Dissertations / Theses on the topic 'Aroma compounds'

Pervaporation is a selective membrane technique in which a liquid feed mixture is separated by means of partial vaporization through a non-porous perm-selective membrane. This method can be used for the recovery of heat sensitive aroma compounds to avoid them from thermal damage in beverage industries. The main objective of this study was to determine the effects of feed temperature (30, 40, 50°
C), composition (different model solutions, strawberry essence), concentration (50, 100, 150 ppm) and permeate pressure (4, 8 mbar) on the recovery of aroma compounds of strawberry by pervaporation in terms of mass flux and selectivity. In addition, it was aimed to optimize the extraction conditions (extraction time, temperature, agitation speed, strawberry matrix) of Solid-phase microextraction (SPME), which is used for the analysis of strawberry aroma compounds. Optimum results for SPME were obtained at 40°
C, 700 rpm for 30 min and no matrix effect was observed. Pervaporation experiments were performed using a hydrophobic membrane, PERVAP 1070 (PDMS). As the feed temperature increased, the mass flux and selectivity increased and the total mass flux followed an Arrhenius type relation. Decreasing downstream pressure increased both total flux and selectivity, while increase in feed concentration led to higher organic fluxes but lower selectivities. In general, PERVAP 1070 showed a higher selectivity towards Methyl butyrate (MTB) than Ethyl butyrate (ETB) and MTB flux was affected negatively by the presence of ETB in the feed solution. Pervaporation experiments were also performed with a strawberry essence and strawberry model solution. The selectivities of MTB and ETB were negatively affected by the presence of other aroma compounds.

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.

Lambic beer is one of the oldest beer styles still being brewed in the western world today and the only beer that is still brewed through spontaneous fermentation. Lambic beers are only produced within a 500 km radius of Brussels because of the natural microflora found within the air in that region. Little is known about the chemical composition of lambic beers. The objective of this research were (1) to compare SPME and SAFE for the isolation of flavor and aroma compounds,  (2) determine the volatile composition and acids of commercially available lambic gueuze using SPME/GC-MS and HPLC, and (3) determine the major aroma compounds of aging lambic beer using GC-O.  Comparing the two extraction methods, both SPME and SAFE were able to identify a similar number of chemical compounds, however SAFE identified a greater number of acid compounds. A total of 50 compounds were identified within nine commercial brands of lambic beer. HPLC was used in the identification and quantification of acetic and lactic acids. The concentration of acetic acid in the commercial products ranged from 723 mg/L â " 1624 mg/L and lactic acid ranged from 995 â " 2557 mg/L. GC-O was used in the analysis of aged (3-28 months) lambic beer samples. As the samples increased in age, the number of aroma compounds detected by the panelists also increased. Panelists were detected nine aroma compounds in the 3 month old sample, while 17 compounds were detected in the 28 month old sample. The research conduct increased the number of volatile and semi-volatile compounds identified in lambic beer from 27 to 50.
Ph. D.

The presence of protein in white wines represents a major problem for the wine industry mainly due to the fact that proteins generate haze in the bottled white wines. Protein instability, which results in wine haze formation, is due to some grape PR-Proteins that thanks to their intrinsic resistance survive the vinification process, pass into the wine where cause the appearance of undesirable haze and deposits, leading to rejection by consumers. Protein hazing of white wines is considered to be a three-step process, involving protein denaturation followed by aggregation into colloidal particles able to scatter the visible light and make the wine turbid. Because of the complexity and the variability of the wine matrix, the factors and mechanisms involved in this process are still largely unknown. Commonly, winemakers prevent haze formation by removing the proteins through the use of bentonite. However, this treatment causes loss of wine and, being unspecific, also the removal of some aroma compounds. It has been calculated that the total cost deriving from bentonite treatments corresponds to a worldwide total amount of 1 billion dollars per year. Therefore, basic and applied research is still needed to solve the problem of protein haze formation in white wines. Firstly, the present thesis faces the problem of the impairment of aroma due to bentonite fining. In particular, the study arises from a previous investigation which suggested the existence of an interaction between proteins and aroma compounds. In this context, the interaction of the main wine protein VVTL1 with some fatty acid ethyl esters (FAEE), which are important fermentative aroma compounds has been investigated. Due to the difficulty to determine this interaction at the molecular level, Synchrotron Radiation Circular Dichroism (SRCD) has been used to study the secondary structure of the wine protein as affected by the interactions with FAEE having different chain lengths. Subsequently, the research continued with the investigation of the role played by tannins in the phenomena leading to protein instability of white wines. To this purpose, the effects of several polyphenols (deriving from wine and not) on the stability of VVTL1 has been investigated using SRCD. In parallel, the capability of tannins to react with the proteins over time in bottled wine has been evaluated by Dynamic Light Scattering (DLS) studies in a model wine system. In addition, the thermal stability of two purified proteins, which are representative of the major classes of proteins in white wine (i.e. a class IV Chitinase and the VVTL1), has been investigated by Differential Scanning Calorimetry (DSC) in the presence of the tannins purified from wine at different times after bottling. Finally, the last part of the research focuses on the possibility to produce good quantities of grape proteins in pure form starting from the in vitro culture of berry pulp tissues. These proteins can be used for molecular and functional characterisation. In particular, with this technique it is possible to label the proteins by cultivating the cellular tissues in the presence of N15 which allows the study of their fine structure and interactions by spectroscopic methods.
La presenza di proteine nei vini bianchi rappresenta un problema di grande importanza per l’industria del vino, principalmente dovuto alla formazione di torbidità nei vini bianchi in bottiglia. L’instabilità proteica, nonché formazione di torbidità, è associata ad alcune proteine di difesa della pianta che per mezzo della loro intrinseca resistenza e stabilità sopravvivono al processo di vinificazione, passando nel vino dove causano la comparsa dell’indesiderata torbidità e di depositi in bottiglia, la quale non incontra le aspettative del consumatore e viene quindi scartata. La torbidità proteica nei vini bianchi è considerata come un processo a tre stadi, che coinvolge la denaturazione delle proteine seguita dall’aggregazione in particelle colloidali capaci di disperdere la luce visibile e far divenire il vino torbido. Data la complessità e la variabilità della matrice vino, i fattori e meccanismi coinvolti in questo fenomeno sono ancora largamente sconosciuti. Normalmente gli enologi prevengono la formazione di torbidità rimuovendo le proteine attraverso l’uso della bentonite. Tuttavia questo trattamento causa la perdita di vino, ed essendo aspecifico, causa anche la rimozione di alcuni composti aromatici. È stato calcolato che il costo totale derivante dall’uso di bentonite corrispondi a 1 miliardo di dollari l’anno. Pertanto è ancora necessaria della ricerca di base e applicata per risolvere il problema della formazione di torbidità proteica nei vini bianchi. La prima parte di questa tesi tratta il tema dell’impoverimento aromatico causato dal trattamento con bentonite. In particolare questo studio prende ispirazione da un precedente lavoro, il quale suggerisce l’esistenza di un’interazione tra le proteine e i composti aromatici. In questo contesto è stata studiata l’interazione della principale proteina dei vini bianchi, la VVTL1, e alcuni esteri etilici degli acidi grassi, i quali sono importanti aromi fermentativi del vino. Data la difficoltà di determinare a livello molecolare questo tipo d’interazione è stata sfruttata la luce di sincrotrone applicata al dicroismo circolare (SRCD) per studiare l’effetto di alcuni esteri etilici di acidi grassi a media catena sulla struttura secondaria della proteina del vino. In seguito, la ricerca è proseguita con lo studio mirato a comprendere l’esatto ruolo dei tannini nel fenomeno che conduce all’instabilità proteica dei vini bianchi. A questo scopo, sono stati studiati tramite sincrotrone gli effetti di diversi polifenoli (derivati o no dal vino) sulla stabilità della VVTL1. In parallelo la capacità nel tempo dei tannini di reagire con le proteine in bottiglia è stata valutata svolgendo degli studi di diffusione dinamica della luce (DLS) in vino modello. Con l’ausilio della Calorimetria differenziale a scansione (DSC), è stato inoltre studiata, la stabilità termica di due tra le più rappresentative proteine del vino (la VVTL1 e la chitinasi classe IV) in presenza di tannini purificati a tempi diversi di evoluzione da vino imbottigliato. Infine, l’ultima parte della ricerca si focalizza nella possibilità di produrre proteine dell’uva in quantità accettabili in forma pura partendo dalla coltivazione in vitro di tessuti di polpa della bacca. Queste proteine possono essere utilizzate per la caratterizzazione strutturale e funzionale. In particolare, con questa tecnica si rende possibile la marcatura delle proteine facendo crescere i tessuti cellulari d’uva in presenza di N15, il quale consentirebbe, per mezzo di metodi spettroscopici, lo studio in dettaglio della loro struttura e delle loro interazioni.

Orientadores: Helena Teixeira Godoy, Rodrigo Ramos Catharino
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O aroma final de um vinho é formando por um grande número de compostos químicos voláteis, presentes em concentrações variáveis e detectáveis pelo sistema olfativo humano. O estudo da fração volátil de vinhos tem sido o objetivo de muitas iniciativas de estudo, sendo que as metodologias de pesquisa de compostos voláteis em vinhos compreendem etapas fundamentais, tais como: a extração dos compostos, a separação das diferentes moléculas por cromatografia gasosa acoplada à um sistema de detecção e posteriormente, a identificação e quantificação dos compostos voláteis. O objetivo deste trabalho foi extrair, identificar e quantificar os compostos voláteis de vinhos Chardonnay produzidos em diferentes regiões brasileiras utilizando microextração em fase sólida no modo de headspace (HS-SPME) e cromatografia gasosa acoplada à espectrometria de massas tandem (GC-MSMS). As amostras foram produzidas nas principais regiões vitícolas brasileiras: Serra Gaúcha e Campanha (Rio Grande do Sul), Serra Catarinense e Região de Altitude (Santa Catarina) e Sul de Minas (Minas Gerais). O método de extração de compostos de aroma foi desenvolvido usando a técnica de HS-SPME e GC-MS/MS e análise multivariada com delineamento composto central rotacional (CCRD) e metodologia de superfície de resposta para obtenção de um ponto ótimo de extração. As condições otimizadas foram temperatura (°C) e tempo (minutos) de extração. A otimização multivariada das condições de extração por HS-SPME permitiu avaliar os efeitos de tempo e temperatura de extração nesta metodologia, permitindo a obtenção de um ponto ótimo de extração, 30°C e 45 minutos, dos representantes das classes dos principais compostos voláteis presentes em vinhos Chardonnay. A análise qualitativa e quantitativa da fração volátil dos vinhos foi realizada usando a técnica de GC-MS/MS. O método foi validado e aplicado para a análise de 31 compostos voláteis nas amostras de vinhos Chardonnay. Destes compostos analisados, 30 foram utilizados para a realização de PCA e HCA visando a diferenciação regional dos vinhos. A Análise de Componentes Principais (PCA) e Análise de Agrupamento Hierárquico (HCA) foram realizadas com os dados obtidos. Os resultados obtidos demonstraram que as amostras produzidas em Minas Gerais e na Serra Gaúcha possuem marcadores olfativos herbáceos e vegetais, enquanto as amostras produzidas na região da Campanha/RS, Serra Catarinense e Região de Altitude/SC possuem marcadores florais e frutados, reforçando o papel da origem geográfica na formação e diferenciação do aroma dos vinhos analisados
Abstract: Wine aroma wine is formed by a large number of volatile chemical compounds, present in variable concentrations and detectable by the human olfactory system.The study of the volatile fraction of wines has been the aim of many research studies and those analytical methods including basic steps, such as: isolation and extraction of matrix, separation and detect by gas chromatography coupled to a detection system and finally, the identification and quantification of the individual components. The aim of this study was to extract, identify and quantify the volatile compounds of brazilian Chardonnay wines, produced in different regions, using solid phase microextraction in headspace mode (HS-SPME) and gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). Samples were produced in the main wine producing regions in Brazil: Serra Gaúcha and Campanha (Rio Grande do Sul state), Santa Catarinense and Região de Altitude (Santa Catarina state) and Sul de Minas (Minas Gerais state). A extraction method of aroma compounds was developed and optimized using HS-SPME and GC-MS/MS and multivariate central composite rotational design (CCRD) with response surface methodology to obtain an optimum extraction condition. The optimized conditions were temperature (°C) and time (minutes) of extraction. Multivariate optimization of extraction conditions for HS- SPME allowed to evaluate the effects of time and temperature of extraction allowing the achievement of the optimum extraction point of compounds representatives of the main classes of volatile compounds in Chardonnay wines. The optimum point observed is at temperature in 30 ° C and time in 45 minutes. A method for qualitative and quantitative analysis of the volatile fraction of wines was carried out using the technique of GC-MS/MS. The method was validated and used for the analysis of 31 volatile compounds in samples of Chardonnay wines. From compounds analyzed, 30 compounds were used to perform PCA and HCA targeting regional differentiation of wines. The Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were performed with the data obtained. The results showed that samples produced in Minas Gerais and Serra Gaúcha shows high concentration of compounds with vegetal and herbaceous notes. Meanwhile, samples of Campanha, Serra Catarinense and Região de Altitude shoes high concentration of compounds with floral and fruity notes. These results reinforce the role of geographical origin in the formation and differentiation of aroma in wines
Doutorado
Ciência de Alimentos
Doutora em Ciência de Alimentos

Orientador: Gláucia Maria Pastore
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Este trabalho de doutorado teve como principal objetivo estudar a produção biotecnológica de compostos de aroma a partir da biotransformação de terpenos, analisando potenciais biocatalisadores, processos bioquímicos diferenciados e a otimização das condições de processo envolvidas na produção destes aromas naturais para possíveis aplicações industriais. Inicialmente, o trabalho prático foi direcionado para uma série de estudos com o fungo Fusarium oxysporum 152B, visando complementar os recentes avanços alcançados pelo grupo de pesquisa na caracterização e otimização da bioconversão de R-(+)-limoneno em ?-terpineol. Desta forma, os resultados foram promissores, demonstrando o grande potencial e versatilidade deste fungo para a área, sendo que este biocatalisador foi capaz de produzir diferentes metabólitos a partir dos substratos S-(?)-limoneno, ?-pineno, ?-terpineno e linalol. Na sequência, o trabalho visou avaliar a bioconversão de S-(?)-limoneno para limoneno-1,2-diol em maiores detalhes. A produção de limoneno-1,2-diol, em condições não otimizadas, chegou a 1,2 g.L-1, sendo que foi detectada a continuação desta via metabólica quando este produto foi posteriormente metabolizado a 1-hidroxi-2- oxolimoneno, sugerindo que o fungo Fusarium oxysporum 152B possui uma via de degradação de limoneno recentemente descoberta. O trabalho prático visou também a otimização da produção de limoneno-1,2-diol por meio de uma estratégia sequencial de experimentos. Com base nas análises estatísticas, a produção deste composto a partir da bioconversão de S-(?)-limoneno pelo fungo Fusarium oxysporum 152B chegou a 3,7 g.L-1, utilizando pH 6,5, 5 g.L-1 de substrato, a 28 oC e 250 rpm de agitação. Além disso, esta foi a primeira descrição da utilização do resíduo agroindustrial conhecido como manipueira, originada ao longo do processamento da mandioca, para a produção de biomassa deste fungo, bem como uma das maiores concentrações de limoneno-1,2-diol reportadas na literatura específica. Além disso, o trabalho conduzido visou realizar uma série de comparações práticas entre a biotransformação de R-(+)-limoneno a ?-terpineol e a bioconversão de S-(?)-limoneno a limoneno-1,2-diol, pela mesma linhagem. Adicionalmente, este foi o primeiro trabalho que analisou as diferenças ultraestruturais causadas no biocatalisador ao longo do processo de bioconversão destes substratos, por meio de microscopia eletrônica de varredura e transmissão. Finalmente, foi estudada a otimização da produção de ?-terpineol, a partir da biotransformação de limoneno, utilizando o biocatalisador reconhecido como Sphingobium sp. Após o trabalho prático e análise estatística dos dados, observou-se que as melhores condições para desenvolvimento deste processo foram pH 7,0, concentração de limoneno de 350 g.L-1, agitação de 200 rpm e 28 oC. Nestas condições, a produção deste álcool monoterpênico chegou a 500 g.L-1, que pode ser considerada como a maior concentração de ?-terpineol já relatada na bibliografia de processos biotecnológicos.
Abstract: This work aimed to study the biotechnological production of aroma compounds from the biotransformation of terpenes, analyzing potential biocatalysts, biochemical processes and optimization of process conditions involved in the production of natural flavors for industrial applications. Initially, the practical work was directed to a series of studies with the fungal strain Fusarium oxysporum 152B, aiming to enhance the knowledge obtained in recent advances achieved by our research group in the characterization and optimization of the biotransformation of R-(+)-limonene into ?-terpineol. Thus, the results were promising, showing the great potential and versatility of this fungus to the specific area, and this biocatalyst was able to produce new metabolites from substrates S-(?)-limonene, ?-pinene, ?-terpinene and linalool. In the sequence, this study aimed at assessing the bioconversion of S-(?)-limonene into limonene-1,2-diol in greater detail. Thus, chapters 3, 4 and 5 were designed to a better characterization of this pathway. The production of this compound under non-optimized conditions, reached 1.2 gL-1, and the continuation of this pathway has been detected when this product was subsequently metabolized to 1-hydroxy-2-oxolimonene, suggesting that the fungus Fusarium oxysporum 152B might have a limonene degradation pathway only recently discovered. The practical work also conducted an extensive optimization of the production of limonene-1,2-diol by means of a sequential strategy. Based on statistical analyzes, the production of this compound from the bioconversion of S-(?)-limonene by the fungus Fusarium oxysporum 152B reached 3.7 gL-1, using pH 6.5, 5 gL-1 of substrate at 28 ° C and 250 rpm agitation. Moreover, this is the first description of the use of agroindustrial residue known as cassava wastewater, for the production of this fungal biomass, as well as one of the highest concentration of biotechnological limonene-1,2-diol reported in the specific literature. In addition, the research conducted aimed to perform a series of comparisons between practical conditions involved in the biotransformation of R-(+)-limonene into ?-terpineol, and the bioconversion of S-(?)-limonene into limonene-1,2-diol, for the same strain. Additionally, this is the first study that reported the ultrastructural differences along the bioconversion process of these substrates by means of scanning and transmission electron microscopy. Finally, this work evaluated the optimization of the production of ?-terpineol from the biotransformation of limonene using the biocatalyst recognized as Sphingobium sp. After the practical work and statistical analysis, it was observed that the best conditions for developing this process were pH 7.0, concentration of limonene 350 g L-1, agitation at 200 rpm and 28 oC. Accordingly, the production of the monoterpe alcohol reached 500 g.L-1, which can be considered as the highest concentration of ?-terpineol already reported in the literature for biotechnological processes.
Doutorado
Ciência de Alimentos
Doutor em Ciência de Alimentos

La maduixa representa el cultiu de baies més consumit a tot el món, amb una producció de 9,2 milions de tones l’any 2017. Els programes de millora tradicionalment s’han centrat en els trets agronòmics, però la qualitat dels fruits s’ha convertit recentment en un objectiu principal. El sabor és el principal factor responsable de la qualitat de la maduixa, sent atractiu per el consumidor. La maduixa diploide (Fragaria vesca) es un model per a el maduixot, gràcies al cicle de vida perenne, genoma reduït, curt període generacional, sistema de transformació genètica senzill i eficient, i als abundants recursos genètics. L’objectiu principal d’aquest treball ha estat estudiar la base genètica del sabor en la maduixa diploide mitjançant una col·lecció de línies isogéniques (NIL) desenvolupada per un encreuament entre un pare recurrent F. vesca i el progenitor donant F. bucharica. En primer lloc, hem millorat la col·lecció NIL desenvolupada anteriorment amb deu noves línies, cobrint en total el 94,8% del parental donant. És una eina genètica altament rellevant per mapar diversos trets de maduixa diploides. Per localitzar els loci dels caràcters quantitatius (QTL)de sabor es van analitzar el pH, l’àcid cítric i ºBrix. Es va mapar un QTL estable associat a l’augment del pH, dos QTL majors per la reducció del pH, dos QTL majors per la disminució dels nivells de l’àcid cítric i un QTL per l’augment dels ºBrix. Per localitzarQTL majors per a compostos volàtils al LG5, hem estudiat en profunditat els compostos aromàtics a les línies del LG5. Es van identificar disset compostos volàtils clau i es van assignar cinc QTL. Els QTL de metil 2-aminobenzoat i mirtenil acetat es van localitzar a LG5:20-35cM,el QTL per disminuir el contingut de metil butanoat a LG5:11-20cM i dos QTL per a compostos volàtils verds (CVV) (Z)-3-hexenil acetat i (E)-2-hexenil acetat a LG5:50-76cM. Onze gens van ser seleccionats percandidats a controlar l’acumulació de compostos volàtils.Es va analitzar el nivell de transcripció F. vesca i les NILs. Finalment, es van verificar tres gens en LG5:0-35cM i tres gens en LG5:50-76cM per presentar diferències significatives en l’expressió entre el pare recurrent i les NILs amb al·lels de F. bucharica en LG5. El gen FvH4_5g29270 que codifica una isomerasa 3Z-2E-enal es va seleccionar com a gen candidat per a CVV en LG5:50-76cM. Per verificar la funció del gen FvH4_5g29270, es va construir el vector de sobreexpressió gènica i es va expressar de forma transitòria en F. vesca i en dos NILs amb l’al·lel F. bucharica de FvH4_5g29270 mitjançant transfecció mediada per agrobacterium. Els resultats de contingut de CVVno van ser els esperats, principalment a causa de la baixa eficiència de la transformació. Un altre caràcter de qualitat important es l’aparença del fruit. Es va analitzar el color de la maduixa amb vuit paràmetres de color per a tres collites en la col·lecció. El color taronja es va observar en algunes NILs i es va mapar en LG5:35-39cM. Es van analitzar divuit gens candidats en aquesta regió per obtenir el nivell d’expressió de la transcripció en fruites madures. El gen FvH4_5g14770 que codifica per una proteïna d’unió al complex de clorofil·la A-B dependent de la llum es va seleccionar com a gen candidat, ja que mostrava un nivell de transcripció significativament superior en fruites de color taronja que en fruites vermelles,iaquest canvi pot estar relacionat amb el procés de reducció del contingut de clorofil·la. Tots aquests resultats van proporcionar una base fonamental per a la millora de l’aroma i el color de la maduixa.
La fresa representa el cultivo de bayas más consumido en todo el mundo, con una producción de 9,2 millones de toneladas en el año 2017. Los programas de mejora se han centrado en caracteres agronómicos, siendo la calidad del fruto un objetivo principal en los últimos años. El sabor es el factor responsable de la calidad en la fresa, actuando directamente sobre el consumidor. La fresa diploide (Fragaria vesca) es modelo para elfresón, debido a su ciclo de vida perenne, pequeño genoma, corto periodo generacional, un sistema de transformación genética simple y eficiente y abundantes recursos genéticos. El objetivo principal de este trabajo es estudiar la base genética del sabor del fruto en la fresa diploide utilizando una colección de líneas isogénicas (NIL) desarrollada por un cruce entre unparental recurrente F. vesca y un parental donante F. bucharica. En primer lugar, incrementamos la colección NIL desarrollada previamente con 10 nuevas líneas, con una cobertura del 94.8% del genoma del parental donante. Para posicionarlos loci de los caracteres cuantitativos (QTL),se analizaron pH, ácido cítrico y ºBrix enfresas maduras. Se mapeó un QTL estable asociado alincrementodel pH, dos QTL asociados con su disminución, dos QTL asociados con la reducción en ácido cítrico y un QTL relacionado con incremento deºBrix. Debido a la localización en LG5 de QTL asociados a compuestos volátiles, estudiamos su acumulaciónen todas las líneas del LG5. Se identificaron diecisiete compuestos volátiles clave y se maparon cinco QTL. Los QTL asociados al metil2-aminobenzoato y mirtenil acetato se localizan en LG5:20-35cM. El QTL asociado a la disminución de metil butanoato se localizó en LG5:11-20cM. Dos QTL ligados a la variación decompuestos volátiles verdes (CVV) (Z)-3-hexenil acetato y (E)-2-hexenil acetato están en LG5:50-76cM. Los genes candidatos que controlan la acumulación de compuestos volátiles aromáticos en LG5fueron once yse analizósu transcripción en frutas completamente maduras de F. vesca y de las líneas NIL de la región LG5.Se identificaron tres genes en LG5:0-35cM y tres genes en LG5:50-76cM que presentan diferencias significativas en la expresión entre el padre F. vesca y las NIL que albergan alelos de F. bucharica. El gen FvH4_5g29270 que codifica una 3Z-2E-enal isomerasa se seleccionó como gen candidato para CVV en LG5:50-76cM. Para verificar la función del gen FvH4_5g29270, se construyó el vector de sobreexpresión génica y se expresó de forma transitoria en F. vesca y en las NILs que contienen el alelo de FvH4_5g29270 en F. bucharica mediante transfección mediada por agrobacterium. Sin embargo, los resultados del contenido de CVVno fueron los esperados, principalmente debido a la baja eficiencia de transformación. La importancia de apariencia externa enfresa es el color,y se analizó aplicando ocho parámetros de color en tres cosechas distintas en la colección. Se observó el color del fruto anaranjado en algunas NILs y se mapeó en LG5:35-39cM. Se analizaron dieciocho genes candidatos en esta región para determinar el nivel de expresión de la transcripción en frutos maduros en plantas de F. vesca y en las NILs que albergan la introgresión LG5:35-39. El gen FvH4_5g14770 que codifica para una proteína de unión al complejo clorofila A-B dependiente de luz se seleccionó como un buen gen candidato ya que mostró un nivel de transcripción significativamente mayor en las fresas de color anaranjado que en las fresas rojas, y dicho cambio puede estar relacionado con la reducción del contenido de clorofila. Todos estos resultados proporcionaron una base fundamental para la mejora del aroma y la variación del color de la fresa.
Strawberry, belonging to Fragaria genus, Rosaceae family, is the most commonly consumed berry fruit crop worldwide, with a production of around 9.2 million tons during 2017. Although traditionally breeding programs have been focused on improving agronomic traits, fruit quality has become a main goal recently. Fruit flavor is the main factor responsible for the fruit quality that is a direct factor attracting customers. The diploid strawberry (Fragaria vesca) serves as an important model plant for cultivated strawberry and Rosaceae family, due to its perennial life cycle, small genome, short generation time, a simple and efficient genetic transformation system and abundant genetic resources. The main goal of this work was to study the genetic basis of fruit flavor in diploid strawberry using a isogenic lines collection (NIL) developed by a cross between the recurrent parent F. vesca and the donor parent F. bucharica. Firstly, we improved the previously developed NIL collection with 10 new lines added. Finally, this population consists of 49 lines with overlapping introgressions covering 94.8% of background of donor parental line. It is a highly relevant genetic tool for mapping a variety of traits for diploid strawberry. Fruit flavor traits, including pH, citric acid and oBrix of ripe fruits of the collection were statistically analyzed for mapping as quantitative traits loci (QTL). One stable QTL was mapped for increasing pH and two major QTL for decreasing pH. Two major QTL were mapped for decreasing citric acid and one QTL for increasing oBrix. Due to locating many major QTL for aroma volatile compounds in LG5, we deeply studied the aroma compounds in the new lines harboring introgressions in LG5. Seventeen key volatile compounds were identified and five QTL were mapped. The QTL for methyl 2-aminobenzoate and myrtenyl acetate were located in the same region LG5:20-35cM.The QTL for decreasing methyl butanoate content was located in LG5:11-20cM. Two QTL for green volatile compounds (Z)-3-hexenyl acetate and (E)-2-hexenyl acetate were located in LG5:50-76cM. In order to investigate the major genes controlling aroma volatile compounds accumulation, eleven genes were selected as important candidate genes to analyze the transcription level in fully ripe strawberry fruits of F. vesca and NILs harboring F. bucharica alleles. Finally, three genes in LG5:0-35cM and three genes in LG5:50-76cM were verified to present significant differences in expression between the recurrent parent F. vesca and the NILs harboring F. bucharica alleles in these LG5 regions. The gene FvH4_5g29270 encoding a 3Z-2E-enal isomerase was selected as candidate gene for green leaf volatile compounds in LG5:50-76cM. For verifying the function of gene FvH4_5g29270, the gene overexpression vector was constructed and transiently expressed in F. vesca and two NILs with the F. bucharica allele of FvH4_5g29270 via agrobacterium mediated transfection. However, the green volatile compounds content results were unexpected, mainly due to the low transformation efficiency. Attending to the important fruit appearance traits, the fruit color was analyzed with eight color parameters for three harvests. Orange fruit color was observed in some NILs and was mapped in LG5:35-39cM. Eighteen candidate genes in this region were analyzed for transcript expression level in fully ripen fruits from the recurrent parent F. vesca and NILs harboring the introgression containing LG5:35-39. The gene FvH4_5g14770 encoding light-harvesting complex chlorophyll A-B binding protein was selected as a good candidate gene since it showed significantly higher transcript level in orange colored fruits than in red fruits that can be related to chlorophyll content reduction. All these results provided fundamental basis for strawberry aroma and color breeding.

By increasing fruit exposure to sunlight and influencing fruit development, leaf thinning in the fruit zone can improve grape quality and lower disease incidence; however, further investigations on the timing, varietal response and intensity are needed to optimize results and to better understand underlying physiologic responses. Fruit zone leaf thinning was applied at different timing and intensities to evaluate its effect on cluster health and fruit composition in Cabernet Sauvignon and Chardonnay. Treatments consisted of control (C), pre-bloom leaf thinning (PB) and two levels of fruit-set leaf thinning (three leaves, PF3 and six leaves, PF6). In an additional project on Cabernet Sauvignon, two levels of hedging (17th node, NH and 12th node, H) were integrated with no leaf thinning (L) and fruit set leaf thinning (LR, three leaves). All leaf thinning treatments consistently reduced disease incidence compared to control vines in both varieties, with the reduction extent varying between 2017 and 2018. Yield was not negatively affected by leaf thinning treatments, even though PB reduced cluster compactness by decreasing the number of berries per cluster of Chardonnay in 2017. Control vines tended to have greater titratable acidity than defoliated vines, while Brix and pH responses varied between seasons. No direct positive correlation was found between sunlight exposure and norisoprenoids concentration. Post fruit set leaf thinning PF6 consistently increase free norisoprenoids at harvest, while pre-bloom defoliation never did. Heterogeneous responses were observed for bound and total norisoprenoids. In Cabernet Sauvignon free, bound and total 1,1,6-trimethyl-1,2-dihydronaphtalene (TDN) was consistently increased by PF3. Hedging negatively influenced Brix and anthocyanins accumulation in 2017, and increased free norisoprenoids while decreasing the bound and total fraction. Results revealed that a high level of stress possiblt caused by excess sunlight and/or reduced photosynthesis might negatively affect norisoprenoids glycosylation.
Master of Science in Life Science

Mestrado em Engenharia Alimentar - Processamento de Alimentos - Instituto Superior de Agronomia
The aim of this study was the analysis of volatile compounds responsible for the flavour of a texturized mango product, namely terpenes, lactones, aldehydes and alcohols, evaluating the impact of the processing applied: thermal treatment to inactivate enzymes, texturization with gellan gum, application of an edible coating and two conservation processes. The analytical method used was gas chromatography combined with the technique of solid phase microextraction. The product under development consists in a bar of mango puree texturized using the hydrocolloid gellan gum, matching 1% of the weight of the mango puree. This percentage is composed by gellan low and high acyl, with the ratio 25/75, respectively. The product also has an edible coating, comprised by 1% of gellan gum which is formed with a 90/10 low acyl/high acyl ratio. The thermal treatment to inactivate enzymes of mango puree caused a decrease of most volatile compounds. The texturized mango puree has exhibited an ability to release the volatiles, similar to mango puree. The edible coating applied to the surface of the texturized product showed retention capacity for a small number of volatiles. The applied conservation processes, high hydrostatic pressures and thermal treatments, showed similar effects and didn’t change the volatile profile of the bar significantly. Microbiologically the product proved to be stable and both conservation treatments applied were effective in microbiological control.

Orientador: Gláucia Maria Pastore
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O aroma é responsável por grande parte do sabor de um alimento, e considerado um dos atributos mais importantes na aceitação do produto pelo consumidor. A preferência dos consumidores por produtos naturais tem incentivado o desenvolvimento de novos processos biotecnológicos para a produção de compostos de aroma naturais. A biotransformação de terpenos é uma alternativa promissora para a produção de aromas, visto que pode contornar alguns problemas associados a síntese química como a baixa geração de resíduos tóxicos e alta regio- e estéreo-seletividade das reações. Ademais, segundo a legislação brasileira, produtos obtidos por métodos microbiológicos podem ser rotulados como 'naturais' (ANVISA, Resolução nº 104, de 14 de maio de 1999). Os processos de biotransformação de monoterpenos frequentemente utilizam bactérias do gênero Pseudomonas como biocatalisadores por se adaptarem facilmente a diferentes substratos e condições extremas devido a sua elevada versatilidade metabólica e sistema enzimático complexo. Assim, o presente trabalho visou a utilização de monoterpenos (limoneno, a-pineno e citronelol) como substratos em processos de biotransformação por micro-organismos do gênero Pseudomonas. O Capítulo 1 apresenta um artigo de revisão atualizado sobre o potencial biotecnológico de micro-organismos do gênero Pseudomonas como biocatalisadores em processos de biotransformação. O Capítulo 2 refere-se aos resultados obtidos pelos experimentos que contemplam o isolamento de bactérias em meio seletivo e diferencial para Pseudomonas, e sua aplicação em processos de biotransformação de monoterpenos (R-(+)-limoneno, a-pineno, citronelol). Das 34 linhagens selecionadas, 9 bactérias foram capazes de biotransformar pelo menos um dos substratos em compostos de aroma de maior valor agregado. R-(+)-limoneno foi convertido a cis- e trans-carveol e carvona por 7 linhagens, atingindo concentrações de até 412 mg/L, 275 mg/L e 209 mg/L, respectivamente. a-Pineno, por sua vez, foi usado como fonte de carbono e energia por 7 linhagens e foi convertido principalmente em cis-verbenol e verbenona a baixas concentrações por 5 linhagens. Outros compostos como limoneno, mirtenol e ß-pineno, também foram identificados como produtos principais da conversão do a-pineno por 2 linhagens. Por outro lado, apenas 3 linhagens foram capazes de metabolizar citronelol em outros compostos derivados, como óxidos de rosa e isopulegol. O efeito da variação do pH do meio de biotransformação e da indução das linhagens, através da pré-exposição do micro-organismo ao substrato, no crescimento celular e formação de produtos de bioconversão foram avaliados com o intuito de otimizar o processo de biotransformação. A produção de óxido de rosa a partir de citronelol foi estudada e detalhada no capítulo final, que configurou um artigo publicado na revista Chemical Engineering Transactions. Em testes de citotoxicidade, a linhagem mostrou alta resistência a maiores concentrações de terpeno, revelando grande potencial para posterior otimização do processo. Os compostos derivados mais notáveis foram destacados no presente trabalho pela alta qualidade sensorial e alto valor agregado. Além das notas sensoriais altamente relevantes industrialmente, a carvona e o carveol são ainda reconhecidos por apresentar algumas funções biológicas. A verbenona e verbenol se destacam pelo alto custo e vasta aplicação nas áreas alimentícia, da agricultura, e da medicina. O óxido de rosa é um dos componentes mais importantes na criação de composições florais em perfumaria, além de possuir um baixo threshold. Sendo assim, a biotransformação de precursores de baixo custo em compostos de alto valor agregado pode ser considerado uma estratégia vantajosa e econômica
Abstract: Aroma compounds influence greatly the flavor of food products and govern their acceptance by consumers and market success. The increasing consumer preference for natural products has encouraged remarkable efforts towards the development of biotechnological processes for the production of natural flavor compounds. Biotransformation of terpenes represents a very promising alternative for production of aromas, since overcome the problems associated with chemical synthesis such as the low generation of toxic waste and high regio-and stereo-selectivity reactions. In addition, products obtained by microbiological methods can be labeled 'natural' under Brazilian law (ANVISA Resolution No. 104 dated May 14, 1999). Studies on monoterpene bioconversion frequently use bacteria from the genus Pseudomonas as biocatalysts since they adapt easily to different substracts and extreme conditions thanks to their high metabolic versatility and complex enzyme system. Therefore, the present work aims to use monoterpenes, such as limonene, a-pinene and citronellol, as substrate for the biotransformation processes by microorganisms from the genus Pseudomonas. The Chapter 1 presents a current review on the biotechnological potencial of microorganisms from the genus Pseudomonas. Chapter 2 refers to the results obtained from laboratory experiments which include the isolation of bacteria in selective and differential medium for Pseudomonas and their application in monoterpene biotransformation processes ((R-(+)-limonene, a-pinene, citronellol). Among 32 selected strains for investigation of biotransformation capability, 9 bacteria were able to biotransform one of the substrates on value-added flavor compounds. R-(+)-limonene was converted to cis- and trans-carveol and carvone by 7 strains, at concentrations of 412 mg/L, 275 mg/L e 209 mg/L, respectively. a-Pinene, in turn, was used as sole carbon and energy source for 7 strains, and yielded especially cis-verbenol and verbenone at low concentrations by 5 strains. Other compounds such as limonene, myrtenol and ß-pinene have also been identified as major products from the conversion of a-pinene by 2 strains. On the other hand, only 3 strains were able to metabolize citronelol on other derivative compounds, such as rose oxides and isopulegol. The effects of different pHof biotransformation medium and induction phase on cell growth and product concentration were evaluated in order to optimize the biotransformation process. The production of rose oxides by strain L1B2P was studied and detailed separately in the third chapter, set as a published article at Chemical Engineering Transactions. In citotoxicity tests, the strain showed impressive terpene resistance, revealing great potential for further process optimization. Most notable compound derivatives were highlighted in the present work due to their high sensory quality and added value. Besides highly industrially relevant sensory notes, carvone and carveol are also recognized by pursuing some biological functions. The verbenone and verbenol stand by the high cost and wide application in food, agriculture, and medicine fields. Rose oxide, in turn, is one of the most important components in creating floral compositions in perfumery, besides its low threshold. Thus, biotransformation of inexpensive precursors into high value added compounds can be considered an economical and advantageous strategy
Mestrado
Ciência de Alimentos
Mestra em Ciência de Alimentos

Orientador: Gláucia Maria Pastore
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A crescente demanda por processos que tenham impacto ambiental reduzido, rendimentos consideráveis e ainda custos minimizados recorrem à biotecnologia como uma ciência que pode tornar essa necessidade uma realidade. Processos biotecnológicos para a produção de compostos de interesse industrial como biocorantes, açúcares funcionais, biosurfactantes e biodiesel são uma tendência no meio científico e ganham destaque pela inovação, resultados e aplicações obtidos. O apelo comercial dos produtos biotecnológicos é outro fator que influencia as pesquisas haja vista que esses produtos são considerados naturais e tem-se comprovado que muitos deles apresentam atividades biológicas relevantes. O aroma é um importante atributo dos alimentos e pode ser produzido via biotecnológica a partir de substratos de baixo custo. A utilização de resíduos agro-industriais com alto teor nesses substratos, como por exemplo, resíduos ricos em monoterpenos são de grande interesse na biotecnologia de produção de aromas. Além disso, a utilização de micro-organismos isolados de diferentes fontes como biocatalisadores na produção desses compostos é outra perspectiva, muito explorada e ainda desafiadora. A biotransformação de monoterpenos para a produção de compostos de aroma é vantajosa quando comparada com a síntese química clássica. Contudo, alguns problemas ainda devem ser minimizados, como sensibilidade dos biocatalisadores à toxicidade dos substratos ou condições operacionais e baixos rendimentos. Assim, a seleção de novos biocatalisadores, como micro-organismos endofíticos, o uso de ferramentas de engenharia genética e a utilização de métodos estatísticos de otimização de processos são promissores e caracterizam-se como a nova vertente na área de bioaromas, contornando as desvantagens até então limitantes dos processos de biotransformação. Eles permitem em alguns casos, o aumento de escala e dessa forma a real aplicação da bioconversão microbiana na indústria. Assim, trabalhos que visem selecionar novos biocatalisadores é ainda uma necessidade e quando aliados a otimização de processos podem fornecer caminhos para que os rendimentos da bioconversão de monoterpenos sejam aumentados. Nesse contexto, o presente trabalho visou selecionar micro-organismos com potencial para a bioconversão de monoterpenos de baixo custo e presentes em grandes quantidades em resíduos agro-industriais, como 'alfa'-pineno e R-(+)-limoneno, para a produção de compostos de aroma valorizados comercialmente, S-(-)-verbenona e 'alfa'-terpineol. O capítulo 1 traz uma revisão sobre os compostos monoterpênicos, abordando suas características químico-estruturais, biossíntese e recentes aplicações terapêuticas, além do seu uso como substratos na biotecnologia de produção de compostos de aroma. O capítulo 2 é dividido em duas partes: a parte I mostra os resultados de um screening realizado com 81 linhagens de fungos isolados de diferentes fontes, entre eles, fungos endofíticos, utilizados no estudo do potencial de biotransformação de R-(+)-limoneno e 'alfa'-pineno. A parte II descreve a otimização do processo de produção de S-(-)-verbenona biotecnológica a partir de ?-pineno por uma linhagem de fungo endofítico, ainda não identificado, isolado da romã (Punica granatum). O uso de um Delineamento Composto Central Rotacional (DCCR) de 4 variáveis proporcionou o estabelecimento de condições mais favoráveis ao processo, elevando a concentração de verbenona que era de 66 mg.L-1 para aproximadamente 108 mg.L-1 em um dos ensaios do DCCR e em torno de 98 mg.L-1 no ensaio de validação. O capítulo 3 aborda a otimização do processo biotecnológico de produção de ?-terpineol a partir de R-(+)-limoneno pela linhagem Bacillus tequilensis isolada de resíduos de processamento de frutas cítricas. Após a otimização e validação do processo nas condições consideradas ótimas através da análise de superfícies de resposta, a concentração de ?-terpineol passou de 25 mg.L-1 para 85 mg.L-1. Portanto, os resultados obtidos no trabalho mostraram um ganho de rendimento nos processos de bioconversão de substratos monoterpênicos e que a otimização de processos é uma importante ferramenta para a melhoria das condições experimentais possibilitando trabalhos futuros que envolvam aumento de escala
Abstract: The growing demand for processes that reduce environmental impacts, have high yields and costs minimized uses biotechnology as a science that can make this need into a reality. Biotechnological processes for the production of industrial interesting compounds, such as biocolorant, functional sugars, biosurfactants and biodiesel, are a trend in the scientific field and are highlighted by innovation, application and results obtained. The commercial appeal of biotechnological products is another factor that stimulates researches since these products are considered natural and has been proven that many of them presents significant biological activities. The flavor is an important attribute of food and can be produced through biotechnological processes from low-cost-substrates. The use of agro-industrial wastes with high content of these substrates, such as residues rich in monoterpenes, is of great interest in biotechnology. Furthermore, the use of micro-organisms isolated from several sources as biocatalysts in the production of flavor compounds is another well-explored and challenging perspective. The biotransformation of terpenes for aroma compounds production is advantageous when compared with chemical synthesis. However, there are still some problems that must be minimized like biocatalysts sensitivity, toxicity of substrates and low yields. Thus, the selection of new biocatalysts, such as endophytic micro-organisms, use of genetic engineering and statistical methods for optimization of processes are promising and characterize a new perspective in bioaromas field. Thereby, works that search for new biocatalysts are necessary and associated with optimization processes can improve the monoterpenes bioconvertion. In this context, the objective of this work was the screening and selection of micro-organisms that are able to realize the biotransformation of low cost monoterpenes, 'alfa'-pinene and limonene, into high-value aroma compounds, such as verbenone and 'alfa-terpineol. The chapter 1 presents a current review on monoterpenes, showing their chemical characteristics, biosynthesis and recent therapeutic applications, besides their potential use as substrates for bioflavor production. The chapter 2 is divides in two parts: the first one shows the results of the screening of 81 strains of fungi isolated from several sources, including endophytic strains, for biotransformation of limonene and ?-pinene. The second part describes the optimization process for biotechnological verbenone production from "alfa'-pinene by a non-indentified fungal strain, isolated from pomegranate (Punica granatum). The use of a central composite rotatable design (CCRD) of 4 variables provided the optimal conditions for this process, increasing the verbenone concentration from 66 mg.L-1 to 108 mg.L-1 in one of the CCRD assays and 98 mg.L-1 after validation. The chapter 3 also shows an optimization study, in this case using limonene as substrate and the product obtained was ?-terpineol. The biocatalyst used was a bacterial strain, identified as Bacillus tequilensis, isolated from citrus fruit processing waste. After the optimization and validation in the best conditions obtained through the surface response analysis, the concentration of ?-terpineol reached was 85 mg.L-1. Therefore, the results obtained in this work show an improve of the yields in the biotransformation of both monoterpenic substrates and that optimization of processes is an important tool for the progress of future works involving scale-up studies
Mestrado
Ciência de Alimentos
Mestra em Ciência de Alimentos

Marlborough is the largest wine region in New Zealand and Sauvignon blanc cultivars represented about 60 % of the vineyard area in 2009. The main compounds responsible for the most intense aromas in Sauvignon blanc wines had been assumed to be methoxypyrazines, responsible for the herbaceous, capsicum and asparagus aromas, and varietal thiols, responsible for fruity aromas such as grapefruit, citrus or passion fruit. However, the aromatic potential of Sauvignon blanc wines is too complex to be limited to just these two families of compounds. This study has shown that further groups of aroma compounds (esters, terpenes, C6- and higher alcohols, fatty acids, C13- norisoprenoids, cinnamates and anthranilates) could also be involved in the characteristic aroma of these wines. Winemakers divide the Marlborough Sauvignon blanc growing area into 7 sub-regions, and ascribe different aroma profiles to wines coming from these regions. An extensive study of the compounds that impact on Sauvignon blanc aroma has been undertaken with over fifty important impact aroma compounds quantified in 54 wines from different sub-regions within Marlborough. Some important variations with regard to the Marlborough sub-regions have been revealed, and the extent to which these differences in chemical composition can impact on wine sensory differences has been assessed. A further aim of this research was to assess the optimum small scale winemaking protocol in order to obtain aroma composition results in research wines comparable to those found in commercial wines. Different conditions in the early stages of the winemaking process were studied: destemming and crushing of the grapes, differences between hand-picked and machine-harvested grapes, and the use of maceration enzymes. The results have shown that both destemming and crushing of the grapes are needed to obtain wines with an aroma composition similar to the commercial wines.

Orientador: Gláucia Maria Pastore
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A biotecnologia é uma área interdisciplinar que tem possibilitado inúmeros avanços no desenvolvimento de processos sustentáveis e na obtenção de novos produtos de interesse industrial. Entre estes exemplos estão os aromas naturais que, atualmente, são preferidos por muitos consumidores e já dominam o mercado em certas regiões como a Europa. Uma das opções biotecnológicas para a obtenção destes compostos ocorre pela biotransformação de terpenos, que são derivados de isoprenos, e substratos amplamente distribuídos na natureza. Estes compostos são classe relevante dentre os compostos de aroma e alguns podem ser obtidos a custos relativamente baixos. Limoneno e ?-pineno, por exemplo, estão disponíveis como subprodutos de indústrias de frutas cítricas e de papel, respectivamente. Estes monoterpenos são considerados substratos ideais para obtenção de derivados oxigenados, os terpenóides, muitos dos quais de grande interesse comercial. Nessa perspectiva, 51 fungos endofíticos das frutas caju (Anacardium occidentale L.), jenipapo (Genipa americana L.), sapoti (Manilkara zapota L.) e camu-camu (Myrciaria dubia), foram isolados durante este trabalho. As linhagens foram também avaliadas quanto ao potencial para biotransformação de limoneno ou ?-pineno como fontes únicas de carbono. A partir deste segundo substrato, destacaram-se dois fungos produtores de verbenona, um dos compostos chave do aroma de alecrim (Rosmarinus officinalis). A produção de verbenona por via biotecnológica mostrou um grande potencial para a continuidade dos estudos, tendo em vista que a concentração obtida foi de, aproximadamente, 200 mg/L após 288 horas de contato com o subtrato ?-pineno. Além disso, outro fungo apresentou produção de álcool perílico a partir da biotransformação de limoneno. Este produto é um terpenóide, cujos mecanismos de proteção contra o câncer já tem sido investigados e reconhecidos, aumentando o interesse na sua pesquisa e produção biotecnológica. No encerramento do trabalho, os três fungos com resultados positivos pra biotransformação de terpenos foram analisados em microscopia óptica para observação de suas morfologias e auxílio na identificação
Abstract: As an interdisciplinary field, biotechnology has allowed several advances for the development of sustainable processes and many achievements concerning production of new compounds with industrial interest. Natural aroma compounds have been frequently prioritized by costumers and dominate a considerable market share in some regions such as Europe, and one of biotechnological routes for obtaining these compounds occurs through the biotransformation of terpenes. These substrates are naturally available compounds derived from isoprene units, and represent a relevant class of aroma compounds, which can be often obtained at relatively low costs. Limonene and ?-pinene, for instance, are available as by-products in citrus and pine industries, respectively. These monoterpenes represent ideal precursors for biotechnological production, leading to their oxygenated derivatives denominated terpenoids, some of which commercially valuable. In this perspective, fifty one endophytic fungi from Brazilian fruits, such as cashew (Anacardium occidentale L.), genipap (Genipa americana L.), sapodilla (Manilkara zapota L.) and camu-camu (Myrciaria dubia) were screened during this work. Furthermore, these fungal strains were tested in the biotransformation of limonene or ?-pinene as sole carbon source. Amongst all fungi, two strains showed noteworthy capability to biotransform ?-pinene into verbenone, key compound of rosemary (Rosmarinus officinalis) aroma. The biotechnological production of verbenone proved to be an interesting strategy with a great future potential, considering that that the concentration of this product reached approximately 200 mg/L after 288 hours of contact with the substrate ?-pinene. Moreover, from the biotransformation of limonene, one fungi produced perillyl alcohol. This important terpenoid presents protection mechanisms against cancer already investigated and recognized. The practical steps achieved in this project also involved the observation of these strains with positive results through optical microscopy, in order to verify their morphology and general characteristics
Mestrado
Ciência de Alimentos
Mestra em Ciência de Alimentos

Recent demands for minimally-processed foods, has led to the exploration of plant-derived essential oil (EO) compounds as an alternative means of preservation. While some of these compounds are effective against foodborne pathogens, their strong aroma and "spicy" flavor are not compatible with the flavor of juice. The purpose of this research was to evaluate the antimicrobial activity of three EO compounds (thymol, eugenol, and trans-cinnamaldehyde) alone and in combination with three naturally-occurring apple aroma compounds (hexanal, trans-2-hexenal and 1-hexanol) in order to identify combinations that lower the concentrations needed to destroy foodborne pathogens in apple juice. The standard agar dilution method (SAD) and the Spiral Gradient Endpoint method (SGE) were compared for their abilities to determine minimum inhibitory concentrations (MIC) of the EO compounds. Both methods produced similar patterns of inhibition; however, the MICs produced by the SGE system were significantly higher than those produced by the SAD method of analysis (P<0.05). Since the results produced by the SAD method were more comparable with those published in literature, this method was selected for further testing. In general, the EO compounds were significantly more effective against the test pathogens (Listeria monocytogenes, Salmonella Typhimurium and Staphylococcus aurues) than were the apple aroma compounds (P<0.05). Cinnamaldehye exhibited the highest degree of activity, followed by thymol and eugenol. Eugenol was the only compound that acted synergistically with the apple aroma compounds. The most effective compounds (cinnamaldehyde, eugenol and trans-2-hexenal) were then used to inactivate L. monocytogens and S. Typhimurium in preservative-free apple juice. In most cases, treatment with 0.05% of each compound resulted in a 5 log CFU/ml reduction in bacterial numbers following one day of storage at 4°C or 25°C. Likewise, treatment with antimicrobial combinations (containing 0.025% of trans-2-hexenal in combination with 0.025% trans-cinnamaldehyde or eugenol) also resulted in a 5 log CFU/ml reduction in bacterial numbers, following one day of storage at 4°C or 25°C. Since these combinations contained half the effective concentration of the essential oil compounds, they may be used to preserve the microbial quality of apple juice, while reducing the likelihood of off flavors in the final juice product.
Ph. D.

Viognier (Vitis vinifera L.) grapes were grown in Northern Virginia for three seasons using three different training systems in a randomized complete block design consisting of Vertical Shoot Positioned (VSP), Smart Dyson (SD), and Geneva Double Curtain (GDC), and evaluated for the effects on grape and wine glycosides and volatile compounds. Fruit was harvested at the same Brix each season, and differences in berry weights were not observed. VSP-trained vines had the lowest crop load and lowest light levels in the fruit zone. Seventeen volatile compounds were analyzed using headspace solid-phase microextraction, GC-MS. Fruit showed differences in linalool, á-terpineol, â-damascenone, and n-hexanol concentrations among the training systems. Wines showed differences in both grape-derived and fermentation-derived volatiles. SD had the highest concentration for most of the free volatiles quantified in both the juice and wine. VSP had lower phenol-free wine glycosides all three seasons and lower phenol-free juice glycosides one season. Triangle difference sensory testing demonstrated differences between GDC and SD in wine aroma and flavor, and differences between VSP and SD in flavor, for two of three seasons.
Master of Science

Thesis (PhD (Chemistry and Polymer Science))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Honeybush tea, also known as “South Africa’s sweetest tea”, is a herbal tea made from the leaves and twigs of Cyclopia spp., indigenous to the fynbos biome in the Western and Eastern Cape provinces of South Africa. The pleasant sweet aroma and taste of fermented honeybush, its low tannin content and the absence of caffeine have led to widespread interest in the commercial cultivation and processing of honeybush tea since the mid-1990s. Although more than 20 species of honeybush grow in the wild, only a few species are commercially exploited for the manufacture of tea. Currently the more prominent species are C. intermedia, C. subternata, C. genistoides, and C. sessiliflora. The present research contributes to a comprehensive honeybush research programme being conducted at the Agricultural Research Council (ARC) Infruitec-Nietvoorbij in South Africa. The first phase of the present study, using C. genistoides as representative species, was aimed at developing the necessary methodology for the analysis of extremely low concentrations of honeybush volatiles. A high-capacity headspace sample enrichment probe was applied successfully in conjunction with gas chromatography-mass spectrometry (GC-MS) to analyse the volatile organic compounds present in dry or infused unfermented and fermented honeybush. A total number of 255 volatile compounds were identified in unfermented and fermented honeybush, the majority of which are terpenoids (138; 54%) comprising mostly terpenes, terpene ketones, terpene alcohols and terpene ethers. Of the other compound classes, the aldehydes are the largest group, followed by esters, hydrocarbons and ketones. The stereochemistry of the identified compounds was determined whenever possible. This is the most comprehensive chemical characterisation of the volatile compounds in a South African herbal plant reported to date. A comparative study of green and fermented honeybush showed that the same compounds are, to a large extent, present in both, albeit in different relative concentrations. Not all of the identified honeybush volatiles are necessarily odour-active compounds contributing to the overall typical honeybush aroma. An important aspect of this research was thus the identification of the 46 odour-active compounds in fermented honeybush by means of gas chromatography-olfactometry (GC-O), using detection frequency and aroma extract dilution analysis methods. Fifteen of these compounds, mainly terpenoids, were singled out as the most intense individual contributors to the honeybush aroma based on consideration of all the relevant GC-O data. The odours of certain compounds, i.e. (6E,8Z)-megastigma-4,6,8-trien-3-one, (6E,8E)-megastigma- 4,6,8-trien-3-one, (7E)-megastigma-5,7,9-trien-4-one, 10-epi- -eudesmol, epi- -muurolol and epi- - cadinol, were perceived by GC-O assessors as typically honeybush-like. The quantitative GC-MS data of seven different Cyclopia samples (including four different species and variants thereof) were compared with respect to all the volatile components and particularly with respect to the odour-active compounds. Interesting variations were found in the concentrations of certain odour-active compounds in the various samples. The quantitative data obtained for the odour-active honeybush volatiles and data obtained from the sensory analysis of eight Cyclopia samples (including four different species and variants thereof) were subjected to statistical analysis and interesting associations between compounds with certain sensory aroma attributes were established. The present study has made a major contribution to the scientific knowledge regarding one of South Africa’s most popular indigenous herbal teas.
AFRIKAANSE OPSOMMING: Heuningbostee, wat ook bekend staan as “Suid-Afrika se soetste tee”, word gemaak van die blare en takkies van Cyclopia spp. wat inheems is en voorkom in die fynbosbioom van die Wes- en Oos-Kaapprovinsies van Suid-Afrika. Die aangename soet smaak en aroma van gefermenteerde heuningbos, die lae tannnien-inhoud en die feit dat die tee kafeïenvry is, het gelei tot belangstelling in die kommersiële verbouing en prosessering van heuningbostee gedurende die 1990s. Meer as 20 heuningbosspesies kom in die natuur voor, maar slegs ‘n paar spesies word kommersieel verbou vir die vervaardiging van heuningbostee waarvan C. intermedia, C. subternata, C. genistoides en C. sessiliflora tans die belangrikste spesies is. Die navorsing maak deel uit van ‘n omvattende heuningbos navorsingsprogram wat onder leiding staan van die Landbounavorsingsraad Infruitec- Nietvoorbij in Suid-Afrika. In die eerste fase van die huidige studie is die nodige analitiese metodologie ontwikkel vir die monsterneming en analise van die vlugtige organiese verbindings wat in uiters lae konsentrasies in heuningbos voorkom, deur van ‘n verteenwoordigende spesie, C. genistoides, gebruik te maak. ‘n Sogenaamde “sample enrichment probe” (SEP) is ontwikkel en suksesvol in kombinasie met gaschromatografie-massaspektrometrie (GC-MS) aangewend vir die analise van die vlugtige verbindings aanwesig in die bodamp van sowel droë plantmateriaal as infusies van ongefermenteerde en gefermenteerde heuningbos. ‘n Totaal van 255 vlugtige verbindings is geïdentifiseer, waarvan die meeste hoofsaaklik terpenoïede is (138, 54%) en terpene, terpeenketone, terpeenalkohole en terpeeneters insluit. Die ander verbindingsgroepe, waarvan die aldehiede die grootste groep is, sluit in esters, koolwaterstowwe en ketone. Indien haalbaar, is die stereochemie van die geïdentifiseerde verbindings ook bepaal. Hierdie studie is die mees omvattende chemiese karakterisering van die vlugtige verbindings in ‘n Suid-Afrikaanse kruieplant wat tot dusver onderneem is. ‘n Vergelykende studie het getoon dat ongefermenteerde en gefermenteerde heuningbos tot ‘n groot mate dieselfde verbindings, hoewel in verskillende relatiewe konsentrasies, bevat. Nie al die geïdentifiseerde vlugtige verbindings in heuningbos is noodwendig aroma-aktiewe verbindings wat ‘n bydrae tot die algehele tipiese heuningbosaroma lewer nie en daarom was die identifisering van die 46 aroma-aktiewe verbindings in geferementeerde heuningbos deur gebruik te maak van gaschromatografie-olfaktometrie (GC-O) deur middel van deteksiefrekwensie en aroma ekstrak verdunningsanalise, ‘n belangrike aspek van die navorsing. Na oorweging van al die tersaaklike GC-O data is 15 van hierdie verbindings, hoofsaaklik terpenoïede, uitgesonder as die verbindings wat die belangrikste bydrae tot die heuningbosaroma lewer. Die reuke van sekere van die verbindings, nl. (6E,8Z)-megastigma-4,6,8-triën-3-oon, (6E,8E)-megastigma-4,6,8-triën-3-oon, (7E)-megastigma-5,7,9-triën-4-oon, 10-epi- -eudesmol, epi- -muurolol, en epi- -cadinol, is deur sommige van die GC-O paneellede as tipies heuningbosagtig beskryf. Die kwantitatiewe GC-MS data van sewe verskillende Cyclopia monsters (insluitende vier verskillende spesies en variante daarvan) is vergelyk met betrekking tot al die vlugtige verbindings, asook veral met betrekking tot die aroma-aktiewe verbindings. Interessante variasies in die konsentrasies van sekere aroma-aktiewe verbindings is in die verskillende monsters waargeneem. Die kwantitatiewe data van die aroma-aktiewe heuningbosverbindings en data verkry uit die sensoriese analise van agt Cyclopia monsters (insluitende vier verskillende spesies en variante daarvan), is onderwerp aan statistiese analises waaruit interessante assosiasies tussen verbindings met sekere sensoriese aroma-eienskappe waargeneem is. Hierdie studie lewer ‘n groot bydrae tot die wetenskaplike kennis aangaande een van Suid- Afrika se mees populêre inheemse kruietees.

Thesis (MScAgric)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Ferdinand de Lesseps grapes have a distinctive fruity varietal character and were often used judiciously by wine-makers to enhance the fruity bouquet of some white table wines. Ferdinand de Lesseps grape juice was investigated to identify the main contributing compounds responsible for its unique and intense aroma. The juice was recovered from grapes under anaerobic conditions and extracted using Freon 11. The concentrated extract was analysed using a combination of capillary gas chromatography-mass spectrometry and gas chromatography-sniffing techniques. Thirty-two compounds were reported. With the aid of GC-sniffing, it was concluded that the hybrid note of the Ferdinand de Lesseps grape was most likely attributed to the presence of 2,5-dimethyl-3(2H)-furanone and 0- aminoacetophenone. Esters, which featured prominently in the juice, consisted mainly of ethyl butanoate, ethyl- and methyl- 3-hydroxy butanoate, and to a lesser extent, ethyl 3- hydroxy hexanoate and ethyl-3-hydroxy propanoate. These esters are most likely responsible for the sweetish pineapple aroma. According to EEC regulations, the use of non- Vinifera grapes in the production of commercial wines is prohibited. The presence of 2,5-dimethyl- 3(2H)-furanone in wine could therefore be interpreted as a labrusca indicator should it be suspected that Ferdinand de Lesseps grapes were used in the blend. The quality of some South African Kerner table wines often is rated inferior owing to an unwanted odour described as "Elastoplast" or "medicinal". This odour is encountered occasionally in wines from other cultivars such as Gewurztraminer, Weisser Riesling, Muscat de Frontignan and Chenin blanc. The identification of the compounds responsible for the offodour was investigated, as well as possible relationships between its occurrence and viniviticultural practices. The compound responsible for the "medicinal" off- odour was identified as p-vinyl guaiacol. It is formed via decarboxylation of ferulic acid by yeast during alcoholic fermentation. lts concentration is directly related to the concentration of ferulic acid and the yeast strain. Other factors affecting p-vinyl guaiacol formation in Kerner wines are region, microclimate, exposure of grapes to sunlight and grape maturity. Wines made from grapes harvested from the warmer climatic regions and exposed to direct sunlight and increased maturity contained higher levels of p-vinyl guaiacol. Viticultural practices that can be applied to limit the formation of the p-vinyl guaiacol precursor in grapes are the use of a canopy manipulation to protect the grapes from direct sunlight and an earlier harvesting date. Various winemaking techniques can be used to lower p-vinyl guaiacol levels in wine. Careful consideration must be given to the choice of yeast, as different yeast strains differ considerably with their ability to form p-vinyl guaiacol. Fining oxidised grape juice with phenol adsorbing agents such as activated charcoal, polyvinylpolypirrollidone, casein, gelatin (particularly in combination with bentonite and "kieselsol") led to decreases in the p-vinyl guaiacol levels. Although fining with activated charcoal was the most effective must treatment for reducing the "medicinal" aroma of Kerner wines, it stripped the wines of colour and character. Excessive oxidation of must followed by fining involves extra costs and time for the winemaker and would not be used in making the reductive style of wines.
AFRIKAANSE OPSOMMING: Ferdinand de Lesseps druiwe en wyne vertoon unieke vrugtige varieteitskarakter en is dikwels deur wynmakers in versnitte gebruik om die geur van sekere wit tafelwyne te verbeter. In hierdie studie is Ferdinand de Lesseps druiwe ondersoek om die belangrikste komponente wat verantwoordelik is vir die prominente, unieke aroma daarvan te identifiseer. Die sap is onder anaerobe toestande herwin en met Freon 11 ekstraheer. Die gekonsentreerde ekstrakte is met behulp van 'n kombinasie van kapillêre gaschromatografie-massaspektrometrie en gaschromatografie-snuif tegnieke analiseer. Twee-en-dertig komponente is gerapporteer. Met die behulp van GC-snuiftegnieke is bevestig dat die hibried karakter in Ferdinand de Lesseps druiwe hoofsaaklik aan die teenwoordigheid van o-amino-asetofenoon en 2,5-dimetiel-3(2H)- furanoon toegeskyf kan word. Die vernaamste esters in die sap was etielbutanoaat, etiel- en metiel-3-hidroksibutanoaat en in 'n mindere mate etiel-3-hidroksiheksanoaat en etiel-3- hidroksipropanoaat. Dié esters is waarskynlik verantwoordelik vir die soet pynappel aroma. Volgens EEG regulasies word die gebruik van nie- Vinifera druiwe vir die produksie van komersiéle wyne verbied. Die teenwoordigheid van 2,5-dimetiel-3(2H)-furanoon in wyn kan dus interpreteer word as 'n labrusca indikator in gevalle waar gebruik van Ferdinand de Lesseps druiwe vermoed word. Die kwaliteit van sommige Suid-Afrikaanse Kerner wyne word dikwels verlaag vanweë die teenwoordigheid van 'n geur wat beskryf word as "Elastoplast" of "medisinaal". Dié reuk word soms waargeneem in wyne van ander kutivars soos Gewurztraminer, Weisser Riesling, Muskaat de Frontignan en Chenin blanc. Die identiteit van die komponent wat vir dié ongewenste reuk verantwoordelik is, asook moontlike verwantskappe met wingerd- en wynkundige praktyke is ondersoek. Die komponent wat vir die medisinale karakter verantwoordelik is, is geïdentifiseer as para-vinielguajakol. Dit word deur dekarboksilasie van feruliensuur deur gisselle tydens alkoholiese gisting gevorm. Die konsentrasie van paravinielguajacol in wyn is direk verwant aan die konsentrasie feruliensuur en die gisras. Ander faktore wat para-vinielguajacol-vorming in Kerner wyn affekteer is streek, mikroklimaat, blootstelling van druiwe aan direkte sonlig en die rypheidsgraad van die druiwe. Wyne wat berei is van ryper druiwe uit warmer streke en wat direk aan sonlig blootgestel was, het hoër konsentrasies para-vinielguajacol bevat. Lowerbestuurspraktyke met behulp waarvan druiwe teen direkte sonligblootstelling beskerm word, is van die vernaamste wingerdkundige praktyke wat aangewend kan word om die vorming van die voorlopers van paravinielguajacol in druiwe te verlaag. Verskeie wynbereidingstegnieke kan gebruik word om die para-vinielguajacol vlakke in wyn te verlaag. Gisraskeuse is van besondere belang omdat gisrasse grootliks verskil in hul vermoë om para-vinielguajacol te vorm. Breibehandelings van geoksideerde sap met middels soos geaktiveerde koolstof, PVPP, kasseïen, gelatien (veral in kombinasie met bentoniet en "kieselsol") het die vlakke van para-vinielguajacol in wyne verlaag. Hoewel geaktiveerde koolstof die doeltreffendste breimiddel vir die verlaging van para-vinielguajacol was, het dit te veel kleur en geur uit die wyn verwyder. Oormatige oksidasie van mos gevolg deur breibehandelings sal meer geld en tyd verg en word nie aanbeveel vir wyne wat in 'n reduktiewe styl berei word nie.

The aroma compounds suitable for food flavouring are mainly of plant origin. It is necessary to take into account the possible allergic effects of some of them. The theoretic part of this thesis is focused on aroma compounds and their biological effects. It also describes methods useful for their isolation and analysis with a nearer intent on the methods used in this thesis - solid phase microextraction and gas chromatography. The aim of the experimental part was to establish, optimise and validate the method for determination of allergic aroma compounds chosen and subsequently to enable its using in wide spectrum of food and other products.

The aim of the diploma thesis was the aroma active compounds assessment and sensory profile determination of various types of sea buckthorn (Hippopha rhamnoides L.). The theoretical part provides the detailed description of this plant, comprising the occurence, taxonomy, chemical composition and use. Experimental part summarizes the data obtained: identification and quantification of aroma compounds using the SPME – GC –FID and GC-MS method and evaluation of complete sensory profile. Various cultivars of sea buckthorn harvested in 2009-2012 were analysed. In total 23 various types of sea buckthorn were measured, 23 alcohols, 10 aldehydes, 11 esters, 14 ketones and 5 acids were found.

Orientador: Helena Teixeira Godoy
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: As pimentas do gênero Capsicum apresentam ampla aplicação em função dos atributos sensoriais de cor, pungência e aroma, decorrentes da presença de carotenoides, capsaicinoides e voláteis. No entanto, variações no conteúdo desses metabólitos secundários dependem de fatores como sazonalidade, ritmo circadiano, desenvolvimento da planta, características edafo-climáticas, ataque de patógenos. Neste estudo foi realizada a caracterização da composição de capsaicinoides e fração volátil de frutos de Capsicum chinense. A caracterização foi realizada através do uso de métodos analíticos cromatográficos (UPLC, UHPLC-MS e GC-MS). Com a execução desta pesquisa, foi possível caracterizar os frutos de C. chinense quanto à composição de metabólitos relacionados aos seus principais parâmetros de qualidade, fornecendo subsídios científicos para o conhecimento e melhoria da qualidade em termos de produção, aplicação industrial, compreensão de rotas metabólicas e melhoramento genético, além de gerar informações a respeito de substâncias potencialmente benéficas, devido a suas ações no organismo humano. Inicialmente foram desenvolvidos e validados os métodos, usando ferramentas estatísticas. De acordo com a etapa de otimização para a extração dos capsaicinoides, 100% de metanol combinado com o uso do banho de ultrassom por 10 minutos foram empregados. O método analítico desenvolvido através do uso de cromatografia em fase líquida de ultra eficiência permitiu a separação de 8 capsaicinoides em 4 minutos de análise cromatográfica e mostrou-se eficiente de acordo com a validação realizada. 9 acessos de pimentas C. chinense foram analisadas. Os teores de capsaicina e dihidrocapsaicina obtidos estavam nas faixas de 156 - 1442 ?g.g?1 e 26 - 478 ?g.g?1 (peso de fruto fresco), respectivamente. A caracterização do perfil de voláteis em pimentas 'Habanero', através do uso de HS-SPME (Headspace Solid-Phase Microextraction), foi realizada após etapa de otimização. Delineamento composto central e ferramenta de desejabilidade foram usadas a fim de avaliar, simultaneamente, duas respostas: soma total da área dos picos e número de compostos extraídos. O resultado maximizado para as duas respostas usando a fibra tripla (DVB/Car/PDMS) indicou o uso das condições de temperatura de extração de 40 °C e 30 min de extração. 82 compostos foram tentativamente identificados na fração volátil da pimenta 'Habanero' e os ésteres foram os compostos mais abundantes. O 2,3-dimetilciclohexanol e longifoleno foram identificados pela primeira vez em pimentas 'Habanero'. Além disso, pimentas 'Habanero' submetidas à diferentes condições de disponibilidade de água durante seu cultivo foram avaliadas quanto a composição de capsaicinoides e voláteis. Maiores quantidades de capsaicina e dihidrocapsaicina foram encontradas na pimentas maduras, alcançando valores entre as faixas de 2,85 - 3,33 mg.g-1 para capsaicina e 1,06 - 1,71 mg.g-1 para dihidrocapsaicina. Os teores de compostos voláteis totais foram maiores nas pimentas verdes (195,47 - 298,94 mg.kg-1), independente do tratamento de estresse hídrico que receberam. Os resultados obtidos com o PCA realizado possibilitaram, inicialmente, separar as pimentas 'Habanero' em função do grau de maturação. Enquanto, PCA aplicado, separadamente, nas pimentas verdes e maduras demonstrou uma separação evidente em função da data de colheita. Isso se deve ao fato de que influências do grau de maturação e época da colheita se sobressaíram no estudo. As pimentas foram agrupadas de acordo com o tratamento de irrigação recebido, mas para uma melhor compreensão dos efeitos dos tratamentos de estresse hídrico sobre a composição das amostras, seria necessário uma estratégia de estudo com monitoramento mais frequente dos frutos formados além de outras investigações sensoriais, como o uso de olfatometria.
Abstract: The Capsicum peppers are largely used due their sensorial properties as color, pungency and aroma. These characteristics occur by the presence of determinate classes of compounds including carotenoids, capsaicinoids and volatiles. These secondary metabolism derivatives compounds are influenced by two factors: genetics and plant-environment interactions and are susceptible to water availability conditions. This study had a proposal of the characterization of the capsaicinoids and volatile composition of the Capsicum chinense pepper fruits. The analysis were carried out by using of chromatographic methods (UPLC, UHPLC-MS and GC-MS). The results obtained and the characterization of the C. chinense fruits regarding the mainly compounds which influenced in the quality of the peppers provide the scientific subsidies for improvement of general quality, agricultural practices, industrial applications and breeding programs and also, the knowledge about substances potentially beneficial to the human organism. Initially, was developed and validated a fast, efficient and reproducible method to analyze capsaicinoids in Brazilian Capsicum chinense fruits by means the use of an optimization strategy to extracted the capsaicinoids. The extracts were obtained following the condition 100% of methanol and 10 min on ultrasound assisted extraction. The analytical method developed in an ultra high performance liquid chromatographic system coupled to a mass spectrometer permits the separation of 8 capsaicinoids in 4 min of time analysis expending only 2 mL of solvent as mobile phase. The method achieved was fully validated and show the effectiveness and satisfactory performance to answer the analytical needs of this research area. Different accessions of C. chinense fruits were analyzed, the contents of capsaicin and dihydrocapsaicin were in the range of 156 - 1442 ?g.g?1 and 26 - 478 ?g.g?1 of fresh fruit, respectively. Characterization of aroma profile of Capsicum chinense peppers 'Habanero' type, using Headspace Solid-Phase Microextraction (HS-SPME), was performed after an optimization step by GC-MS. Central composite design and Derringer's desirability function strategies were used for this optimization, in order to evaluate simultaneously the 'total sum peak areas' and 'number of extracted compounds' responses. The maximized results for both responses were obtained using PDMS/Car/DVB fiber, temperature of 40 °C and extraction time of 30 min. Eighty-two compounds were tentatively identified in the volatile fraction of the 'Habanero' pepper and the most abundant were hexyl isovalerate, cis-hexenyl isovalerate, hexyl 3-methylbutanoate, 3,3-dimethylcyclohexanol, longifolene, and 2-methyl-1-tetradecene. The compound 2,3-dimethylcyclohexanol and longifolene were reported for the first time in 'Habanero' pepper. In addition, Brazilian 'Habanero' peppers grown under different condition of water availability were evaluated regarding their capsaicinoids and volatiles composition. The higher values of capsaicin and dihydrocapsaicin were found in the mature peppers, achieving ranges from 2.85 - 3.33 mg.g-1 for capsaicin and 1.06 - 1.71 mg.g-1 for dihydrocapsaicin. Total volatile compounds presented higher values for the peppers in green stage of maturity with ranges from 195.47 - 298.94 mg.kg-1, whereas in the mature samples the range was from 68.74 - 118.50 mg.kg-1. According the results obtained with the PCA performed was possible separe the 'Habanero' peppers in function of the degree of maturity. While, PCA apllied, separately, for green and mature peppers showed a clear separation in function of the harvest date. This fact can be justified by the large influence of maturity degree and harvest date on the peppers composition. The peppers were clustered in function of the water availability treatments received, but to better understand the effects of water availability on the peppers composition, another strategy of evaluation, with more frequent monitoring would be interesting, as well as the inclusion of sensorial studies, as the olfactometry technique.
Doutorado
Ciência de Alimentos
Doutora em Ciência de Alimentos

Orientador: Glaucia Maria Pastore
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Este trabalho teve por objetivos o estudo da biotransformação de terpenos e avaliação de suas propriedades biológicas. A biotransformação de limoneno foi realizada por uma linhagem de Fusarium oxysporum cultivada em manipueira e transferida para meio mineral, sendo o óleo essencial de laranja, um resíduo da indústria do suco de laranja, a fonte do limoneno. Rendimentos da ordem de 450 mg/L de a-terpineol, o principal produto obtido, foram alcançados. Da mesma forma, a biotransformação do citronelol foi conduzida por uma linhagem de Penicillium sp. cultivada em manipueira e transferida para meio mineral. O principal produto obtido foi o cis-óxido de rosa em concentrações da ordem de 70 mg/L. A seleção de microrganismos biotransformadores de limoneno (cuja fonte foi óleo essencial de laranja) a- e b-pinenos (cuja fonte foi terebentina, resíduo industrial da indústria de papel) foi realizada por microextração em fase sólida (MEFS). A técnica mostrou-se eficaz para a recuperação de voláteis presentes no 'heapspace¿ de culturas esporuladas de superfície para a biotransformação. Os microrganismos selecionados por MEFS foram submetidos à biotransformação em cultura líquida e produção da ordem de 50 mg/L de verbenol por Mucor sp. 2276 e 70 mg/L de verbenona resultaram da biotransformação de a- e b-pinenos. Aspergillus sp. 2357 e Penicillium sp. 2360 produziram aproximadamente 90 e 10 mg/L de a-terpineol e álcool perílico respectivamente. A biotransformação de a-farneseno por linhagens de Aspergillus niger gerou compostos nunca relatados anteriormente na literatura. Quatro produtos principais foram obtidos. Apenas um composto pôde ser identificado por meio de CG-EM como 6-OH-farneseno. Análises de CG-olfatometria descreveram 6-OH-farneseno como aroma cítrico impactante. Estudos 'in vitro¿ e 'in vivo¿ com extrato da biotransformação de limoneno por Fusarium oxysporum e com padrões de monoterpenos revelaram o potencial desses compostos em atuarem como antioxidantes, gerando uma possibilidade para esses compostos serem utilizados industrialmente como aromas funcionais
Abstract: The biotransformation of terpenes and their functional properties were investigated in this study. The biotransformation of limonene was done by a Fusarium oxysporum strain grown in cassava waste water and transferred into a mineral medium for biotransformation. The limonene source was an orange essential oil from a orange juice industry. The main biotransformation product was a-terpineol, reaching around 450 mg/L. Similarly, biotransformation of citronellol was conducted by a Penicillium sp. strain grown also in cassava waste water and transferred into a mineral medium. The main product was cis-rose oxide, reaching concentrations higher than 70 mg/L. The screening of microrganisms for biotransformation of limonene (from orange essential oil) and a-, b-pinenes (from turpentine oil, residue from pulp industry) was done by solid phase microextraction (SPME). The technique was effective for the recovery of the volatiles from the headspace of sporulated surface biotransformation cultures. Liquid culture biotransformation experiments performed with the SPME screened strains resulted in the production of 50 mg/L of verbenol by Mucor sp. 2276 and 70 mg/L of verbenone form a-, b-pinenes and 90 mg/L of a-terpineol by Aspergillus sp. 2357 and 10 mg/L of perillyl alcohol by Penicillium sp. 2360. The biotransformation of a-farnesene by Aspergillus niger strains resulted in compounds never described in the literature before. Four main new compounds were obtained. Only one of them could be identified by GC-MS as 6-OH-farnesene. CG-O experiments revealed the impactant citrus aroma of 6-OH-farnesene. 'In vitro¿ and 'in vivo¿ experiments with the limonene biotransformation extract by Fusarium oxysporum and with the monoterpene standards present in the extract revealed the antioxidant potential of these compounds, which open a new perspective for the utilization of these compounds as functional aroma compounds
Doutorado
Doutor em Engenharia de Alimentos

Mestrado em Viticultura e Enologia - Instituto Superior de Agronomia
The present work is intended investigate the incidence of defect reduction in the several types of wine and the effect of treatments made with copper metal in their composition. For this purpose, was made a retrospective analysis of incidence of the defect between 2004 and 2010, in the various types of wine produced in the winery under study. The wines seem more susceptible to be the red, while the rosé, whose production has increased significantly in recent years, also showed high incidences Merely on an empirical basis, the defect appears be associated with harvest earliest and longer fermentation. As a result of the addition of copper sulphate, in order to resolving the problem of reduced wines, the only metal whose concentration was significantly increased copper. However, this increase did not change the general balance of the remaining metals in wine. Thus, for the multielement analysis we observed that treatment with copper did not affect the composition of metals in order to prevent a breakdown of samples according to the origin.

Heritage apple cultivars for cider-making are often distinguished by a high concentration of tannins (phenolic compounds), and/or acid. The phenolic content of some cider apples far exceeds that of white wine, however most cider fermentation practices are directly taken from white winemaking, not accounting for effects of high concentrations of phenolic compounds on yeast fermentation. The objective of this study was to determine the impact of ferulic acid, p-coumaric acid, and chlorogenic acid—at concentrations reported in apples—and their interactions with yeast assimilable nitrogen (YAN) on fermentation kinetics and cider aroma. Our hypothesis was that the phenolic compounds present in high-tannin cider apples would negatively impact fermentation kinetics, but not alter the aroma, and that added YAN would reduce these effects. Ferulic acid negatively affected fermentation performance (p < 0.05), but p-coumaric acid and chlorogenic acid did not. p-Coumaric acid led to the greatest changes in cider aroma. Differences were also detected for different concentrations of ferulic acid. Chlorogenic acid did not affect aroma. Yeast strain influenced fermentation performance and cider aroma. Finally, addition of exogenous YAN improved fermentation performance for the low concentration ferulic acid condition, but not for the high concentration. Adding YAN also changed cider aroma in the presence of p-coumaric acid.
Master of Science in Life Sciences

Mestrado Vinifera Euromaster - Instituto Superior de Agronomia - UL / Universitá degli Studi di Torino
In recent years, New Zealand in terms of production has expressed a certain respect and attention towards Sauvignon Blanc for its organoleptic chemical characteristics. In this project seven different amino acids were used, two blend of amino acids and the DAP (di-ammonium phosphate) used in wineries, with the aim of investigating the effect of different levels of nitrogenous source of musts from the two different vineyards ( Omaka Vineyard and Gifford's Creek Vineyard), on the progress of fermentation and the impact on the aromatic component, with the use of the "New Zealand Grape and Wine Research Program" winemaking protocols, with all the processes being reduced to improve the aromatic quality of Sauvignon Blanc in the Marlborough region of New Zealand. This experimental thesis project with the title "Influences of amino acids during alcoholic fermentation and the development of aromatic compounds in Sauvignon Blanc", observes the single amino acids added starting from two musts, with different initial nitrogenous source, and examines the reaction from the yeast during alcoholic fermentation, with reference to the type of free aromatic compounds and their concentration
N/A

The thesis deals with the issue of potentially allergenic aroma compounds in food. Aroma compounds suitable for food flavouring are mainly of plant origin. These aroma compounds and their mixtures have a lot of positive and negative effects on human organism. Although the aroma compounds are not among main groups of allergens, several studies indicate a potential risk of allergenic effects of some aroma compounds. The theoretic part of this thesis focuses on aroma compounds of plant origin and gives an overview of 26 the most important fragrance allergens according to the European cosmetic legislation. The aim of the experimental part was to verify the selectivity and appropriateness of the currently used methodology of headspace solid-phase microextraction and gas chromatography-flame ionization detection (HS-SPME-GC-FID) for determination of selected allergenic aromatic active compounds. Secondly, it is to use a proven method for a wide spectrum of real food products.

This diploma thesis is focused on aromatic substances occurring in food. The theoretical part describes their characteristics, the use of fragrances of plant origin for flavoring of food and points to their possible negative effects on the human organism. Twenty six potentially allergenic fragrances are thus identified in European legislation and an overview of their physical properties, occurrence and use is also processed in this work.The aim of the experimental part was to develop and validate a method for assessment of fragrances using headspace solid phase microextraction combined with gas chromatography (HS-SPME-GC-FID). The following parameters have been validated : repeatability, linearity, limits of detection (LOD) and limits of quantification (LOQ). The HS-SPME-GC-FID method was finally applied to selected samples of foods commonly available on the market.

The subject of this thesis was the determination of the profile of volatile aromatic compounds of varietal wines and the subsequent statistical analysis, aiming to find varietal markers of selected varieties – individual substances, their typical contents, mutual correlations. The HS-SPME-GC-MS technique was used to determine the aromatic profile. The main focus of this work was the analysis of the varieties Pálava, Hibernal, Johanniter and Solaris. The data obtained were statistically processed using descriptive and multivariate statistical methods. On the basis of these analyses, models were developed which, from the contents of 14 selected compounds of the aromatic profile, allow the Piwi varieties Hibernal, Johanniter and Solaris to be distinguished from each other and from the Pálava variety and, for the Pálava variety wines, to determine their origin in the Znojmo or Mikulov wine sub-region.

This master thesis deals with the assessment of selected fragrant substances in cosmetic products. At the beginning the occurrence, methods of acquiring and application of these substances is described. They are essential components primarily in cosmetic products, however they can cause an allergic reaction in sensitive individuals. The theoretical part of this thesis also describes the cause and the progress of these undesirable effects. There are several hundreds, up to thousands fragrant substances used in the perfume industry, from which 26 was designated by the European regulations as potential allergenic fragrant substances and their content is regulated. Legislative standards as well as the overview of the methods used to determine these fragrances are given here. The experimental part is focused on the development and validation of the separation methods for determining of the selected and potentially allergenic fragrant substances. The method used was a solid phase microextraction using Carboxen/Polydimethylsiloxane fiber combined with a gas chromatography coupled to a flame ionization detector (SPME-GC-FID). Amylcinnamyl alcohol, benzyl alcohol, cinnamyl alcohol, coumarin, lyral, ?-isomethyl ionone were among the determined allergenic substance. Consequently, this method was also applied on 10 selected cosmetic products specimens, in which the linalool and geraniol were identified as the most common allergenic substances.

The effects of crop load and berry exposure on the composition of Marlborough Sauvignon blanc grapes and wine from the Brancott vineyard, Blenheim, were explored. Commercially grown, 2-cane and 4-cane Sauvignon blanc vines were used with a row orientation of north-south. Two exposure treatments were imposed in the following manner: complete leaf removal was undertaken in the fruit zone and 50% shade cloth was erected to give a uniform shading treatment to half the trial vines. Weekly thirty-berry and whole bunch samples were taken from each of the 32 plots with the exception of the veraison period when two samples per week were taken. Vine vigour was assessed using pruning and leaf area per vine data. Harvest occurred on different dates for 2-cane and 4-cane pruned vines so that fruit attained from both treatments had similar °Brix. Fruit was processed at the Lincoln University winery. Must analysis and wine analysis were undertaken. As expected, 4-cane vines had almost double the yield of 2-cane vines. Higher crop load significantly reduced leaf area per shoot and shoot thickness. Lower leaf area to fruit ratio for 4-cane berries resulted in delayed onset of veraison and slowed the rate of sugar accumulation. Crop load, which limited leaf area to fruit ratio, appeared to be the dominant factor in determining timing of grape physiological ripeness as expressed by °Brix over other factors such as fruit exposure. Malic acid, tartaric acid, IPMP (iso-propylmethoxypyrazine) and IBMP (iso-butyl-methoxypyrazine) were lower at equivalent °Brix in 4-cane compared with 2-cane berries. Significantly higher concentrations of quercetin were found in exposed compared to shaded berries. Must analysis showed a significant influence of crop load on berry titratable acidity and pH, reflecting berry ripening results. Exposure significantly increased the concentrations of nitrogenous compounds in 4-cane must yet showed no influence on 2-cane must. After wine processing lower malic acid concentrations in wines made from 100% exposed fruit became evident in lower wine titratable acidity but showed no influence on wine pH. Bentonite addition to wines had a small but statistically significant influence on wine by reducing pH, titratable acidity and alcohol. Bound sulphur concentrations were significantly higher in 4-cane versus 2-cane wines. At harvest, methoxypyrazine levels in grapes and wines were very low; IBMP concentrations where significantly lower than those normally found in Sauvignon blanc wines from Marlborough. This was attributed to the absence of basal leaves from the shoots of ripening berries. The results suggest that leaf area to fruit ratio is a powerful determinant of grape and wine quality.

The effects of crop load and berry exposure on the composition of Marlborough Sauvignon blanc grapes and wine from the Brancott vineyard, Blenheim, were explored. Commercially grown, 2-cane and 4-cane Sauvignon blanc vines were used with a row orientation of north-south. Two exposure treatments were imposed in the following manner: complete leaf removal was undertaken in the fruit zone and 50% shade cloth was erected to give a uniform shading treatment to half the trial vines. Weekly thirty-berry and whole bunch samples were taken from each of the 32 plots with the exception of the veraison period when two samples per week were taken. Vine vigour was assessed using pruning and leaf area per vine data. Harvest occurred on different dates for 2-cane and 4-cane pruned vines so that fruit attained from both treatments had similar °Brix. Fruit was processed at the Lincoln University winery. Must analysis and wine analysis were undertaken. As expected, 4-cane vines had almost double the yield of 2-cane vines. Higher crop load significantly reduced leaf area per shoot and shoot thickness. Lower leaf area to fruit ratio for 4-cane berries resulted in delayed onset of veraison and slowed the rate of sugar accumulation. Crop load, which limited leaf area to fruit ratio, appeared to be the dominant factor in determining timing of grape physiological ripeness as expressed by °Brix over other factors such as fruit exposure. Malic acid, tartaric acid, IPMP (iso-propylmethoxypyrazine) and IBMP (iso-butyl-methoxypyrazine) were lower at equivalent °Brix in 4-cane compared with 2-cane berries. Significantly higher concentrations of quercetin were found in exposed compared to shaded berries. Must analysis showed a significant influence of crop load on berry titratable acidity and pH, reflecting berry ripening results. Exposure significantly increased the concentrations of nitrogenous compounds in 4-cane must yet showed no influence on 2-cane must. After wine processing lower malic acid concentrations in wines made from 100% exposed fruit became evident in lower wine titratable acidity but showed no influence on wine pH. Bentonite addition to wines had a small but statistically significant influence on wine by reducing pH, titratable acidity and alcohol. Bound sulphur concentrations were significantly higher in 4-cane versus 2-cane wines. At harvest, methoxypyrazine levels in grapes and wines were very low; IBMP concentrations where significantly lower than those normally found in Sauvignon blanc wines from Marlborough. This was attributed to the absence of basal leaves from the shoots of ripening berries. The results suggest that leaf area to fruit ratio is a powerful determinant of grape and wine quality.

Vineyards in New Zealand suffer bird damage caused by several avian species, including blackbirds and silvereyes. The introduced European Blackbird takes whole grapes which reduces yield. The self-introduced Australasian Silvereye pecks on grapes, leaving them on the vine to be further attacked by fungi and bacteria, and the subsequent off-odours can cause grapes to be refused by the winery or to suffer a price-reduction. Bird control methods remain primitive and largely ineffective during the long ripening period of wine grapes. An ecologically sound method to manage and reduce bird pressure requires deeper understanding of why some birds eat grapes, especially since grapes are not particularly nutritious. This work investigated the extent to which blackbirds and silvereyes are attracted by various compounds in ripening grapes. Since in natural grapes these compounds develop and change simultaneously, I developed an artificial grape in which a single parameter could be investigated. Artificial grapes (and sometimes nectar) were presented on a bird feeder table and the responses of birds to hexose sugars, the aromas 2-3-isobutylmethoxypyrazine and geraniol, tartaric and malic acids, grape tannins, and purple and green colour were recorded on timelapse video and analysed.

Odor detection thresholds of hexanal, 2-heptenal, 2-pentanone, and 2,4-nonadienal were determined in spring water, high temperature short time (HTST) 2% fat milk, and extended shelf life soymilk. The efficacy of odor scavenger's beta-cyclodextrin, D-sorbitol, and nylon 6 in removing these odors was also determined. The odor thresholds of the different odor and media combinations were as follows: hexanal in spring water, milk, and soymilk were 585, 339, and 536 ppb respectively; 2-heptenal in spring water, milk, and soymilk were 2,092, 2,322, and 3,184 ppb respectively; 2-pentanone in spring water, milk and soymilk were 24,925, 29,255 and 33,271 ppb respectively; and 2,4-nonadienal in spring water, milk, and soymilk were 164, 326, and 243 ppb respectively. These amounts reference the initial spiked concentration that was added directly to the media. Both hexanal and 2,4-nonadienal had lower thresholds than 2-heptenal and 2-pentanone in all of the media. The odor detection thresholds of 2-heptenal, 2-pentanone, and 2,4-nonadienal did show a significant difference between soymilk and water, but not for milk. The efficacy of the odor scavengers were determined by use of solid phase micro-extraction gas chromatography (SPME-GC) and sensory evaluation. Hexanal, 2-heptenal, 2-pentanone, and 2,4-nonadienal were spiked at 1,000, 3,000, 30,000, and 300 ppb respectively in all three media. Beta-cyclodextrin, D-sorbitol, and nylon 6 were added at a level of 0.1% w/v and 1.0% w/v. In all of the media, beta-cyclodextrin was found to significantly reduce hexanal, 2-pentanone, 2-heptenal, and 2,4-nonadienal at both 0.1% w/v and 1.0% w/v. Nylon 6 was not found beneficial.
Master of Science

In recent years, there have been a number of studies looking at inhibition of microorganisms by spices, herbs or their extracts.  Many of these products have been shown to have antimicrobial activity against foodborne pathogens.  The purpose of this research was to evaluate the antimicrobial activity of three essential oil (EO) compounds (thymol, eugenol, and trans-cinnamaldehyde) alone and in combination with three naturally-occurring apple aroma (AA) compounds (hexanal, trans-2-hexenal and 1-hexanol) to identify the minimum inhibitory concentrations necessary to inhibit E. coli O157:H7.  Three commercial apple juice flavoring mixtures (natural apple cinnamon, natural apple spice and natural red apple) were additionally tested alone for antimicrobial activity against E. coli O157:H7.
The standard agar dilution method (SAD) and checkerboard assay were used to evaluate the efficacy of the nine compounds, alone and in combination against E. coli O157:H7.  In general, the EO compounds were significantly more effective against E. coli O157:H7 than the AA compounds (P<0.05).  Cinnamaldehye, with an MIC of 0.2 mg/mL, exhibited the highest degree of activity, followed by thymol, eugenol and trans-2-hexenal, which each had individual MIC values of 1.6 mg/mL.  No synergism was found in the combinations of EO compounds with AA compounds.
Master of Science in Life Sciences

[EN] Fruits both produce and emit volatile chemical compounds. These are short-chained low polarity molecules involved in many processes, and they are responsible of our perception of fruit aroma and of most of their flavour. This thesis is focused on the study of volatile compounds in the tomato fruit, which is one of the most important horticultural worldwide and is a model system for the study of fruit development and ripening. Some of the analytical methods more frequently used for the analysis of tomato fruit volatiles were systematically compared. Results revealed that the observed volatile profile is highly dependent on the precise analytical method used, both for sample processing and for the technique used for volatile acquisition. It was concluded that the method of election for the comparison of large sets of samples from a multi-omics approach consists on flash freezing the biological material with liquid nitrogen at the selected ripening stage and the use of headspace solid phase microextraction coupled to gas chromatography and mass spectrometry for the subsequent analysis. This method was implemented and was used for the determination of volatile compounds in selected NILs harbouring QTLs for characters related to flavour and aroma introgressed in different genetic backgrounds. The results allowed the association of several of the organoleptic characters previously identified with modified levels of several volatiles. It was also observed that the genetic background has a major effect on the production of such metabolites. Correlation analysis between the levels of volatiles and primary metabolites led to the conclusion that the production of volatile compounds is generally not determined by the levels of their precursors. Its regulation is most likely to be due to downstream processes such as the availability of either precursors or intermediate metabolites, the variability in specific processes leading to the concersion of precursors into volatiles, or to other still unknown regulatory mechanisms. Volatile compounds were also studied in a RIL population derived from a cross between Solanum pimpinellifolium accession TO-937, the closest species to cultivated, which produces red fruits, and S. lycopersicum cv. 'Moneymaker', a tomato variety for the fresh market. This allowed the identification of 102 QTLs for 39 different volatile compounds, 76 of which had not been previously described. All these QTLs were mapped along the 12 tomato chromosomes by means of the SOLCAP SNPs molecular market map. Most of the QTLs identified were subsequently evaluated on introgression lines (ILs) generated from the same original genotypes. It was observed that almost half of the QTLs previously identified retained their effect after introgression in the 'Moneymaker' genetic background. Additionally, 12 new QTLs were identified in this IL population. Based on the existing knowledge about the effect of volatile compounds on our perception of flavour and aroma and also on their ability to maintain their effect after introgression in the cultivated tomato, some of the QTLs identified are good candidates to be used in tomato flavour breeding programs. Eventually, the comparison of the localization in the genome of the QTLs identified in the different populations studied with those already described in the literature revealed a very low degree of co-localization between the different QTLs. This implies that there exists a wide range of variability in the wild species related to tomato available for breeding tomato flavour and aroma.
[ES] Los frutos producen y emiten compuestos químicos volátiles. Estos son moléculas en general poco polares y de cadena corta que cumplen diversas funciones, y son las responsables de que percibamos el aroma y buena parte del sabor de los frutos. Esta tesis está centrada en el estudio de los volátiles del fruto del tomate, que es uno de los cultivos hortícolas más importantes y un sistema modelo para el estudio del desarrollo y maduración del fruto. Se compararon de forma sistemática los métodos analíticos más comúnmente utilizados para el análisis de volátiles en fruto de tomate, y se observó que el perfil de volátiles detectado está fuertemente condicionado por el método analítico utilizado, tanto por el proceso de preparación de la muestra como por la técnica de adquisición de los volátiles. Finalmente se concluyó que la técnica más adecuada para la comparación de grandes grupos de muestras desde una aproximación multi-ómica consiste en congelar con nitrógeno líquido el material vegetal una vez alcanzado el momento idóneo de recolección, y su análisis posterior mediante microextracción en fase sólida (SPME) acoplada a cromatografía de gases y espectrometría de masas. Se puso a punto esta técnica y se utilizó para la determinación de los compuestos volátiles en varias líneas NILs portadoras de QTLs de caracteres relacionados con el sabor y el aroma en distintos fondos genéticos. Los resultados permitieron asociar varios de los caracteres organolépticos identificados con alteraciones en los niveles de algunos volátiles. Igualmente se observó que el fondo genético tiene un efecto importante sobre la producción de estos metabolitos. Los análisis de correlación entre los niveles de volátiles y metabolitos primarios permitieron concluir que la producción de compuestos volátiles, en general, no está determinada por los niveles de sus precursores, sino que su regulación debe encontrarse más bien en procesos posteriores, tales como la disponibilidad de los precursores o de metabolitos intermedios, variabilidad en procesos específicos relacionados con la conversión de los precursores en volátiles, o algún otro mecanismo regulador aún desconocido. También se estudiaron los volátiles en una población de RILs derivada de un cruce entre Solanum pimpinellifolium entrada TO-937, la especie más próxima al tomate cultivado, la cual produce frutos rojos, y S. lycopersicum cv. "Moneymaker", una variedad de tomate para el mercado en fresco. Esto permitió identificar 102 QTLs para 39 volátiles diferentes, 76 de las cuales no se habían descrito previamente, las cuales se mapearon a lo largo de los 12 cromosomas del tomate utilizando el mapa de marcadores moleculares de SNPs SOLCAP. Posteriormente se evaluaron la mayoría de las QTLs identificadas mediante la determinación de los volátiles en líneas ILs generadas a partir de los mismos materiales. Se observó que casi la mitad de estas QTLs mantuvieron su efecto al ser introgresadas en el fondo genético "Moneymaker", al tiempo que 12 nuevas QTLs se identificaron en esta población de ILs. Algunas de las QTLs identificadas, en base al conocimiento existente sobre el efecto de los compuestos volátiles en nuestra percepción del sabor y el aroma, y en base a su capacidad para mantener su efecto tras su introgresión en el tomate cultivado, resultan ser candidatos prometedores para su utilización en la mejora genética del sabor del tomate. Finalmente, el análisis de la localización en el genoma de las QTLs analizadas en las distintas poblaciones objeto de estudio en esta tesis, junto con las descritas en la bibliografía disponible, puso de relieve el bajo grado de co-localización existente entre las distintas QTLs, lo cual implica que en las especies silvestres relacionadas con el tomate existe un amplio rango de variabilidad genética susceptible de ser utilizado para la mejora de su sabor y su aroma.
[CAT] Els fruits produixen i emitixen compostos químics volàtils. Estos són molècules en general de baixa polarita i cadena curta que tenen diverses funcions, i són les responsables de la nostra percepció de l'aroma i de bona part del sabor dels fruits. Esta tesi està centrada a l'estudi dels volàtils del fruit de la tomata, que és un dels cultius hortícoles més importants i un sistema model per a l'estudi del desenvolupament i la maduració del fruit. Es van comparar de forma sistemática els mètodes analítics més habituals per a l'anàlisi de volàtils en fruits de tomata, i es va observar que el perfil de volàtils detectat està fortament condicionat per el mètode analític utilitzat, tant per el procés de preparació de la mostra com per la técnica d'adquisició dels volàtils. Es va concluir que la t`cnica més adequada per a la comparació de grans grups de mostres desde una aproximació multi-òmica consistix en congelar en nitrògen líquid el material vegetal en el momento idoni de recolecció, i analitzar-lo posteriorment per microextracció en fase sòlida (SPME) acoplada a cromatografía de gasos i espectrometría de masses. Es va posar a punt esta técnica i es va utilitzar per a la determinació dels composteos volàtils en varies línies NILs portadores de QTLs de caràcters relacionats en el sabor i l'aroma en fons genètics diversos. Els resultats van permetre associar alguns dels caràcters organolèptics identificats en alteracions en els nivells d'alguns compostos volàtils. També es va observar que el fons genètic té un efecte important sobre la producción d'estos metabolits. Els anàlisi de correlació entre els nivells de volàtils i els metabolits primaris ens van permetre concluir que la producción de compostos volàtils, en general, no està determinada per els nivells dels seus precursors. La seua regulació és deguda a procesos posteriors, com la disponibilitat dels precursors o de metabolits intermediaris, la variabilitat en processos específics relacionats en la conversió dels precursors en volàtils, o en algún altre mecanisme regulador encara desconegut. També es van estudiar els volàtils en una población de RILs rerivada d'un creuament entre Solanum pimpinellifolium entrada TO-937, la espècie silvestre més próxima a la tomatera cultivada i que produix fruits rojos, i S. lycopersicum cv. Moneymaker, una varietat de tomata per al mercat en fresc. Açò va permetre l'identificació de 102 QTLs de 39 volàtils diferents, 76 de les quals no s'havien descrit prèviament, i que es van mapejar al llarg dels 12 cromosomes de la tomatera mitjançant el mapa de marcadors moleculars de SNPs SOLCAP. Posteriorment es van evaluar la majoria de les QTLs identificades mitjançant la determinació dels volàtils en línies ILs generades a partir dels mateixos materials. Es va observar que quasi la meitat de estes QTLs van mantindre el seu efecte al ser introgressades en el fons genètic "Moneymaker". Adicionalment, 12 noves QTLs es van identificar en esta población d'ILs. Algunes de les QTLs identificades, en base al coneiximent existent respecte a l'efecte dels volàtils en la nostra percepció del sabor i l'aroma, i tenin en cónter la seua capacitat de mantindre el seu efecte al ser introgressats en la tomata cultivada, són candidats prometedors per a ser utilitzats en la millora genética del sabor de les tomates. Finalment, l'anàlisi de la localització en el genoma de les QTLs analitzades en les distintes poblacions objecte d'aquesta tesi, junt a les descrites en la bibliografía disponible, va evidenciar que existix una baixa freqüència de co-localització entre les distintes QTLs. Açò implica que existix molta variabilitat genética en les espècies silvestres relacionades en la tomatera, que pot ser utilitzada per a la millora del sabor i l'aroma dels seus fruits.
Rambla Nebot, JL. (2016). GENES AND GENOMIC REGIONS RELATED TO THE PRODUCTION OF VOLATILE COMPOUNDS IN THE TOMATO FRUIT [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61768
TESIS
Premiado

Hibiscus sabdariffa (H. sabdariffa) is a rich source of bioactive phytochemicals with potential health benefits. Based on its reported use in folk medicine for the treatment of hypertension and recently type 2 diabetes, the plant has gained considerable commercial importance as a functional food. Three varieties of H. sabdariffa were analyzed for their phytochemical content and inhibitory potential on carbohydrate-digesting enzymes as a basis for selecting a variety for wine production. The dark red variety was chosen as it was highest in phenolic content and partially inhibited α–glucosidase (maltase), with delphinidin 3-O-sambubioside, cyanidin 3-O-sambubioside and 3-O-caffeoylquinic acid accounting for 65 % of this activity. None of the varieties significantly inhibited α–amylase. Regarding H. sabdarifa wine, the effect of fermentation (20 and 30 ˚C) and ageing (6, 15 and 30 °C) temperatures on the phytochemical composition and bioactivity of Roselle wine was investigated for a period of 40 days and 12 months respectively. The main change in phytochemical composition observed during fermentation was the hydrolysis of 3-O-caffeolquinic acid and the concomitant increase of caffeic acid irrespective of fermentation temperature. Wine fermented at 20 ˚C was slightly more active for α–glucosidase inhibition with more fruity aromas, but there were more flowery notes at 30 ˚C. Although ageing temperature did not produce an obvious trend in concentrations of most of the volatiles analysed, its impact was clearly demonstrated in the final concentrations of diethyl succinate which increased with higher ageing temperature. The data on the overall quality rating of roselle wine by a sensory panel showed that neither fermentation nor ageing temperatures produced any significant difference (p ≥ 0.05) in the wine samples. This study demonstrates that processing of H. sabdariffa into wine represents a promising alternative to expanding the functional properties of this crop.

Echinacea purpurea is a rich source of biologically active compounds, which are attributed to several therapeutic effects. This thesis is focused on the optimization of the extraction process, considered these substances. Maceration was chosen as a type of extraction, due to its simplicity. The Optimal conditions were solid-solvent ratio 1: 5, 40% ethanol, 60 °C, 60 minutes. An extract from the dried flowers of Echinacea purpurea was prepared by this optimized process. The content of total phenolic compounds was set on 365.18 ± 3.51 [mg_GAEl^(-1)] and the antioxidant activity was 507.50 ± 6.89 [mg_TEACl^(-1)]. The percentage of radical scavenging activity ABTS•+ was 95.45 ± 1.30. The antimicrobial activity of the extract was tested against 3 microorganisms – Escherichia coli, Bacillus cereus and Candida glabrata. There was not detected inhibitory effect on the growth of microorganisms. In a total, 39 volatile compounds were identified, and the biggest group were represented sesquiterpenes (70.9%). The obtained results confirm that Echinacea extract has antioxidant activity and contains several interesting aromatic substances, In the future, it can be used as a natural resource of nutritionally and sensory valuable compounds, e.g., into various foods and delicacies.

Capes; CNPq
O aroma de café é constituído por uma mistura de componentes voláteis, os quais podem ser encontrados no óleo obtido na torração do café. O café solúvel, em função das condições de processamento, perde parte do seu aroma que, a princípio, poderia ser reincorporado ao produto pela adição do óleo de café diretamente sobre o produto final. Contudo, alguns trabalhos apontam para dificuldades de se obter bons resultados quando se trabalha com o óleo in natura devido à sua volatilidade e à degradação dos aromas. A incorporação de nanocápsulas contendo o óleo de café pode ser uma alternativa para minimizar estas perdas, pois o polímero encapsulante pode agir como barreira de proteção e promover sua liberação de forma controlada. A proposta do trabalho é a obtenção de nanocápsulas de poli(L-ácido lático)(PLLA), contendo o óleo de café torrado pela técnica de miniemulsificação/evaporação do solvente e validar o método para quantificação do óleo de café por espectrofotometria UV-Vis. Os estudos de validação possibilitaram garantir a qualidade dos resultados obtidos. Formulações com diferentes concentrações de PLLA, óleo e tipos de surfactantes foram valiadas quanto ao tamanho e índice de polidispersão frente à recuperação de óleo. Todas as formulações testadas apresentaram valores de recuperação de óleo acima de 80% mostrando que a técnica é adequada e pode ser considerada uma alternativa promissora para produzir nanocápsulas contendo óleo de café. As análises da composição do aroma das nanocápsulas, que indica a qualidade da porção de óleo encapsulada, mostraram que as nanocápsulas contêm frações de todos os compostos encontrados no óleo.
Coffee flavor is a mixture of volatile components found in the oil obtained from the roasted coffee. Soluble coffee loses part of its aroma due to processing conditions which could be reintroduced to the product by the direct addition of the coffee oil. However, some studies point out difficulties in obtaining good results when working with in natura oil because of the high volatility and the flavor degradation. The incorporation of nanocapsules containing coffee oil could be an alternative to minimize these losses, because the encapsulating polymer could act as a protecting barrier and promote its controlled release. The aim of this work is obtain PLLA nanocapsules containing roasted coffee oil by the miniemulsification/solvent evaporation technique. The spectrophometric UV- Vis method used to quantify the coffee oil was validated. Formulations with different PLLA and oil concentrations and kind of surfactant were evaluated in respect to the average nanocapsules size and polydispersion index and oil recovery. All formulations demonstrated oil recovery values above 80 % demonstrating that the technique is adequate and can be consider a promising alternative to coffee oil nanocapsules production. Flavor analysis nanocapsules composition, which indicates the quality of the encapsulated oil, showed that the nanocapsules contain fractions of all the substances found on the in natura oil.

This diploma thesis deals with the comparison of aroma compounds in goat and cow milk. The content of aroma active substances in samples of goat and cow milk, which were analyzed by HS-SPME-GC-MS method, is monitored. In the theoretical part, milk is characterized, its formation in the dairy mammary gland is described, its composition including an overview of aromatic active substances, physical and chemical as well as its sensory properties, milk defects and differences between cow's and goat's milk are described. Further, gas chromatography in combination with solid phase microextraction technique in tandem with mass spectrometry is described. In the experimental part, 13 samples of goat and 8 samples of cow milk were subjected to HS-SPME-GC-MS analysis. Samples were analyzed fresh and after heat treatment. Altogether, 78 volatile substances were identified in the samples. Ketones were the most numerous, aldehydes, alcohols and carboxylic acids were the most abundant chemical groups. There were differences between samples in number as well as in content of the identified compounds, 64 in goat milk and 48 in the cow milk.