tesi di dottorato abbiamo sviluppato due sistemi per la produzione di esteri con proprietà aromatiche: 1) Un metodo biocatalitico per la preparazione enzimatica di diversi esteri con proprietà aromatiche da alcoli primari (isoamilico, n-esilico, geranilico, cinnamilico, 2-feniletilico e benzilico) ed esteri etilici naturalmente disponibili (formiato, acetato, propionato e butirrato). Le biotrasformazioni sono catalizzate da un'aciltransferasi di Mycobacterium smegmatis (MsAcT) e precedute da eccellenti rese (80- 97%) e brevi tempi di reazione (30-120 minuti), anche quando sono state utilizzate concentrazioni di substrato elevate (fino a 0,5 M). Questa strategia enzimatica rappresenta un'alternativa efficace all'applicazione delle lipasi nei solventi organici e un miglioramento significativo rispetto ai metodi già noti in termini di uso ridotto di solventi organici, aprendo la strada a una preparazione sostenibile ed efficiente degli agenti aromatizzanti naturali. 2) Un metodo biocatalitico con la lipasi legata al micelio liofilizzato di Aspergillus oryzae che catalizza l'esterificazione diretta di alcoli e acido acetico in solvente organico. Ha mostrato un'elevata stabilità verso substrati e prodotti. L'acqua prodotta durante l'esterificazione non ha influenzato in modo significativo l'equilibrio della reazione, consentendo conversioni elevate. Queste caratteristiche sono state sfruttate per preparare esteri con proprietà aromatiche dell'acetato (isoamil e cinnamil acetato) in sistemi in batch e continui. È stato sviluppato un continuous stirred tank membrane reactor (CST-MR) ovvero un reattore continuo a membrana sotto agitazione per garantire una buona produttività e un'elevata stabilità del biocatalizzatore. Entrambi i sistemi di produzione sono promettenti, rappresentano due diverse alternative e possono essere ulteriormente ottimizzati e scalati per gli interessi del settore.
Esters play a significant role in the food industry, they are among the most important and versatile components of natural flavours and fragrances in food, drinks and cosmetics Their preparation starting from natural substrates and using bioprocesses (e.g., fermentation or enzymatic reactions) is appealing, since the final product can be labelled and commercialized in EU and USA as natural. Therefore, new biotechnological approaches for obtaining flavours are highly demanded as long as they are efficient and sustainable. Many flavour/fragrance esters can be enzymatically obtained using lipases that catalyse esterification, transesterification or interesterification reactions. In this PhD thesis we studied two systems for production of flavours esters: 1) A straightforward biocatalytic method for the enzymatic preparation of different flavour esters starting from primary alcohols (e.g., isoamyl, n-hexyl, geranyl, cinnamyl, 2-phenethyl, and benzyl alcohols) and naturally available ethyl esters (e.g., formate, acetate, propionate, and butyrate) was developed. The biotransformations are catalysed by an acyltransferase from Mycobacterium smegmatis (MsAcT) and preceded with excellent yields (80-97%) and short reaction times (30-120 minutes), even when high substrate concentrations (up to 0.5 M) were used. This enzymatic strategy represents an efficient alternative to the application of lipases in organic solvents and a significant improvement compared to already known methods in terms of reduced use of organic solvents, paving the way to a sustainable and efficient preparation of natural flavouring agents. 2) Mycelium-bound lipase of dry mycelium of Aspergillus oryzae catalysed direct esterification of alcohols and acetic acid in organic solvent, showing high stability towards substrates and products. Water produced during the esterification did not significantly affect the equilibrium of the reaction, allowing for high conversions. These features were exploited for preparing flavour-active acetate esters (e.g., isoamyl and cinnamyl acetate) in batch and continuous systems. A continuous stirred tank membrane reactor (CST-MR) was developed securing good reactor productivity and high biocatalyst stability. Both production systems are promising, represent two different alternatives and can be further optimized and scaled up for the interests of the industry.

Thesis (PhD)--Stellenbosch University, 2004.
ENGLISH ABSTRACT: Yeasts produce a broad range of aroma-active volatile esters and higher alcohols during alcoholic fermentation. Some of these esters and higher alcohols are important for the fruity flavors and therefore the final quality of wine and other fermented beverages. Esters are produced and hydrolyzed by alcohol acetyltransferases and esterases, respectively. In yeast, ester-synthesizing activities are represented by two alcohol acetyltransferases encoded by the ATFI and ATF2 genes, and by an ethanol hexanoyl transferase encoded by the EHTI gene. Atfl p and Atf2p appear responsible for the production of ethyl acetate and isoamyl acetate, while Ehtl p synthesizes ethyl hexanoate from ethanol and hexanoyl-CoA. Although a fair amount of information is available regarding the ATF 1 gene, limited information is available on the remaining alcohol acetyltransferases. Only two genes that code for esterases have been identified in yeast, namely lAHI and TIPI. It has also been shown that the balance between alcohol acetyltransferases and esterases is important for the net rate of ester accumulation. Higher alcohols are synthesized from the a-keto-acids in the branched-chain amino acid metabolic pathway by decarboxylation and reduction. The transamination of the amino acid to the respective a-keto-acid is catalyzed by mitochondrial and cytosolic branched-chain amino acid transferases, which are encoded by the BATI and BAT2 genes, respectively. In recent years, a strong scientific and industrial interest in the metabolism of flavoractive compounds has emerged, but information regarding the roles of specific enzymes and the physiological relevance of their metabolism remains limited. The aim of this project was to investigate the physiological and metabolic consequences of changes in the expression levels of some of the key enzymes involved in aroma compound production. The consequences of these changes on the chemical composition and the fermentation bouquet of wines and distillates were also investigated. The first part of the section on the results in this dissertation reports on the role and relative importance of the Saccharomyces cerevisiae enzymes involved in ester metabolism, namely Atflp, Atf2p, Ehtlp, Iahlp and Tiplp. The corresponding genes were overexpressed in a laboratory strain of S. cerevisiae, BY4742, and in a widely used commercial wine yeast strain, VIN13. Table wine and base wines for distillation were prepared with these VIN13 transformed strains. The ester concentrations and aroma profiles of the wines and distillates were analyzed and compared. The data indicated that the overexpression of ATF 1 and ATF2 increased the concentrations of ethyl acetate, isoamyl acetate, 2-pheylethyl acetate and ethyl caproate, while the overexpression of JAHI resulted in a significant decrease in the concentrations of ethyl acetate, isoamyl acetate, hexyl acetate and 2-phenylethyl acetate. The overexpression of EHTI resulted in a marked increase in the concentrations of ethyl caproate, ethyl caprylate and ethyl caprate, while the overexpression of TJP1 did not decrease the concentrations of any of the esters. In most cases, there was a correlation between the increase in esters and the decrease in higher alcohols. The data suggest that yeast balances the amount of different esters produced through alcohol acetyltransferases and esterases, and that, in some cases, these enzymes appear to overlap in function and/or influence each other's activity. In the second part of the results section, the consequences of the deletion and the overexpression of two genes, BATl and BAT2, which encode transaminases that contribute to the metabolism of higher alcohols, were investigated. The genes were both disrupted in a S. cerevisiae BY4742, and overexpressed in both this laboratory strain and in the VIN13 wine yeast strain. The effects of these modifications on the general physiology of the corresponding yeast strains and on higher alcohol metabolism were assessed in a range of growth conditions, including aerobic and anaerobic growth conditions, in the presence of glucose or raffinose as sole carbon source and growth in the presence of various concentrations of amino acids. Table wine and base wines for distillation were prepared with the modified industrial strains and the concentrations of the higher alcohols and the aroma profiles of the wine and distillates were analyzed and compared. Batl deletion seemed to be lethal under the conditions that were created, and therefore only the bat2!:!.strain, together with the BATI and BAT2 overexpression strains, were investigated. These modifications did not appear to significantly affect the general physiology of the strains. The results obtained indicated that the overexpression of BATI increased the concentrations of isoamyl alcohol and isoamyl acetate, and, to a lesser extent, the concentrations of isobutanol and isobutyric acid. The overexpression of the BAT2 gene resulted in a substantial increase in the levels of isobutanol, isobutyric acid and propionic acid production, and a modest increase in the level of propanol and isovaleric acid. Interestingly, the overexpression of BAT2 led to a decrease in isoamyl alcohol and isoamyl acetate concentrations. Sensory analyses indicated that the wines and distillates produced with the strains in which the BATl and BAT2 genes were overexpressed had more fruity characteristics (peach and apricot aromas) than the wines produced by the wild-type strains. This study offers new prospects for the development of wine yeast starter strains with optimized ester and higher alcohol-producing capability that could assist winemakers in their efforts to consistently produce wine to definable specifications and styles and a predetermined flavor profile.
AFRIKAANSE OPSOMMING: Gedurende fermentasie produseer giste 'n wye verskeidenheid vlugtige aromatiese esters en hoër alkohole. Sommige van hierdie esters en hoër alkohole is belangrik vir die vrugtige geure en dra dus by tot die finale kwaliteit van wyn en ander gefermenteerde drankies. Esters word onderskeidelik deur alkoholasetieltranferases en esterases geproduseer en gehidroliseer. In giste word die ester-sintetiserende aktiwiteite deur twee alkoholasetieltransferases verteenwoordig wat deur die ATFI-en ATF2-gene, asook 'n etanolheksanoïeltransferase wat deur die EHTl-geen, gekodeer word. Dit blyk dat ATFlp en ATF2p verantwoordelik is vir die produksie van etielasetaat en isoamielasetaat, terwyl Ehtl p-etielheksanoaat vanaf etanol en heksanoïel-CoA sintetiseer. Alhoewel daar 'n redelike hoeveelheid inligting t.o.v die ATF I-geen beskikbaar is, is daar weinig inligting oor die res van die aloholasetieltransferases. Slegs twee gene wat vir esterases kodeer, is in gis geïdentifiseer, naamlik IAHI en TIPI. Daar is ook bewys dat 'n balans tussen die alkoholasetieltransferases en esterases baie belangrik is vir die netto-tempo van ester-akkumulasie. Hoër alkohole word gesintetiseer vanaf a-keto sure in die vertakte-ketting aminosuur metaboliese pad deur dekarboksilasie en reduksie. Die transaminasie van die aminosuur na die onderkeidelike a-ketosuur word deur vertakte-ketting aminosuur transferases, geleë in die mitochondrion en sitosol, en gekodeer deur BATl- en BAT2-gene, gekataliseer. In die laaste paar jare het daar 'n sterk wetenskaplike, asook industrïele, belangstelling in die metabolisme van aroma-aktiewe komponente te voorskyn gekom, maar inligting in verband met die rol van spesifieke ensieme en die fisiologiese belangrikheid van hul metabolisme is egter beperk. Die doel van hierdie projek was om die fisiologiese en metaboliese gevolge van veranderinge in die ekspressievlakke van sommige sleutelensieme betrokke by aromakomponent-produksie te ondersoek. Die gevolge van hierdie veranderinge op chemiese vlakke, asook hoe die fermentasie-aroma van die wyne en distillate beïnvloed word, is ook bestudeer. Die eerste gedeelte van die resultate rapporteer oor die rol en relatiewe belangrikheid van die Saccharomyces cerevisiae-ensieme betrokke by estermetabolisme, naamlik Atfl p, Atf2p, Ehtlp, Iahlp en Tiplp. Die gene was ooruitgedruk in 'n laboratoriurnras van S. cerevisiae, BY4742, asook in 'n kommersïele wyngisras, VIN13. Tafelwyne en basiswyne vir distillasie is gemaak met die getransformeerde VIN13-rasse. Die esterkonsentrasies en aromaprofiele van die wyne en distillate is ontleed en vergelyk. Die data het gewys dat die ooruitdrukking van ATFI- en ATF2-gene 'n verhoging in etielasetaat, isoamielasetaat, 2-fenieletielasetaat en etielkaproaat veroorsaak het, terwyl ooruitdrukking van !AHI 'n betekenisvolle afname in etielasetaat-, isoamielasetaat-, heksielasetaat- en 2-fenieletielasetaat-konsentrasies veroorsaak het. Die ooruitdrukking van EHTI het 'n duidelike verhoging in etielkaproaat, etielkaprilaat en etielkapraat veroorsaak en die ooruitdrukking van TIPIhet geen van die esterkonsentrasies verander nie. In die meeste gevalle was daar nie 'n korrelasie tussen die toename in esters en afname in hoër alkohole nie. Die data stelook voor dat die gis 'n balans tussen die verskillende esters handhaaf deur middel van die alkoholasetieltrasferases en esterases, en in sommige gevalle blyk dit dat die ensieme dieselfde funksies het en/of mekaar se aktiwiteit beïnvloed. In die tweede gedeelte van die resultate is die oorsake van delesie en ooruitdrukking van twee gene, BAT1 en BAT2, wat kodeer vir transaminases wat tot hoër alkohol metabolisme bydra, bestudeer. Die gene is uitgeslaan in S. cerevisiae BY4742 en ooruitgedruk in BY4742 en in die wyngisras VIN13. Die effekte van hierdie modifikasies op die algemene fisiologie van die verskillende gisrasse en op hoëralkoholmetabolisme is onder 'n verskeidenheid kondisies bestudeer, naamlik aërobies en anaërobiese groeikondisies, in die teenwoordigheid van glukose of raffinose as die enigste koolstofbron, asook in die teenwoordigheid van 'n verskeidenheid konsentrasies aminosure. Tafelwyne en basiswyne vir distillasie is gemaak met die gemodifiseerde industrïele rasse en die konsentrasies van die hoër alkohole en aromaprofiele van die wyne en distillate is ontleed en vergelyk. Bat1-delesie was dodelik onder die kondisies, daarom is slegs die batlts-tes tesame met die BAT1 en BAT2 wat in die rasse ooruitgedruk is, bestudeer. Die modifikasies het nie 'n beduidende effek op die algemene fisiologie van die rasse getoon nie. Die data het wel getoon dat die ooruitdrukking van BAT1 'n verhoging in isoamielalkohol- en isoamielasetaatkonsentrasies, en tot 'n mindere mate isobutielalkohol- en isobottersuur-konsentrasies, veroorsaak het. Die ooruitdrukking van BAT2 het 'n beduidende toename in isobutanol-, isobottersuur- en propioonsuurkonsentrasies en 'n kleinere toename in propanol- en isovaleriaansuur veroorsaak. Die ooruitdrukking van BAT2 het ook gelei tot 'n afname in isoamielalkohol- en isoamielasetaatkonsentrasies. Sensoriese analises het getoon dat die wyne en distillate wat geproduseer is met die rasse waarin die BAT1 en BAT2 gene ooruitgedruk is meer vrugtige eienskappe (perske- en appelkoos-aromas) getoon het as die wyne wat deur die wildetipe rasse geproduseer is. Die studie lewer nuwe vooruitsigte vir die ontwikkeling van wyngiste met geoptimiseerde ester en hoër alkohol produserende eienskappe wat die wynmakers in staat kan stelom wyne te produseer met gedefinieerde spesifikasies en style en 'n voorafbepaalde aromaprofiel.

Estery and woody flavour notes are important characteristics of distilled spirit flavour. It has been reported for malt whisky that the estery character of mature whiskies typically declines relative to that of the new make spirit, even though the analytical concentrations of esters remain broadly constant. One potential explanation for this observation would be a sensory interaction between mature and estery characters. The work described in this thesis was designed to test this hypothesis and to further explore the nature of the congeners responsible for eliciting these characteristics across different spirit types, as influenced by their maturation conditions (time, temperature, cask provenance etc.). In the research described in Chapter 2, four pairs of non-mature and mature spirits (tequila, bourbon and 2 malt whiskies) were characterized by instrumental analysis with the aim of defining the key aroma compounds that determined the mature character in each spirit. According to PLS analysis of the full data set, concentrations of 17 congeners were positively correlated with ageing time and might thus influence the mature character of the aged spirits. In Chapter 3, the same eight spirit samples were analysed by GC-Olfactometry using the AEDA (aroma extraction dilution analysis) approach. Aged spirits presented a more complex aroma than new make spirits, and contained more compounds with the highest FD-factors. Whilst a full GC-O characterisation was completed, the main focus was on identifying compounds which contributed to the estery and woody/mature characters of each spirit. In Chapter 4 we attempted to reproduce these characters for each spirit through aroma recombination, based on blends of the odiferous compounds identified at high FD factors and their analytical concentrations in the actual samples as reported in Chapter 3. It soon became apparent that relatively simple mixtures of esters on the one hand and maturation-linked compounds on the other did not adequately reproduce the nature of these characteristics in the spirits themselves. This implied either that our analysis had missed some significant compounds contributing to these characteristics, or that the complexity of the full spirit matrix is required to give the groups of compounds the nuanced flavour that they lacked in isolation. The latter hypothesis was tested by adding in additional blends of compounds to increase the complexity of the recombinant aroma mixtures. It was concluded that the authenticity of the aroma blends overall was improved by both the addition of a cocktail of ‘low boiling compounds’ (those analysed by a separate direct injection GC technique) and the introduction of a ‘structuring’ compound (ethyl hexadecanoate) at a concentration that would cause agglomeration within the whisky (micellar structures) thus influencing aroma partitioning and release. It was concluded that these modifications produced recombinant aromas which were close enough to the authentic spirit characters to use them in sensory interaction studies (Chapter 5). As opposed to interaction effects there was simply a tendency for the woody/mature characters to suppress the corresponding estery character of mature spirits, particularly at the higher concentrations of added wood extractives. Because the woody/mature compounds which characterised maturation were broadly similar across the spirit types, but differed in concentration according to the maturation conditions, we decided finally to investigate the extraction kinetics of wood-derived compounds from oak sticks as a function of ageing time, temperature, spirit type and alcohol content (Chapter 6). Temperature and alcohol content were the most significant factors that determined the extraction rate and final concentrations of all 18 wood-extractive compounds (P < 0.05) analysed. Not surprisingly, extraction rates increased with increasing temperature, but the trend in terms of alcoholic strength depended on the particular compound. Overall this thesis has improved knoEstery and woody flavour notes are important characteristics of distilled spirit flavour. It has been reported for malt whisky that the estery character of mature whiskies typically declines relative to that of the new make spirit, even though the analytical concentrations of esters remain broadly constant. One potential explanation for this observation would be a sensory interaction between mature and estery characters. The work described in this thesis was designed to test this hypothesis and to further explore the nature of the congeners responsible for eliciting these characteristics across different spirit types, as influenced by their maturation conditions (time, temperature, cask provenance etc.). In the research described in Chapter 2, four pairs of non-mature and mature spirits (tequila, bourbon and 2 malt whiskies) were characterized by instrumental analysis with the aim of defining the key aroma compounds that determined the mature character in each spirit. According to PLS analysis of the full data set, concentrations of 17 congeners were positively correlated with ageing time and might thus influence the mature character of the aged spirits. In Chapter 3, the same eight spirit samples were analysed by GC-Olfactometry using the AEDA (aroma extraction dilution analysis) approach. Aged spirits presented a more complex aroma than new make spirits, and contained more compounds with the highest FD-factors. Whilst a full GC-O characterisation was completed, the main focus was on identifying compounds which contributed to the estery and woody/mature characters of each spirit. In Chapter 4 we attempted to reproduce these characters for each spirit through aroma recombination, based on blends of the odiferous compounds identified at high FD factors and their analytical concentrations in the actual samples as reported in Chapter 3. It soon became apparent that relatively simple mixtures of esters on the one hand and maturation-linked compounds on the other did not adequately reproduce the nature of these characteristics in the spirits themselves. This implied either that our analysis had missed some significant compounds contributing to these characteristics, or that the complexity of the full spirit matrix is required to give the groups of compounds the nuanced flavour that they lacked in isolation. The latter hypothesis was tested by adding in additional blends of compounds to increase the complexity of the recombinant aroma mixtures. It was concluded that the authenticity of the aroma blends overall was improved by both the addition of a cocktail of ‘low boiling compounds’ (those analysed by a separate direct injection GC technique) and the introduction of a ‘structuring’ compound (ethyl hexadecanoate) at a concentration that would cause agglomeration within the whisky (micellar structures) thus influencing aroma partitioning and release. It was concluded that these modifications produced recombinant aromas which were close enough to the authentic spirit characters to use them in sensory interaction studies (Chapter 5). As opposed to interaction effects there was simply a tendency for the woody/mature characters to suppress the corresponding estery character of mature spirits, particularly at the higher concentrations of added wood extractives. Because the woody/mature compounds which characterised maturation were broadly similar across the spirit types, but differed in concentration according to the maturation conditions, we decided finally to investigate the extraction kinetics of wood-derived compounds from oak sticks as a function of ageing time, temperature, spirit type and alcohol content (Chapter 6). Temperature and alcohol content were the most significant factors that determined the extraction rate and final concentrations of all 18 wood-extractive compounds (P < 0.05) analysed. Not surprisingly, extraction rates increased with increasing temperature, but the trend in terms of alcoholic strength depended on the particular compound. Overall this thesis has improved knowledge of the chemical and sensory changes that accompany spirit maturation and has highlighted some of the factors that cause differences in mature character across spirit types.
Moreover, it concludes that the sensory perception of woody/mature generally suppresses the fresh estery characteristic of new make spirits, even though analytically the esters are still there in similar concentrations. wledge of the chemical and sensory changes that accompany spirit maturation and has highlighted some of the factors that cause differences in mature character across spirit types. Moreover, it concludes that the sensory perception of woody/mature generally suppresses the fresh estery characteristic of new make spirits, even though analytically the esters are still there in similar concentrations.

Thesis (MSc)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: No single chemical constituent can be accredited with giving wine and brandy their overall aroma and flavour. The aroma and flavour of wine and brandy are rather attributed to a number of chemical constituents reacting together and it is these reactions that give the beverage its character. Certain chemicals within wine and brandy do, however, make larger contributions to the flavour. These include the esters, terpenes and volatile acids, although others also exist. Esters are a large group of volatile compounds with variable aroma and flavour characteristics, including banana-like (isoamyl acetate), apple-like (ethyl caproate) and chemical/solvent-like (ethyl acetate). Esters are produced as secondary metabolites during the conversion of sugar to ethanol and are formed when an alcohol binds with a fatty acid. Chemically, ester metabolism is well documented and understood; however, much work still needs to be done on a genetic level. The yeast strain used during fermentation is one of the most important factors contributing to the type and quantity of esters produced. This is due to differences in genetic makeup. The metabolism of esters is controlled largely on a genetic level, with numerous genes being involved. The alcohol acetyltransferase genes are involved in ester anabolism, whilst esterase genes are involved in ester catabolism. Esterases have a negative effect on the overall level of esters within an alcoholic beverage, as they are capable of reducing the number of esters and are thus capable of altering the beverage's aroma and flavour profile. The IAH1 and the TIP1 gene products are believed to encode for two such esterases. The objective of this study was to investigate the contribution of the IAH1 and TIP1 genes to the level of esters in both wine and brandy. This was accomplished by using two approaches. Firstly, the above genes were disrupted using a polymerise chain reaction (PCR)-generated disruption cassette homologous to either the IAH1 or the TIP1 gene. These cassettes were integrated into the industrial wine yeast, Saccharomyces cerevisiae strain VIN13. The integrations were verified by Southern blot analysis to produce yeasts VIN13-~IAH1 and VIN13-~TIP1; however, only a single copy of each was disrupted. Secondly, the IAH1 and the TIP1 genes were cloned from S. cerevisiae using PCR into plasmid pj between the phosphoglycerate kinase gene (PGK1) promoter and terminator, producing plasmids pJ-IOE1 and pJ-TOE1. The PGK1 promoter has previously been shown to constitutively express genes at high levels. These new constructs were then used as template for PCR to produce two overexpression cassettes, one for IAH1 and the other for TlP1. These cassettes were integrated into S. cerevisiae VIN13 and verified by Southern blot analysis to produce strains VIN13-IOE1 and VIN13-TOE1. The above yeast strains including VIN13 were used for the production of wines and base wines from Colombard must. Reverse-transcriptase (RT-PCR) confirmed that the VIN13-IOE1 and VIN13-TOE1 strains overexpressed the appropriate gene at a higher level than the control VIN13 strain. The VIN13-AIAH1 disrupted strain showed no difference in expression level to that of the control strain, whilst VIN13-ATIP1 showed lower levels of expression than that of the control strain. VIN13-IOE1 behaved as expected, with a decrease of between 30% and 60% in the total ester level in the wine and base wine respectively, a 30% decrease in the total acid level and no change in the higher alcohol level. The VIN13-AIAH1 strain showed no difference to the control wine, most likely as this strain still expressed the IAH1 gene at levels consistent with the control strain. VIN13-TOE1 behaved in an unexpected manner - instead of hydrolysing esters, it appeared to produce them. This increase in the total ester level was most noticeable during distillation, when a 20% increase took place. Another unexpected occurrence was a large decline in the total acid level, with acetic acid being the most significant contributor, decreasing by up to 78%. This is a very favourable finding, as acetic acid is a known spoilage molecule and is a cause of sluggish/stuck fermentations. VIN13-ATIP1 behaved in an opposite manner to VIN13-TOE1, with higher total acid levels and slightly decreased total ester levels, especially during distillation. Neither affected the total higher alcohol levels. Sensorially, the only significant difference in the wine samples was for the fruity flavour. A panel of judges distinguished that VIN13-TOE1 was fruitier than the other wines, with VIN13-ATIP1 being the least fruity. This study again proves the significant impact that a single gene can have on the chemical makeup of wine and brandy. The relatively simple genetic alteration of an organism can drastically change and improve not only the organoleptic properties of the organism, but its viability as well. These alterations can produce more favourable organisms with more desirable characteristics for the fermenting beverage industry to produce products of higher quality and better suitability.
AFRIKAANSE OPSOMMING: Geen chemiese komponent kan uitgesonderword as die produseerder van aroma en geur in wyn of brandewyn nie. Die aroma en geur van wyn en brandewyn word eerder toegeskryf aan die interaksie tussen 'n groot aantal chemiese komponente om aan die drank sy karakter te gee. Enkele van hierdie chemiese komponente sluit in esters, terpene en vlugtige sure, om maar 'n paar te noem. Esters is "n groot groep van vlugtige verbindings wat beskik oor 'n verskeidenheid van aroma- en geurkenmerke, soos piesangagtig (isoamielasetaat), appelagtig (etielkaproaat) en chemies/oplosmiddelagtig (etielasetaat). Esters word as sekondêre metaboliete geproduseer wanneer suikers na etanolomgeskakel word en word gevorm wanneer "n alkohol met "n vetsuur verbind. Estermetabolisme is chemies goed beskryf en verstaan, maar op "n genetiese vlak is daar nog heelwat aspekte wat nagevors moet word. Die gisras betrokke gedurende fermentasie word beskou as een van die grootse bydraes tot die tipe en die hoeveelheid esters wat geproduseer word. Dit word toegeskryf aan verskille in die genetiese saamestelling van die gisras. Ester metabolisme word grootliks deur genetiese faktore beheer en verskeie gene is betrokke. Dit is hoofsaaklik die alkoholasetieltransferasegene wat vir esterkatabolisme verantwoordelik is, terwyl die esterasegene vir esteranabolisme verantwoordelik is. Esterases het 'n negatiewe effek op die totale estervlak binne alkoholiese dranke deurdat hulle in staat is om die aantal esters drasties te verminder en sodoende die drank se aroma- en geurprofiel te verander. Daar is voorgestel dat die IAH1- en die TlP1-geen produkte is wat vir twee sulke esterases kodeer. Die doel van hierdie studie was om die IAH1- en die TIP1-gene se bydrae tot die totale estervlak in wyn en brandewyn te ondersoek. Dit is deur twee benaderings uitgevoer. Eerstens is die bogenoemde gene d.m.V. disrupsiekassette wat homoloog aan die IAH1- of die TlP1-gene was, uitgeslaan. Die disrupsiekassette is deur die polimerasekettingreaksie (PKR) geproduseer. Hierdie kassette is in die industriële wyngis, Saccharomyces cerevisiae VIN13, geïntegreer. Die integrasies is deur Southernkladanalise bevestig en het die giste VIN13-~IAH1 en VIN13-~TIP1 gelewer. Net 'n enkele kopie van elke geen is egter uitgeslaan. Tweedens is die IAH1- en TIP1-gene d.m.V. PKR vanaf S. cerevisiae binne in plasmied pJ gekloneer, tussen die fosfogliseraatkinasegeen (PGK1) se promotor en termineerder, om plasmiede pJ-IOE1 en pJ-TOE1 te produseer. Die PGK1-promotor is al tevore geïdentifiseer as "n hoë-vlak konstitutiewe uitdrukker van gene. Hierdie twee nuwe konstrukte het vervolgens gedien as templaat vir PKR om twee ooruitdrukkingskassette, een vir IAH1 en die ander vir TIP1, te produseer. Hierdie kassette is in S. cerevisiae VIN13 geïntegreer en bevestig deur Southernkladanalise. Hierdie integrasies het die giste VIN13-IOE1 en VIN13-TOE1 geproduseer. All die nuwe gisrasse, tesame met VIN13, is gebruik vir die produksie van wyne sowel as rebatwyne vanaf Colombard-mos. Omgekeerde-transkriptase polimerasekettingreaksie (OT-PKR) het bewys dat die VIN13-IOE1 en VIN13-TOE1 rasse die geskikte geen ooruitgedruk het, met hoêr vlakke as van die kontrole VIN13-ras. Dit het ook aangedui dat die VIN13-i\IAH1-ras, waarvan die geen uitgeslaan was, geen verskil in uitdrukking gehad het in vergelyking met die kontroleras nie, terwyl VIN13-i\TIP1 'n lae uitdrukkingsvlak getoon het. VIN13-IOE1 het teen verwagting opgetree, met 'n afname van tussen 30% en 60% in die totale estervlak in beide die wyne en rebatwyne. 'n Afname van 30% in die totale suurvlak, asook geen waarneembare verskil in die hoêr alkoholvlak, in vergelyking met die kontroleras, is ook opgemerk. Die VIN13-i\IAH1-ras het glad nie van die kontroleras verskil nie, heel waarskynlik omdat hierdie ras die IAH1-geen teen dieselfde vlak as die kontroleras kon uitdruk. Die VIN13-TOE1-ras het teen verwagting opgetree deurdat dit esters geproduseer het i.p.v. om esters te hidroliseer. Hierdie toename in die totale estervlak is die meeste waarneembaar tydens distillasie, met tot 'n 20% toename. Nog 'n onverwagte effek was die groot afname in die totale suurvlak. met asynsuur wat die betekenisvolste bydrae gelewer het deurdat dit 'n afname van tot 78% getoon het. Hierdie bevinding is baie voordelig, aangesien asynsuur, 'n bekende bederfmolekuul, veral vir slepende/gestaakte fermentasies verantwoordelik is. VIN13-i\TIP1 het op die teenoorgestelde wyse opgetree as VIN13-TOE1, met 'n hoêr totale suurvlak en 'n klein afname in die totale estervlak. Weereens is dit meer gedurende distillasie waargeneem. Beide rasse het egter geen effek op die hoêr alkoholvlak gehad nie. Die proepaneel het, met betrekking tot die vrugtige geur, een betekenisvolle geurverskil tussen die wyne gevind. VIN13-TOE1 was meer vrugtig as al die ander wyne en VIN13-i\TIP1 was die minste vrugtig. Die studie het weereens bewys dat 'n enkele geen 'n betekenisvolle effek op die chemiese samestelling van wyn en brandewyn kan hê. Die relatief eenvoudige genetiese verandering van 'n organisme kan die organoleptiese eienskappe asook die lewensvatbaarheid van "n organisme, drasties verander en verbeter.

L'elaboration de boissons alcoolisees par fermentation de produits vegetaux est une pratique tres ancienne qui a connu de reels progres depuis les travaux de pasteur au 19#e siecle. La selection des varietes de raisin, le choix du meilleur moment pour la vigne ainsi que le controle du procede fermentaire, sont autant de facteurs d'amelioration du vin. Pour notre part, nous nous sommes interesses a la phase de fermentation alcoolique qui contribue tant sur le plan quantitatif que qualitatif a la formation des aromes du vin. Bien que ces composes soient tres nombreux, les principaux ont ete identifies et il est admis que sept d'entre eux se partagent plus de la moitie des aromes dans le vin : cinq alcools superieurs (propanol-1, isobutanol, 2-methyl et 3-methyl butanol-1, phenyl-2 ethanol) et deux esters (acetate d'ethyle et lactate d'ethyle). Les parametres, influant sur la synthese de ces composes, ont fait l'objet de nombreuses etudes mais a des resultats contreverses. D'autre part toutes les investigations entreprises, ont porte sur des evaluations de bilans entree-sortie et aucune ne decrit de facon precise les cinetiques de production. Afin de combler partiellement cette lacune et grace a un chromarographe de type espace-tete, nous avons etabli les cinetiques de production des aromes, ce qui constitue l'originalite de notre travail. Nous avons egalement souhaite preciser par notre etude, le role de parametres importants sur les cinetiques de production. En effet, nous nous sommes interesses a l'effet de la : temperature, la souche de levure, le taux de biomasse initial et a la composition du milieu en acides amines. Par un souci de reproductibilite, nous avons, dans une premiere etape, realise nos experiences sur un milieu modele, puis dans un deuxieme temps, nous avons valide les resultats obtenus sur un mout de raisin naturel : du mauzac.

La réalité sensorielle de l'existence d'un arôme fruité spécifique aux vins rouges a été démontrée par le biais de dégustations affranchies de toute subjectivité liée aux caractéristiques visuelles des vins. Ces dégustations ont également mis en évidence une typicité olfactive des vins rouges de Bordeaux. L'étude de la genèse de cet arôme fruité propre aux vins de Vitis vinifera L. Cv. Merlot et Cabernet-Sauvignon a ensuite révélé le rôle clé de la fermentation alcoolique d'une part et de la présence de constituants pelliculaires extractibles à l'éthanol d'autre part. L'intensité de l'expression fruitée est par ailleurs modulée par la présence de composés soufrés - thiols en particulier - et/ou de leurs précurseurs. La mise au point d'une méthode de fractionnement par HPLC d'extraits aromatiques a permis d'isoler le caractère fruité des vins rouges dans des fractions spécifiques, préservant fidèlement les nuances aromatiques correspondantes. Suite à l'étude GC-olfactométrique des fractions ainsi obtenues, de nombreuses zones odorantes, spécifiques ou non des vins rouges, ont pu être mises en évidence. Les composés aromatiques responsables de vingt d'entre elles ont été identifiés. Ils correspondent à des esters éthyliques et acétates, dont les teneurs dans des vins rouges ainsi que les seuils de perception dans la même matrice ont été déterminés. Ces composés ne présentent pas d'impact direct sur l'arôme fruité des vins rouges. Des tests de reconstitutions aromatiques ont en revanche clairement établi leur impact en mélange, reflet d'interactions perceptives entre ces composés et pouvant conduire à des nuances fruitées de type fruits rouges ou fruits noirs.

Les fermentations alcoolique (FA) et malolactique (FML) sont deux étapes importantes de la vinification en rouge dans l’établissement de l’arôme fruité des vins. Afin d’étudier l’importance relative des microorganismes fermentaires, nous étudions l’influence de six couples levures/bactéries lactiques (BL) - trois souches de levures, deux de BL - sur la modulation des notes fruitées de différents vins rouges de Bordeaux. Une première approche analytique montre l’influence prédominante de la souche de levures sur la concentration de plus de 70 marqueurs potentiels de la note fruitée. L’étude particulière des esters montre que l’effet levures observé dès la fin de la FA persiste au cours du temps malgré la FML et les modifications engendrées par le vieillissement du vin. L’étude sensorielle conforte l’influence majeure des levures sur la modulation de l’arôme fruité des vins rouges à différents temps d’élevage. Néanmoins, les résultats obtenus suggèrent l’implication d’autres composés aromatiques dans la modulation de la note fruitée des vins, non quantifiés dans la première partie de cette étude. Un travail de fractionnement d’extraits de vin par HPLC permet par la suite l’identification d’une fraction d’intérêt impliquée dans des variations aromatiques liées à la souche de levures. L’analyse de cette fraction par chromatographie en phase gazeuse n’a pas permis d’identifier le ou les composés impliqués. Nous avons néanmoins mis en évidence une thiophénone qui pourrait agir en tant que masque de l’arôme fruité, ainsi qu’un ester hydroxylé qui pourrait s’avérer être un marqueur intéressant de l’activité bactérienne et dont l’effet exhausteur de notes fruitées est également envisagé comme perspectives
Alcoholic (AF) and malolactic (MLF) fermentations are important steps in red winemaking for the revelation of wine fruity aroma. To investigate the relative importance of fermentative microorganisms, we studied the influence of six yeasts/lactic acid bacteria (LAB) combination - three yeast strains, two LAB - on Bordeaux red wines fruity notes modulation. A first analytical approach showed the predominant influence of yeast strain on the concentration of more than 70 potential fruity note markers. Special study of esters showed a yeast strain effect since the end of FA that persists over time, despite MLF and changes caused by wine aging. Sensory studies also highlighted the major influence of yeasts on red wines fruity aroma modulations at different aging steps. Nevertheless, results suggested the role of other aromatic compounds in fruity note modulation, not quantified in the first part of this study. The study of fractions made by HPLC with wine organic extracts enables the identification of an interested fraction involved in aromatic variations related to the yeast strain. Analysis of this fraction by gas chromatography has not allowed identifying compounds involved in these organoleptic variations. However, we highlighted a thiophenone that could act as a mask of fruity aroma and a hydroxylated ester that could be an interesting marker of bacterial activity. Its role as enhancer of fruity esters aroma is also considered

L’objectif de cette thèse est d’étudier le rôle de la fermentation malolactique (FML) sur l’arôme fruité des vins rouges. Les bactéries lactiques (BL) modifient la composition du vin mais il n’existe pas de consensus concernant spécifiquement cette famille aromatique. Contrairement aux idées empiriques sur la FML, ce travail a démontré l’absence à court terme d’un " masque lactique ", cependant l’apparition d’une telle interaction olfactive pourrait être plus tardive. Par contre, il est montré l’existence d’un masque proche de la note de réduction, de type fumé/grillé, dont la caractérisation n’a pas été effectuée dans cette étude.Le suivi des principaux marqueurs fruités du vin (70 molécules) a été rendu possible par le développement des méthodes d’analyse chromatographique en phase gazeuse couplée à la microextraction sur phase solide (esters, C13-norisoprénoïdes, lactones, thiols). En particulier, une " base de données esters " (32 composés) a rendu plus robuste l’ensemble des variations constatées au cours du développement des BL. En effet, les modifications des teneurs en esters sont démontrées comme un processus majeur de la balance de la note fruitée au cours de la FML. Cette fermentation permet à court terme, aussi bien la synthèse que l’hydrolyse des esters grâce aux activités estérases et, à plus long terme, la formation tardive d'esters éthyliques d'acides branchés issus du catabolisme de certains acides aminés. La spécificité des estérases vis-à-vis de la nature et de la longueur de la chaîne carbonée des esters est mise en évidence, ainsi que l'importance de la disponibilité des substrats, liée en partie à l'activité des levures.L’étude de l’influence des souches de BL et de la co-inoculation levures/bactéries a permis de confirmer le rôle clé des interactions entre les microorganismes, ainsi que l’importance de la composition de la matrice vin
The aim of this thesis is to study the role of malolactic fermentation (MLF) on the fruity aroma of red wines. Lactic acid bacteria (LAB) modify wine composition but there is no consensus concerning this aromatic group specifically. In opposition to empirical ideas on MLF, this work has demonstrated the absence, in short-term, of a “lactic-mask” although this kind of olfactory interaction may still occur in a later stage of wine development. Nevertheless, the existence of a smoked/toasted reduction-like mask note was proved. Its characterization has not been done in this work. The survey of the main fruity markers of wine (70 compounds) was made possible by the development of several gas chromatography coupled with solid-phase microextraction analytical methods (esters, C13-norisoprenoids, lactones, thiols). In particular, the creation of an “ester database” (32 compounds) has improved the detection of variations during LAB development in terms of analysis robustness. In fact, changes on esters contents are proved to be responsible for a major part of fruity notes evolution during MLF. Initially, this fermentation allows both synthesis and hydrolysis of esters as a consequence of esterase activity. Moreover, it promotes late-production of ethylic esters through the catabolism of certain aminoacids. Esterases specificity towards nature and size of the esters carbon chain is pointed out along with substrates availability, partially related to yeast activity.The study of the influence of both LAB strains and yeast/bacteria co-inoculation has confirmed microorganisms interactions and wine matrix composition to be of the great importance

L’expression aromatique fruitée des vins rouges a été le sujet de nombreuses études qui démontrent qu’au moins une composante de cette expression est le reflet d’interactions perceptives impliquant des esters. La plupart des travaux concernant les interactions perceptives jusqu’à ce jour se sont avérés descriptifs, très peu ayant cherché à déterminer leurs origines. Dans ce but, un outil analytique a été développé afin d’apprécier les changements de volatilité d’esters représentatifs de l’arôme fruité des vins rouges. Ainsi, les coefficients de partage de 9 esters ont pu être déterminés aussi bien dans une solution hydroalcoolique que dans un vin rouge désaromatisé. L’application de cet outil analytique aux interactions perceptives préalablement mises en évidence a permis d'observer des changements de volatilité des esters lors de leur mise en mélange avec d'autres composés volatils en solution. Ces changements de volatilité, synonymes de potentiels effets pré-sensoriels, vont dans le même sens que ceux observés lors de l’analyse sensorielle. L’utilisation d’un verre de dégustation possédant deux compartiments a permis de mettre en lumière le fait que certaines modifications sensorielles pouvaient être expliquées, pour partie au moins, par des effets pré-sensoriels. L'impact olfactif de 5 alcools supérieurs ainsi que de 15 composés issus du bois de chêne a pu être démontré grâce à de nombreuses reconstitutions aromatiques, et leur rôle de masquage de l’arôme fruité des vins rouges a pu être souligné. Le calcul des coefficients de partage des esters a permis de montrer que des changements de volatilité ont lieu au sein de la solution. Ces modifications peuvent être corrélées aux résultats obtenus lors de l’analyse sensorielle. Ainsi, il est possible d’expliquer, en partie, les effets de masquage de l’arôme fruité observés grâce aux seuils de détection et aux profils sensoriels, du fait de la diminution de la présence d’esters dans l’espace de tête venant stimuler le dégustateur. Globalement, nos travaux ont permis de mettre en évidence que la mise en mélange en solution de composés volatils pouvait se traduire par la modification de la volatilité des constituants du mélange et que certaines de ces interactions pré-sensorielles pouvaient conditionner l'expression aromatique fruitée due aux esters
A lot of studies highlighted the perceptual role of esters in fruity aromatic expression of red wines, demonstrating that at least partially it was due to perceptive interactions. Indeed, a lot of synergistic and masking effects have been brought to light in the past. However, the origin of these interactions remains unknown, although some authors suggested several levels where they can take place. In this goal, an analytical tool was developed to study the possible occurrence of esters volatility modifications. The application of this tool allowed determining partition coefficients of 9 esters in dilute alcohol solution and in dearomatized red wine. Thanks to perceptive interactions previously demonstrated by various authors, the application of this analytical tool highlighted modifications of esters volatility when compounds were mixed together in the solution. These modifications support the observations made with sensory analysis, indicating the existence of pre-sensorial effects. The use of a new tool consisting in a tasting glass with 2 compartments, reveals that these volatility changes may led to true sensorial modifications. Masking effect of fruity aroma due to 5 higher alcohols but also 15 wood by-products was highlighted using various aromatic reconstitutions. Esters partition coefficients calculation showed volatility modifications from the matrix to the gas phase. These data may be correlated with sensorial analysis results. Thus, it is possible to explain, at least partially, fruity aroma masking effect highlighted through detection threshold and sensory profile thanks to decrease in esters presence in headspace, and so a decline of taster’s olfactory stimulation. To conclude, our work showed that the mixture of volatile compounds in solution may result in modification of molecules volatility, and furthermore highlighted that these pre-sensorial interactions may impact fruity aromatic expression related to esters

La plupart des composés volatils connus et impliqués dans les mécanismes de l’expression fruitée des vins rouges sont présents à des teneurs inférieures ou proches de leurs seuils de perception individuels. Compte tenu de phénomènes d’interactions perceptives entre eux, il est très complexe de déterminer leur impact réel sur l’arôme du vin. Au vu des difficultés rencontrées pour reconstituer fidèlement l’arôme des vins à partir uniquement de composés purs, nous avons développé une méthodologie permettant d’aborder cette reconstitution aromatique à partir de fractions issues du vin lui-même, afin de pouvoir évaluer l’importance relative de ces différentes fractions aromatiques vis-à-vis de l’arôme global du vin. Grâce à l’analyse sensorielle, et en s’attachant à quelques descripteurs particuliers, nous avons pu mettre en évidence, quelques interactions perceptives particulières comme des effets de contributions marquées ou de masquage. La caractérisation des composés présents dans les fractions concernées et à l’origine de ces effets notables a été mise en œuvre. Nos résultats soulignent le rôle indirect du 2-hydroxy-4-méthylpentanoate d’éthyle, un ester éthylique élué dans la fraction à l’origine d’une contribution marquée aux notes de fruits noirs frais qui, en provoquant la diminution du "seuil de perception" du pool fruité des vins rouges et l’augmentation de l’intensité de leurs notes de fruits noirs et de fruits frais, agit comme un exhausteur naturel de ces notes fruitées. Nous sommes aussi parvenus à mettre en évidence, le rôle direct du diacétyle, mais aussi le rôle indirect de l’acétoïne, de l’acide acétique et de la γ-butyrolactone, malgré leurs concentrations infraliminaires, sur la diminution de l’intensité globale et l’intensité du caractère de fruits frais. Ces résultats soulignent leur fort caractère, seuls ou en mélanges, de "réducteurs" de l’intensité de ces notes, et ce, même à des concentrations infraliminaires. Enfin, le comportement particulier, au sein d’un mélange fruité, du 3-hydroxybutanoate d’éthyle, de l’acétate de 2-méthylpropyle, du propanoate d’éthyle et de l’acétate de butyle, présents à des concentrations infraliminaires a été mis en évidence. La présence en mélange des deux premiers provoque la baisse notable du "seuil de perception" du pool fruité et celle des trois derniers augmente l’intensité des notes de fruits frais et fruits noirs traduisant l’effet exhausteur d’arômes dû à ces composés, effet comparable de celui du 2-hydroxy-4-méthylpentanoate d’éthyle qui présente quelques analogies structurales avec ces composés
Most of volatiles involved in red wines’ fruity expression are present at levels below or close to their individual perception thresholds. Given the existence of perceptive interactions between them, it is very difficult to determine their real impact on wine aroma. Rather than assessing the olfactive behavior of mixtures prepared from pure products, the main goal of this work was to highlight and study the impact of perceptive interactions on wine fruity aroma expression using various aromatic reconstitutions prepared from wine fractions. Sensory profile analyses identified significant differences among aromatic reconstitutions for the intensity of some descriptors, as particular "additive" or "masking" effects. The composition of the involved fractions was then studied by instrumental methods. The final target was to investigate the impact of fraction components on fruity aroma by preparing aromatic reconstitutions and using sensory reconstitution tests, to assess the role of these compounds on the perceptive interactions previously observed. Further analysis revealed that ethyl 2-hydroxy-4-methylpentanoate, eluted in fraction which had an "additive" effect on the black-berry and fresh fruity aroma, does not play a direct role as a key compound in red wine aroma. In contrast, our findings highlighted its indirect contribution to wine aroma, showing that this ester contributed to a synergistic effect, enhancing the perception of fruity character. Finally, it was clearly demonstrated that this compound acts as a natural enhancer for black-berry and fresh fruit notes in red wine. It was also established that diacetyl, acetoin, acetic acid and γ-butyrolactone together played the same hypo-additive role as fractions of which they were eluted, presenting a "masking" effect on fresh fruity aroma. The impact of the last three compounds was demonstrated conclusively, even at subthreshold concentrations. These findings highlighted the existence of new remarkable perceptual interactions impacting overall and fresh-fruit aroma perception. The particular behavior, in a fruity mixture, of ethyl-propanoate, ethyl-3-hydroxybutanoate, butyl acetate and 2-methylpropyl acetate, present at subthreshold concentrations, was demonstrated. The presence of ethyl-3-hydroxybutanoate and 2-methylpropyl acetate in mixture led to a significant decrease of the olfactory threshold of fruity pool confirming their synergistic effect in the overall increase intensity. These compounds with close chemical structures, participate, both quantitatively and qualitatively, in the modulation of red wines’ fruity aromas acting as natural enhancers of black-berry and fresh-fruit aromas

碩士
長庚大學
化學工程研究所
88
Microbial lipase from Candida cylindracea was immobilized by entrapment in sol-gel matrix to catalyze the formation of flavor esters (ethyl butyrate and geranyl acetate) by esterification in hexane. The research can be divided into three parts. I. Using the sol-gel process for entrapment of lipase in sol-gel and magnetite (Fe3O4)-containing sol-gel matrices. The immobilized enzyme was used in ethyl butyrate formation by esterification between butyric acid and ethanol. We have optimized the immobilization conditions first and then discussed the effect of water content in the reaction solution, the effect of adding resin at the beginning of esterification, inhibition of reactants, heat stability and storage stability of free and immobilized enzyme. II. To simplify the processes of recovery, the sol-gel has been coated on macroporous supports, including non-woven sheet, chitosan bead, PU biomass support, Celite 545, and ion-exchange resin. From the results, the activity of lipase entrapped in sol-gel was only least affected by coating on the non-woven sheet, which was chosen as the best support. Properties of the immobilized enzyme was characterized as in part I. III. Using lipase entrapped in sol-gel coated on nonwoven sheet , we carry out esterification between geranol and acetic acid for producing geranyl acetate and characterize properties of the immobilized enzyme as in part I. From the results, the specific activities of lipase entrapped in sol-gel materials are greater than that of free enzyme. Powder formPowder form( magnetite )Nonwoven sheetsupport Ethyl butyrate2.5 fold1.4 fold 1.5 fold Geranyl acetate 1.5 fold After entrapment in the sol-gel matrices, the heat and storage stability of immobilized lipase was increased remarkably. The inhibitory effects of substrates and water content in the reaction mixture on the activity of enzyme are alleviated after also enzyme immobilization.

During fermentation, the yeast Saccharomyces cerevisiae produces a broad range of aroma-active esters that are important for the desirable complex flavour of beer. The sensory threshold levels of these esters in beer are low, ranging from 0.2 ppm for isoamyl acetate to 15-20 ppm for ethyl acetate. Although esters are only present in trace amounts in beer, they are extremely important as minor changes in their concentration may have dramatic effects on beer flavour. Therefore, optimization of the concentrations of these aroma-active esters in beer is of interest in beer brewing. The number and concentration of esters in beer may be influenced by the fermentation parameters, nutritional composition of fermentation medium and yeast strain type. Therefore, this study investigated the influence of fermentation temperature, pH, and wort nutritional supplements (amino acids and zinc) on the production of yeast-derived ester compounds. In addition, the overall fermentation performance was evaluated based on the reducing sugar and Free Amino Nitrogen (FAN) utilization, ethanol production and yeast cell density. These parameters were analysed using the Dinitrosalicyclic acid method, Ninhydrin assay, Gas Chromatography and standard spread plate technique. The concentration and stability of ethyl acetate, isoamyl acetate, phenyl ethyl acetate, ethyl hexanoate, ethyl decanoate and ethyl octanoate was monitored during storage at 4 °C and room temperature (RT), in the final beer by Chromatography. The expression levels of the ester synthetase genes under conditions that resulted in the highest increase in ester production were quantified by Real-Time PCR. For the lager beer, the best fermentation performance was achieved at RT (±22.5°C), resulting in the utilization of the highest amount of nutrients and production of 4.86% (v/v) ethanol. This was accompanied by the highest production of acetate and ethyl esters, which were 40.86% and 87.21%, respectively, higher than that of the control. Spent yeast density ranged from 2.492 to 3.358 mg/ml for all parameters tested, with the highest yield produced when wort was supplemented with 0.120 g/l zinc sulphate. Fermentations at 14 °C yielded the highest foam head stability and spent yeast viability with a foam head rating of 2.67 and a spent yeast viability of 3.85 × 107 cfu/ml. Ester compounds were relatively stable at 4 °C than at room temperature decreasing by only 7.93% after three months. Of all the volatile esters produced, ethyl decanoate was the least stable, with a 36.77% decrease in concentration at room temperature. For the ale beer, the best fermentation performance which resulted in the highest nutrient utilization was achieved when wort was supplemented with 0.75 g/l L-leucine resulting in the utilization of the highest amount of nutrients (51.25% FAN and 69.11% reducing sugar utilization) and production of 5.12% (v/v) ethanol. At the optimum fermentation pH of 5, 38.27% reducing sugars and 35.28% FAN were utilized, resulting in 4.32% ethanol (v/v) production. Wort supplemented with 0.12 g/l zinc sulphate resulted in 5.01% ethanol (v/v) production and 54.32% reducing sugar utilization. Spent yeast density ranged from 1.985 to 2.848 mg/ml for all parameters tested with the highest yield produced when wort was supplemented with 0.120 g/l zinc sulphate. This was also accompanied by the highest yeast viability of 2.12 × 107 cfu/ml achieved on day 3 of fermentation. Supplementation with 0.75 g/l L-leucine yielded the highest foam head stability with a rating of 2.67. Overall, ester compounds were relatively more stable at 4 °C than at RT decreasing by only 6.93% after three months, compared to a decrease of up to 16.90% observed at RT at the same time. Of all the volatile esters produced, ethyl octanoate was the least stable, with a 32.47% decrease in concentration at RT, phenyl ethyl acetate was the most stable ester at RT, decreasing by 9.82% after three months. Wort supplemented with 0.75 g/l L-leucine resulted in an increase in isoamyl acetate and phenyl ethyl acetate production by 38.69% and 30.40%, respectively, with a corresponding high expression of alcohol acetyltransferases, ATF2 (133.49-fold higher expression than the control). Elevation of fermentation temperature to RT resulted in the upregulation of ATF2 (27.11-fold), and producing a higher concentration of isoamyl acetate. These findings indicate that ester synthesis during fermentation is linked to both substrate availability and the regulation of gene expression. Therefore, it would be possible to manipulate the expression of certain ester synthestase genes to create new yeast strains with desirable ester production characteristics. Results from this study also suggest that supplementing wort with essential nutrients required for yeast growth and optimizing the fermentation conditions could be effective in controlling aroma-active ester concentrations to a desired level in beer.
Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

The potential for malodour in wine caused by the accumulation of ethylphenols has been widely studied with respect to the breakdown of the hydroxycinnamic acids, p-coumaric and ferulic acid, by D. bruxellensis. The presence of esterified hydroxycinnamate conjugates in grapes and wine is well established and they account for a large proportion of the hydroxycinnamate content. There exists the possibility that these conjugates could also provide the potential for spoilage, though they have never been linked to the direct formation of ethylphenols. The research highlighted within this thesis examines the potential role of a number of esterified conjugates in the production of ethylphenols by D. bruxellensis. Two classes of berry derived esters, the tartaric acid and glucose bound hydroxycinnamates, as well as the vinification formed ethyl esters, were synthesised and used for model fermentation experiments. Chapter 2 describes the preparation of a number of protected hydroxycinnamic acid derivatives that were used in the synthesis of the hydroxycinnamoyl tartrate esters (7 and 8) for the first time. Coupling 1-O-chloroacetyl protected p-coumaric and ferulic acids (21 and 22) with di-tert-butyl-L-tartrate (34) followed by selective hydrolysis of the tert-butyl esters yielded p-coumaroyl tartrate (7) and feruloyl tartrate (8). Hydroxycinnamoyl glucose esters (9 and 10) were prepared using the same hydroxycinnamates (21 and 22), esterifying with a prepared trichloroacetimidate glucosyl donor sequence, though purification of the glucose esters resulted in undesired chemical transformations. It was found that photoisomerisation of the glucose esters could be prevented via synthesis under red light, which gave trans-9 and 10, however migration of the hydroxycinnamoyl moiety around the glucose ring, which yielded mainly the 2-O-α- and 6-O-α-esters, was a product of submitting the esters to non-aqueous solvents and could not be avoided. The acyl migration of the glucose esters that was observed in Chapter 2 has been researched at a DFT B3LYP 6-31G* theoretical level in Chapter 3 with respect to both the thermodynamics and kinetics of the transformations. The desired 1-O-β-esters were thermodynamically favoured only in water, while in any other solvent studied the 2-O-α- and 6-O-α-esters would prevail. Kinetically, migration to the 3-O-position involved lower energy barriers which can be equated to a more rapid process, although the ring-flipped conformation needed to achieve the migration would promote subsequent migration to the 6-O-position. Step-wise migration, from the 1-O- to the 2-O-position, was found to be thermodynamically less favoured than other migrations investigated. This effect can be rationalised by the formation of a 5-membered cyclic intermediate in comparison to the 6- membered intermediate produced during 1-O- to 3-O-migration. However, the energy barriers involved in 1-O-β- to 2-O-β-migration better explain the comparative extent of migration observed between the p-coumaroyl and feruloyl glucose esters. The possibility of multiple glucose esters existing in wine was the focus of a brief study, finding two separate p-coumaroyl glucose esters in red and white wine, while a lesser extent of migration in feruloyl glucose limited observation to concentrated wine alone. However, due to co-elution of feruloyl glucose (10) with suspected p-coumaroyl anthocyanin derivatives in red wine, HPLC-MRM was required to detect it, which is the first report of this compound in red wine. Theoretical studies into observed photoisomerisations and the synthesis of cis-hydroxycinnamates are described in Chapter 4. The cis-ethyl hydroxycinnamates were isolated and hydrolysed to give a mixture of cis/trans-hydroxycinnamic acids (3 and 4), which could be separated by flash chromatography, though the pure cis-isomers isomerised rapidly under ambient conditions and slowly under red light back to the trans-isomers. Stable isomeric mixtures were achieved by irradiation with ultra-violet light giving mixtures of 40-50% of the cis-isomer which could be used further in fermentation studies. Computational evidence suggested that isomerisation of the hydroxycinnamic acids was favoured with greater resonance throughout the molecule. Those with deprotonated phenolic moieties possessed the most intramolecular electron movement, decreasing the HOMO-LUMO gap and promoting photoisomerisation. Smaller solvent and substrate effects were also noted, though the nature of the phenol and carboxyl clearly played the most important role in determining stability of each isomer. Fermentation in the presence of the synthesised trans-hydroxycinnamoyl esters (7-12) and investigation into the stereospecificity of D. bruxellensis enzyme activities was performed as detailed in Chapter 5. In Australia, three genetic groups of D. bruxellensis account for 98% of isolates, with the largest of these groups making up 85%. AWRI 1499 is a representative of the largest genetic group, with AWRI 1608 and AWRI 1613 belonging to the two remaining significant genetic groups. In the presence of AWRI 1499, the transethyl- esters (11 and 12) were metabolised to varying extents with the preference for breakdown of ethyl coumarate (11) over ethyl ferulate (12). This selectivity was investigated further and found to be common for both AWRI 1499 and AWRI 1608, while AWRI 1613 was unable to breakdown either ester. The preference for formation of 4- ethylphenol (1) over 4-ethylguaiacol (2) from the ethyl esters could accentuate the ratio of these compounds as seen in wine, initially thought to be brought about by the relative concentration of the precursor acids. Of the berry derived esters, the tartrate esters (7 and 8) were not metabolised by AWRI 1499, and subsequent fermentations with AWRI 1608 and 1613 yielded the same result. This confirmed that the tartrate esters cannot contribute directly to the formation of ethylphenols during exposure to D. bruxellensis. The glucose esters were metabolised by AWRI 1499 to a moderate extent (35% conversion), providing information that these can contribute to the accumulation of ethylphenols during barrel ageing. Furthermore, the isomerisation of the glucose esters lead to studies into the stereoselectivity of D. bruxellensis enzyme activities, whereby the decarboxylase as well as the ethyl esterase showed selectivity for the trans-isomers and that the cis-hydroxycinnamate content of grapes and wine are not important in the accumulation of ethylphenols. The experimental procedures employed throughout Chapters 2-5 are outlined in Chapter 6.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2012

碩士
國立清華大學
化學系
92
A method for determining the flavor compounds – esters (ethyl acetate, isobutyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, hexyl acetate, benzyl acetate, ethyl octanoate, 2-phenylethyl acetate) in wines by headspace solid-phase microextraction (HSSPME) combined with gas chromatography-ion trap mass spectrometry (GC/ITMS) was developed. Several parameters of extraction and desorption procedure such as types of fibers, extraction technique, extraction temperature, extraction time, desorption temperature, desorption time, stir rate, sample’s volume, salt concentration, ethanol content, were studied and optimized. The method shows good linearity in two orders for most of analysts. Good precisions (3.6% ~10.2%) are obtained using 4-methyl-2-pentanol, ethyl heptanoate, and 2-octanol as internal standards. Finally, the method was successful applied to analyze esters in two kinds of Taiwan’s wines – Rice Wine and Sorghum Wine.