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 (PhD(Agric) (Horticulture))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Plums with good taste, aroma and eating quality lead to repeat purchases and sustained demand. Taste includes non-volatile compounds, e.g. sugars and acids, and has been well researched to meet the consumers’ preferences. Plum aroma, however, has not enjoyed the same attention. Limited literature is available on the aroma of Japanese plums and none could be found on the effects of relatively long cold storage on the profiles. The main aim of this study is to investigate the changes in aroma compounds of Japanese plums throughout maturation and ripening and the effects of commercial cold storage regimes. Near infra-red (NIR) spectroscopy was also evaluated as a non-destructive method to determine plum quality parameters aimed at minimising sample variability. In Paper 1, NIR spectroscopy was used to develop prediction models for total soluble solid (TSS), total acidity (TA), sugar-to-acid ratio, firmness and weight in three cultivars (Pioneer, Laetitia and Angeleno) and a multi-cultivar model. Samples were collected for seven consecutive weeks and repeated over two seasons. TSS results showed excellent predictability (R2 = 0.817-0.955; RMSEP= 0.453-0.610 % Brix) but the TA models did not perform well. The sugar-to-acid ratio models had results comparable to that of TSS. Both the firmness and weight models had acceptable results. The models of ‘Pioneer’ and ‘Laetitia’ had a better predictability capacity than the ‘Angeleno’ model. Although the multi-cultivar models outperformed the single cultivar models on R2 values it had higher prediction errors. The robustness of all the TSS, TA and firmness models is high in terms of seasonality, range and cultivar. Papers 2 and 3, the main focus of the study, are concerned with the aroma profile dynamics of Japanese plums. HS-SPME was used in both papers to extract the aroma compounds followed by GC-TOFMS for separation and identification. In Paper 2, the aroma volatile compounds of three cultivars (Pioneer, Laetitia and Angeleno) were determined for a seven week period including samples from three maturity stages (immature, harvest and tree-ripe). A total of 35 compounds were identified of which ten were generic. Each cultivar had five unique compounds resulting in different aroma profiles for each of the maturity stages and distinct separation patterns using discriminant analysis. The study was extended in Paper 3 where the aroma volatile compounds of six cultivars (Pioneer, Sapphire, Laetitia, Songold, Larry Anne and Angeleno) and one plumcot (Flavor King) were determined at three functional stages (commercial harvest, tree-ripe fruit and cold stored fruit). A total of 62 compounds were identified and classified into three groups (‘unique’ (31), ‘generic’ (11) and ‘frequent’ (20)) based on their frequency of occurrence. The aroma profiles of ‘Larry Anne’ and ‘Flavor King’ are the most affected by cold storage conditions and ‘Pioneer’ appears to be the least affected. All the cultivars have significantly different aroma profiles at all three of the functional stages with ‘Sapphire’, ‘Larry Anne’ and ‘Flavor King’ showing the largest differences. ‘Flavor King’, a plumcot, presented a ripe aroma profile that was much diverged from that of the true plums. / AFRIKAANSE OPSOMMING: Pruime met ‘n goeie smaak, aroma en eetkwaliteit lei tot herhaalde verkope en volhoubare aanvraag. Smaak sluit die nie-vlugtige stowwe (suikers en sure) in en is goed nagevors om die verbruikersvoorkeure te bevredig. Pruim aroma het egter nie dieselfde aandag geniet nie. Daar is beperkte literatuur beskikbaar wat handel oor die aroma van Japanese pruime en geen kon gevind word oor die effekte van lang koelopberging op die aromaprofiele nie. Die hoof doel van hierdie studie is om die veranderinge in die aromatiese komponente van Japanese pruime te ondersoek tydens die volwassewording- en rypwordingsprosesse asook die effekte van kommersiele koelopberging. Naby infrarooi (NIR) spektroskopie is ook geevalueer as ‘n nie-destruktiewe manier om pruim kwaliteitsparameters te bepaal met die doel om monstervariasie te beperk. In Artikel 1 is NIR spektroskopie gebruik om voorspellingsmodelle vir totale oplosbare suikers (TOS), totale suur (TS), suiker-tot-suur verhouding, fermheid en gewig te bepaal in drie kultivars (Pioneer, Laetitia en Angeleno) asook ‘n multi-kultivar model. Monsters is vir sewe opeenvolgende weke versamel en herhaal oor twee seisoene. TOS resultate toon uitstekende voorspelbaarheid (R2 = 0.817-0.955; RMSEP= 0.453-0.610 % Brix) maar TS modelle het egter nie so goed gevaar nie. Die suiker-tot-suur verhoudingsmodelle se resultate was vergelykbaar met die van TOS. Beide die fermheid- en gewigsmodelle het aanvaarbare resultate opgelewer. Die modelle vir ‘Pioneer’ en ‘Laetitia’ het ‘n beter voorspelbaarheidskapasiteit getoon as die van ‘Angeleno’. Alhoewel die multi-kultivar model beter presteer het as die enkel kultivar modelle op die R2-waardes was daar meer voorspellingsfoute. Hoe robuustheid is gevind i.t.v. seisoene, datagrense en kultivar vir al die TOS, TA en fermheidsmodelle. Artikels 2 en 3, die fokuspunt van die studie, handel oor die dinamika van die aromaprofiel van Japanese pruime. HS-SPME is in beide artikels gebruik on die aromatiese verbindings te ekstraeer gevolg deur GCTOFMS vir skeiding en identifikasie. In Artikel 2 is die aromatiese stowwe van drie kultivars (Pioneer, Laetitia en Angeleno) bepaal vir sewe opeenvolgende weke en sluit monsters van drie volwassenheidsstadiums in (onvolwasse, oes en boom-rypgemaakte pruime). ‘n Totaal van 35 verbindings is geidentifiseer waarvan tien as generies beskou kan word. Elke kultivar het vyf unieke komponente gehad en het gelei tot verskillende aromaprofiele vir elk van die volwassenheidsstadiums en diverse skeidingspatrone tydens die gebruik van diskriminant analise. Die studie is uitgebrei in Artikel 3 waartydens die aromatiese vlugtige stowwe van ses kultivars (Pioneer, Sapphire, Laetitia, Songold, Larry Anne en Angeleno) en een plumcot (Flavor King) bepaal is tydens drie funksionele stadiums (oes, boom-rypgemaak en koelopgebergde pruime). ‘n Totaal van 62 verbindings is geidentifiseer en in drie groepe geklassifiseer (‘uniek’ (31), ‘generies’(11) en ‘gereeld’ (20)) gebaseer op voorkomsfrekwensie. Die aromaprofiele van ‘Larry Anne’ en ‘Flavor King’ is die meeste deur die koelopberging geaffekteer en ‘Pioneer’ die minste. Al die kultivars het kenmerkend verskil t.o.v. hul aromaprofiele in al drie die funksionele groepe en ‘Sapphire’, ‘Larry Anne’ en ‘Flavor King’ het die grootste verskille getoon. ‘Flavor King’, die plumcot, het ook ‘n ryp aromaprofiel gehad wat baie van die van die egte pruime verskil het.

Les gommes d’Acacia (AG, E414EC) sont largement utilisées par ses propriétés stabilisantes, émulsifiantes et filmogènes. Le but de ce projet de thèse consiste à caractériser les propriétés interfaciales et émulsifiantes des gommes d’Acacia. Les études des propriétés interfaciales liquide-liquide ont confirmé que l’A. senegal permettait une plus grande diminution de la tension interfaciale et une formation plus rapide de films à l’interface. Ceci est en accord avec sa teneur plus élevée en AGP de masse molaire élevée riche en protéine, la meilleure accessibilité de la partie protéique et la plus grande flexibilité moléculaire par rapport à A. seyal. De même, ces avantages biochimiques et structuraux de l’A. senegal se sont avérés être impliqué dans les propriétés émulsifiantes des gommes. Les résultats d'une approche innovante visant à contrôler la teneur en AGP de masse molaire élevée riche en protéines dans le milieu et la concentration totale de gomme par le mélange de deux fractions bien caractérisées isolées chez A. senegal ont confirmé la synergie fonctionnelle entre la quantité des AGP de masse molaire élevée riche en protéines et la concentration totale en gomme. De plus, en présence de quantités élevées d'AGP de masse molaire élevée riches en protéines, les émulsions étaient stables contre la floculation/coalescence en raison de la forte teneur en protéines permettant une répulsion électrostatique entre les gouttelettes et la viscosité apparente élevée du milieu. Celles-ci étaient en accord lorsque les propriétés émulsifiantes de A. senegal et A. seyal ont été comparées. En effet, A. senegal contient une plus grande quantité d'AGP de masse molaire élevée riches en protéines et d'acides uroniques, et sa dispersion présente une viscosité apparente supérieure à celle d'A. seyal. En outre, lorsque les gommes d'Acacia étaient utilisées sous forme de films séchés, la grande teneur en AGP riches en protéines de masse molaire élevée et la bonne accessibilité des fragments protéiques d'A. senegal permettaient aux films d’avoir une surface homogène lisse avec des propriétés hydrophobes caractéristiques. En revanche, les films de A. seyal étaient irréguliers et constitués d'une organisation répétitive comme de nombreuses grosses particules uniformément réparties sur la surface par rapport au faible degré de ramification, une teneur élevée en arabinose favorisant les liaisons hydrogènes intra et inter moléculaires et une forte capacité d'hydratation chaînes polysaccharidiques de A. seyal. Selon les résultats, cette thèse apporte de nouvelles connaissances sur la relation entre la composition biochimique, les propriétés structurales et physico-chimiques, pour la première fois, dans les deux aspects de la gomme, à savoir sous forme de dispersion liquide et de films séchés.Mots clés : gomme d’Acacia, propriétés interfaciales et émulsifiantes, arabinogalactane-protéines, arômes / Acacia gums (AG, E414 EC) are widely used for its stabilizing, emulsifying and film-forming properties. The aim of this project is to characterize the interfacial and emulsifying properties of Acacia gums. The main results from liquid-liquid interfacial properties study confirmed that A. senegal showed a faster decrease of interfacial tension and a more rapid interfacial film formation. These were in agreement to its greater content of high molar mass-protein AGPs content, the more accessibility of proteinaceous moieties, and the higher molecular flexibility compared to A. seyal. In the same vein, these biochemical and structural advantages of A. senegal were found to be involved in the emulsifying properties of gums. The results from an innovative approach aiming to control high molar mass protein-rich AGPs content in bulk and the total concentration of gum by mixing two well characterised fractions isolated from A. senegal confirmed the functional synergism between the amount of high molar mass protein-rich AGPs and the total gum concentration. Moreover, in the presence of high molar mass protein-rich AGPs in high amount, the emulsion became stable to flocculation/coalescence due to the great protein content allowing electrostatic repulsion between droplets and the high bulk apparent viscosity. These were in agreement when the emulsifying properties of A. senegal and A. seyal were compared. Indeed, A. senegal containing a greater amount of high molar mass protein rich AGPs and uronic acids and its dispersion having a higher apparent viscosity than A. seyal allowing the former to form emulsion with a more stability. Besides, when Acacia gums were used in the form of dried film, the great content of high molar mass protein-rich AGPs and the good accessibility of proteinaceous moieties of A. senegal allowed the smooth homogeneous surface with a hydrophobic characteristic properties of A. senegal dried films. In contrast, A. seyal films was irregular and composed of a repetitive organization as numerous large particle uniformly distributed on surface in the relation to the low degree of branching, high arabinose content favoring intra and inter molecular hydrogen bonding and high hydration ability causing aggregation of polysaccharide chains of A. seyal. According to the results, this thesis brings new knowledge of the relationship between biochemical composition, structural and physicochemical properties, for the first time, across both aspects of gum, i.e. in the form of liquid dispersion and dried films.Keywords: Acacia gums, interfacial and emulsifying properties, arabinogalactan proteins, aroma compounds

O objetivo desta pesquisa foi identificar os voláteis importantes para o aroma da carne de frangos suplementados com diferentes concentrações de beta-ácidos do lúpulo. Para isso, 1440 frangos de corte Cobb 500 foram alimentados ad libitum durante 42 dias com dietas basais suplementadas com 0 (controle negativo), 30, 60 e 120 mg kg-1 de &beta;- ácidos do lúpulo. Após 42 dias de experimento, os animais foram abatidos e o músculo peitoral maior (peito do frango) foi retirado, separado do tecido conjuntivo, da pele e da gordura subcutânea. Os filés de peito foram imediatamente congelados a -80 °C até o momento das análises. Nos filés foi determinado o perfil de ácidos graxos por cromatografia gasosa acoplada a detector de ionização em chama. E a carne dos animais suplementados com 30 mg kg-1 de &beta;-ácidos foi a que apresentou a maior quantidade dos ácidos graxos insaturados de cadeia longa araquidônico (C20:4-n6) e docosaexaenoico (C22:6-n3), cujas propriedades antioxidantes e anti-inflamatórias são benéficas para saúde humana. Para a caracterização dos voláteis da carne assada foi desenvolvido e otimizado um procedimento de extração dos voláteis da carne por microextração em fase sólida de headspace (HS-SPME) e cromatografia gasosa acoplada à espectrometria de massa (GC-MS). Para isso, foram testados cinco tipos de fibras comerciais de SPME quanto a sua capacidade de extração dos voláteis da carne assada de frango e, para fibra que apresentou maior capacidade de extração (CAR/PDMS), foram otimizadas as condições de temperatura e tempo de extração por meio de delineamento composto central rotacional. As condições ótimas de extração foram: temperatura de extração de 81°C e tempo de extração de 45 min. Foram identificados 70 compostos voláteis na carne assada de frango por GC-MS majoritariamente aldeídos, alcanos, álcoois, ésteres e pirazinas. A análise de componentes principais (PCA) foi capaz de separar as amostras em função da suplementação com beta ácidos, o que indica que os diferentes níveis de suplementação podem originar diferentes voláteis na carne assada. O tratamento controle negativo (0 mg kg-1 de &beta;-ácidos) foi caracterizado principalmente por aldeídos e ácidos como octanal, decanal, benzaldeido e ácido n-hexanóico, o tratamento com 30 mg kg-1 de &beta;-ácidos foi caracterizado por pirazinas (2, 6 - dimetil prirazina e 3- etil, 2, 5 dimetil pirazina), o tratamento com 60 mg kg-1 de &beta;-ácidos foi caracterizado por uma maior quantidade de álcoois, aldeídos e furanos (hexanal, 1-octen-3 ol e pentilfurano), enquanto que o tratamento com 120 mg kg-1 de &beta;-ácidos foi caracterizado pela presença docomposto 2- metil pentanal.<br /> / The aim of this research was to identify volatiles that are important for the aroma of chicken meat supplemented with different concentrations of hops &beta;-acids. To carry out this research, 1440 Cobb 500 broilers were fed ad libitum during 42 days with basal diets supplemented with 0 (negative control), 30, 60, 120 and 240 mg kg-1 of hops &beta;-acids. After 42 days, the animals were slaughtered and the pectoralis major muscle (chicken breast) was removed, separated from the conjunctive tissue, the skin, and the subcutaneous fat. The breast fillets were immediately frozen at -80° C until the time of analysis. The fatty acid profile in the steaks was determined by gas chromatography with flame ionization detector (GC-FID). The meat of the animals supplemented with 30 mg kg-1 of &beta;-acids presented the highest amount of long-chain unsaturated fatty acids such as arachidonic acid (C20:4-n6) and docosaexaenoico (C22:6-n3), which have antioxidant and anti-inflammatory properties benefical to human health. For the characterization of the roasted meat volatiles, an extraction procedure was developed and optimized by headspace solid-phase microextraction (HS-SPME) and gas chromatography coupled to mass spectrometry (GC-MS). Six different types of SPME commercial fibers were tested regarding their ability to extract volatiles from chicken roasted meat, and to the fiber that showed the highest extraction capacity (DVB/CAR/PDMS), temperature and time of extraction were optimized through central composite design. The optimum extraction conditions were: extraction temperature of 81 °C and extraction time 45 minutes. It was possible to characterize 70 volatiles in the volatile fraction of roasted chicken meat by GC-MS, The majority of them were aldehydes, alkanes, alcohols, esters and pyrazines. The Principal Component Analysis (PCA) was able to separate the samples according to the supplementation with hops &beta;-acids, which indicates that different levels of supplementation may yield different volatile compounds on roasted chicken meat. The negative control treatment (0 mg kg-1 of &beta;-acids) was characterized mainly by aldehydes and acids like octanal, decanal, benzaldehyde and n-hexanoic acid, the treatment with 30 mg kg-1 of &beta;-acids was characterized by pyrazines (2,6 - dimethyl pyrazine, and 3-ethyl, 2,5- dimethyl pyrazine, the treatment with 60 mg kg-1 of &beta;-acids was characterized by a major quantity of alcohols, aldehydes and furans (hexanal, 1-octen-3-ol and pentylfuran), whereas the treatment with 120 mg kg-1 of &beta;-acids was characterized by the presence of 2 the compound 2-methyl pentanal.

O objetivo desta pesquisa foi identificar os voláteis importantes para o aroma da carne de frangos suplementados com diferentes concentrações de beta-ácidos do lúpulo. Para isso, 1440 frangos de corte Cobb 500 foram alimentados ad libitum durante 42 dias com dietas basais suplementadas com 0 (controle negativo), 30, 60 e 120 mg kg-1 de &beta;- ácidos do lúpulo. Após 42 dias de experimento, os animais foram abatidos e o músculo peitoral maior (peito do frango) foi retirado, separado do tecido conjuntivo, da pele e da gordura subcutânea. Os filés de peito foram imediatamente congelados a -80 °C até o momento das análises. Nos filés foi determinado o perfil de ácidos graxos por cromatografia gasosa acoplada a detector de ionização em chama. E a carne dos animais suplementados com 30 mg kg-1 de &beta;-ácidos foi a que apresentou a maior quantidade dos ácidos graxos insaturados de cadeia longa araquidônico (C20:4-n6) e docosaexaenoico (C22:6-n3), cujas propriedades antioxidantes e anti-inflamatórias são benéficas para saúde humana. Para a caracterização dos voláteis da carne assada foi desenvolvido e otimizado um procedimento de extração dos voláteis da carne por microextração em fase sólida de headspace (HS-SPME) e cromatografia gasosa acoplada à espectrometria de massa (GC-MS). Para isso, foram testados cinco tipos de fibras comerciais de SPME quanto a sua capacidade de extração dos voláteis da carne assada de frango e, para fibra que apresentou maior capacidade de extração (CAR/PDMS), foram otimizadas as condições de temperatura e tempo de extração por meio de delineamento composto central rotacional. As condições ótimas de extração foram: temperatura de extração de 81°C e tempo de extração de 45 min. Foram identificados 70 compostos voláteis na carne assada de frango por GC-MS majoritariamente aldeídos, alcanos, álcoois, ésteres e pirazinas. A análise de componentes principais (PCA) foi capaz de separar as amostras em função da suplementação com beta ácidos, o que indica que os diferentes níveis de suplementação podem originar diferentes voláteis na carne assada. O tratamento controle negativo (0 mg kg-1 de &beta;-ácidos) foi caracterizado principalmente por aldeídos e ácidos como octanal, decanal, benzaldeido e ácido n-hexanóico, o tratamento com 30 mg kg-1 de &beta;-ácidos foi caracterizado por pirazinas (2, 6 - dimetil prirazina e 3- etil, 2, 5 dimetil pirazina), o tratamento com 60 mg kg-1 de &beta;-ácidos foi caracterizado por uma maior quantidade de álcoois, aldeídos e furanos (hexanal, 1-octen-3 ol e pentilfurano), enquanto que o tratamento com 120 mg kg-1 de &beta;-ácidos foi caracterizado pela presença docomposto 2- metil pentanal.<br /> / The aim of this research was to identify volatiles that are important for the aroma of chicken meat supplemented with different concentrations of hops &beta;-acids. To carry out this research, 1440 Cobb 500 broilers were fed ad libitum during 42 days with basal diets supplemented with 0 (negative control), 30, 60, 120 and 240 mg kg-1 of hops &beta;-acids. After 42 days, the animals were slaughtered and the pectoralis major muscle (chicken breast) was removed, separated from the conjunctive tissue, the skin, and the subcutaneous fat. The breast fillets were immediately frozen at -80° C until the time of analysis. The fatty acid profile in the steaks was determined by gas chromatography with flame ionization detector (GC-FID). The meat of the animals supplemented with 30 mg kg-1 of &beta;-acids presented the highest amount of long-chain unsaturated fatty acids such as arachidonic acid (C20:4-n6) and docosaexaenoico (C22:6-n3), which have antioxidant and anti-inflammatory properties benefical to human health. For the characterization of the roasted meat volatiles, an extraction procedure was developed and optimized by headspace solid-phase microextraction (HS-SPME) and gas chromatography coupled to mass spectrometry (GC-MS). Six different types of SPME commercial fibers were tested regarding their ability to extract volatiles from chicken roasted meat, and to the fiber that showed the highest extraction capacity (DVB/CAR/PDMS), temperature and time of extraction were optimized through central composite design. The optimum extraction conditions were: extraction temperature of 81 °C and extraction time 45 minutes. It was possible to characterize 70 volatiles in the volatile fraction of roasted chicken meat by GC-MS, The majority of them were aldehydes, alkanes, alcohols, esters and pyrazines. The Principal Component Analysis (PCA) was able to separate the samples according to the supplementation with hops &beta;-acids, which indicates that different levels of supplementation may yield different volatile compounds on roasted chicken meat. The negative control treatment (0 mg kg-1 of &beta;-acids) was characterized mainly by aldehydes and acids like octanal, decanal, benzaldehyde and n-hexanoic acid, the treatment with 30 mg kg-1 of &beta;-acids was characterized by pyrazines (2,6 - dimethyl pyrazine, and 3-ethyl, 2,5- dimethyl pyrazine, the treatment with 60 mg kg-1 of &beta;-acids was characterized by a major quantity of alcohols, aldehydes and furans (hexanal, 1-octen-3-ol and pentylfuran), whereas the treatment with 120 mg kg-1 of &beta;-acids was characterized by the presence of 2 the compound 2-methyl pentanal.

Chocolate is one of the most important products for the food industry, being of economic interest all over the world. The cocoa quality depends directly on the post-harvest processing, being the cocoa-pulp fermentation a crucial step for chocolate quality development. The aim of this work was to study the diversity of aroma-producing yeasts associated with cocoa beans fermentation and to select suitable yeast starter culture to cocoa flavor modulation. A total of 39 cocoa-derived yeast isolates were screened for their capacity to produce volatile aroma compounds in a cocoa pulp simulation medium. The seven highest aroma-producing yeasts were identified by ITS-rRNA gene sequencing as belonging to Pichia kudriavzevii, in spite of exhibiting different metabolic profiles. A computer-assisted analysis of rep-PCR genomic fingerprints of Pichia kudriavzevii strains clearly differentiated the upper aroma-forming yeast strains (G1 group; P. kudriavzevii LPB06 and P. kudriavzevii LPB07) from the other strains (G2 group). This demonstrates the potential of rep-PCR technique as a promising genotypic tool for rapid and reliable speciation of aromatic yeast strains. In the second stage of this study, two strains with superior aroma production, namely P. kudriavzevii LPB06 and P. kudriavzevii LPB07, were used in cocoa beans fermentation at laboratory scale. They were able to establish an accelerated fermentation process with efficient yeast growth, sugars consumption and ethanol formation compared to the spontaneous process. The resulting cocoa beans were analyzed by diverse chemical analysis methods, including SPME-GC/MS, FTIR spectroscopy and metal and colorimetric analysis. All together, the results indicated that inoculated fermentations generated cocoa beans with better color development and richer aroma composition, suggesting that cocoa-associated yeast diversity at strain level can be exploited for flavor modulation of cocoa beans. / Atualmente, o chocolate é um dos produtos mais importantes para a indústria de alimentos, sendo de interesse econômico em todo o mundo. A qualidade do cacau depende diretamente do processamento pós-colheita, sendo a fermentação da polpa um passo crucial para o desenvolvimento da qualidade do chocolate. O objetivo deste trabalho foi estudar a diversidade de leveduras aromáticas associadas à fermentação de cacau e selecionar uma cultura iniciadora com potencial para modular o flavor de chocolate. Um total de 39 leveduras foram isoladas e caracterizadas quanto à formação de compostos aromáticos. As sete melhores produtoras foram identificadas através do sequenciamento do gene ITS-rRNA como Pichia kudriavzevii, apesar de apresentarem diferentes perfis metabólicos. Análise de impressões digitais (fingerprints) dos isolados pela técnica de rep-PCR claramente distinguiu as cepas com maior produção de compostos aromáticos, demonstrando o potencial desta técnica como uma ferramenta para rápida e confiável seleção de leveduras. Na segunda etapa deste estudo, duas cepas com superior formação de aroma (P. kudriavzevii LPB06 e P. kudriavzevii LPB07) foram testadas como culturas iniciadoras para fermentações de cacau em escala laboratorial. Estas duas cepas foram capazes de estabelecer um acelerado processo fermentativo, com eficiente consumo de açúcares e formação de etanol, em comparação ao método natural. As amêndoas de cacau resultantes destes processos foram analisadas por diferentes métodos químicos, incluindo SPME-GC/MS, espectroscopia FTIR e análises de metal e calorimetria. Os resultados indicaram que as fermentações inoculadas desenvolveram amêndoas de cacau com melhor cor e composição de aroma, sugerindo que a diversidade de levedura em fermentações de cacau pode ser explorada para a modulação do flavor de chocolate.

L’hydroperoxyde lyase (HPL) est une enzyme issue de la voie de la lipoxygénase, voie métabolique très représentée chez les végétaux, impliquée dans la production de composés aromatisants (l’hexanal, le 3Z-hexenal et le 2E-hexenal). Ces composés sont responsables de l’odeur fraîche de l’herbe coupée dite « note verte » et sont très utilisés par les industries cosmétiques et agroalimentaires. Leur biosynthèse résulte de l’oxydation des acides gras polyinsaturés en hydroperoxydes par la lipoxygénase, puis de leur clivage par l’hydroperoxyde lyase (HPL). Les procédés actuels de production de ces composés présentent certains inconvénients, ils sont notamment très polluants et peu performants, aussi l’utilisation d’enzymes recombinantes dans de tels procédés permettrait d’obtenir ces molécules de manière plus efficace tout en bénéficiant du label "naturel". L’ADNc codant pour l’hydroperoxyde lyase (HPLwt) a été isolé au laboratoire à partir d’olives noires. Afin d’améliorer la solubilité de l’enzyme, une HPL dépourvue de son peptide de transit chloroplastique (HPLdel) a également été produite. Les deux enzymes ont été exprimées chez E.coli, purifiées par chromatographie d’affinité puis caractérisées biochimiquement. Elles agissent exclusivement sur les 13-hydroperoxydes (13-HPL) à un pH et une température optimum de 7,5 et 25°C. De plus l’évaluation des paramètres cinétiques de l’enzyme montre qu’elles ont une meilleure efficacité catalytique (kcat/Km) sur les 13-hydroperoxydes d’acide linolénique (3,68 s-1.µM-1) que sur les 13-hydroperoxydes d’acide linoléique (0,54 s-1.µM-1). La bioconversion des 13-hydroperoxydes d’acide linoléique et linolénique en hexanal et 3Z-hexénal par l’action de l’HPLwt et l’HPLdel a été étudiée. Des taux de conversion maximum atteignant 93 % et 68 % pour la production d’hexanal et 73 % et 45% pour la production d’3Z-hexénal ont été obtenus quand l’HPLwt et l’HPLdel sont utilisées respectivement. La stabilité de l’enzyme a ensuite été étudiée. Des essais de conservation montrent que l’utilisation de glycérol à 10% (v/v) permet le maintien de la totalité de l’activité de l’HPLwt et de l’HPLdel durant cinq semaines de stockage à -80°C. De plus, l’ajout de composés chimiques tels que le KCl, le NaCl, le Na2SO4, la glycine et le glycérol permettent d’augmenter l’activité enzymatique des deux enzymes et d’améliorer les conditions de synthèse de l’hexanal et du 3Z-hexénal en diminuant la quantité d’enzyme nécessaire à leur production. / The hydroperoxide lyase (HPL) derives from a metabolic pathway named lipoxygenase pathway widely represented in plants and involved in the production of flavoring compounds (hexanal, 3Z-hexenal and 2E-hexenal). These volatile compounds are responsible for the fresh odor of cut grass known as "green note" and have a particularly interest for flavor and food industries. Their biosynthesis results from the oxygenation of linoleic and linolenic acids by lipoxygenase action to form fatty acid hydroperoxides, then of their cleavage by hydroperoxide lyase action. The processes of production currently used are highly polluting or lead to a low yield. To overcome these drawbacks, the use of recombinant enzymes in such processes constitutes an attractive alternative because they would allow producing these molecules in a more effective way, while benefiting from the "natural" label.A cDNA encoding for HPL (HPLwt) from black olive fruit was isolated, and in order to improve the enzyme solubility, the HPL deleted of its chloroplast transit peptide (HPLdel) was then produced. Both enzymes were expressed into E. coli (M15), purified by affinity chromatography, and characterized. They act exclusively on 13-hydroperoxide (13-HPL) and display an optimum pH at 7.5 and an optimum temperature at 25 °C. The bioconversion of 13-hydroperoxides of linoleic and linolenic acids in hexanal and 3Z-hexenal respectively, using HPLwt or HPLdel was studied. Conversion yields reach a maximum of 93 % and 68 % for hexanal production, and 73 % and 45 % for 3Z-hexenal when reactions were performed by HPLwt and HPLdel respectively.The enzyme stability was then studied. Conservations tests using 10 % glycerol (v/v) allows the maintenance of the entire activity of HPLwt and HPLdel during five weeks of storage at -80°C. Furthermore, the addition of chemical compounds such as KCl, NaCl, Na2SO4, glycine, and glycerol can increase the efficiency of both enzymes and improve the synthesis of hexanal and 3Z-hexenal by decreasing the amount of enzyme required to produce them.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2015-09-09T23:59:26Z No. of bitstreams: 1 TESE - VERS?O FINAL - HILANA SALETE SILVA OLIVEIRA.pdf: 2650992 bytes, checksum: c49b570e274076a6845058a800581217 (MD5) / Made available in DSpace on 2015-09-09T23:59:26Z (GMT). No. of bitstreams: 1 TESE - VERS?O FINAL - HILANA SALETE SILVA OLIVEIRA.pdf: 2650992 bytes, checksum: c49b570e274076a6845058a800581217 (MD5) Previous issue date: 2015-06-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / A recurring problem in the chocolate industry is the low quality of cocoa beans, which influences the chocolate aroma. One possibility to contribute to solve this problem is the application of commercial exogenous enzymes on slate. This work proposed detection and sequencing, by mass spectrometry, of the peptides present in slates subjected to treatment with microbial enzyme proposed by Oliveira et al. (2011). Associated to the analysis of peptides was studied the concentration of phenolic compounds and antioxidant activity since the reduction of phenolics in cocoa processing steps is known to cause reduction of astringency and bitterness contributing to the formation of important compounds for the chocolate aroma. The results indicate that although the qualitative standard of the hydrophobic amino acids present in the peptides responsible for the mixture of chocolate aroma is more important than the quantitative standard, the presence of the amino acids leucine, alanine, phenylalanine, and tyrosine in appropriate amounts is essential for obtaining desired aroma. The proposed treatment caused a reduction in the phenolics concentration, which is desirable for a chocolate aroma, and in antioxidant activity. In general, the profile peptide, the phenolic content and antioxidant activity of treated slate resembled more good quality almonds suggesting the effectiveness of the proposed treatment. Research the market and trends indicated favorable conditions for diffusion of the proposed treatment in the process industries. / Um problema recorrente na ind?stria de chocolate ? a baixa qualidade das am?ndoas de cacau, o que influencia o aroma do chocolate. Uma forma de contribuir para solucionar este problema ? a aplica??o de enzimas ex?genas comerciais sobre a ard?sia. Esse trabalho prop?s a detec??o e sequencimento, por espectrometria de massas, dos pept?deos presentes em ard?sia submetidas ao tratamento com enzima microbiana proposto por Oliveira et al. (2011). Associado ?s an?lises de pept?deos, foi feito um estudo da concentra??o de fen?licos e atividade antioxidante, uma vez que a redu??o de fen?licos nas etapas de processamento do cacau ? conhecida por causar redu??o da adstring?ncia e amargor, contribuindo para a forma??o de compostos importantes para o aroma de chocolate. Os resultados indicam que embora o padr?o qualitativo dos amino?cidos hidrof?bicos presentes na mistura de pept?deos respons?veis pelo aroma de chocolate seja mais importante que o padr?o quantitativo, a presen?a dos amino?cidos leucina, alanina, fenilalanina e tirosina em propor??es adequadas ? essencial para a obten??o do aroma desejado. O tratamento proposto causou redu??o na concentra??o de fen?licos, o que ? desej?vel para o aroma de chocolate, e na atividade antioxidante. De modo geral, o perfil de pept?deos, o teor de fen?licos e a atividade antioxidante de ard?sia tratada foi muito mais semelhante ao das am?ndoas de boa qualidade sugerindo a efici?ncia do tratamento proposto. Pesquisas de mercado e tend?ncias indicaram condi??es interessantes ? difus?o do tratamento proposto nas ind?strias de processamento.

La mise au point d’une méthode d’analyse par UPLC-MS/MS et dilution isotopique nous a permis d’identifier pour la première fois dans un moût le S-3-(hexan-1-ol)-<gamma>-glutamyl-cystéine (<gamma>GluCys-3MH). Suite à cette découverte, l’étude des interconversions entre les glutathionylés, dipeptides et cystéinylés S-conjugués au 3-mercaptohexan-1-ol (3MH) ou à la 4-mercapto-4-méthyl-pentan-2-one (4MMP), a permis de mettre en évidence deux nouvelles formes de précurseurs. Le suivi de l’évolution de ces différents composés au cours de fermentations alcooliques à l’échelle laboratoire a permis de clarifier leur rôle de précurseurs et d’établir les éventuels mécanismes d’interconversion. Par ailleurs, à travers plusieurs expérimentations viticoles et œnologiques conduites à l’échelle pilote en Languedoc et Val de Loire pendant trois années consécutives, des éléments nouveaux ont pu être apportés quant à l’impact de la maturation du raisin et son hétérogénéité, ainsi qu’à l’importance des opérations pré-fermentaires comme la macération pelliculaire ou la stabulation sur bourbes. / The development of a method using UPLC-MS/MS and stable isotope dilution assay allowed us to identify for the first time the S-3-(hexan-1-ol)-<gamma>-glutamyl-cysteine (<gamma>GluCys-3MH) in musts. Following this discovery, the study of interconversions between glutathione, dipeptides and cysteine S-conjugates to 3-mercaptohexan-1-ol (3MH) or 4-mercapto-4-methyl-pentan-2-one (4MMP) highlighted two new forms of precursors. The evolution monitoring of these compounds during alcoholic fermentations at laboratory scale allowed us to clarify their role as precursors and to establish the possible interconversion mechanisms. In addition, through several viticultural and oenological experiments carried out at pilot scale in Languedoc and Val de Loire during three consecutive years, new elements could be … about the impact of grape ripening and its heterogeneity, as well as the importance of prefermentative processes, such as skin maceration and must conservation on lees.

gamma-Lactones are found in a wide variety of food and beverage products, in particular grapes and wine. This thesis details the work completed on some gamma-lactones in wine: their synthetic preparation, development of quantification methodologies and sensory studies. Chapter 1 outlines the history of the Australian wine industry from the arrival of the first vines on the First Fleet in 1788 with Captain Arthur Philip. This chapter provides: an overview of Australia’s position in the world of grape and wine production; an analysis of the export arm of the industry; and a look at the different wine producing regions around the country. The latter part of the chapter focuses on the different volatile compounds found in wine. Part A: Chapter 2 provides an overview on the history of barrel manufacture and the use of oak wood in cooperage, with an emphasis on oak’s well known ability to impart desirable characteristics to wine through the extraction of volatile aroma compounds. This chapter provides a summary of these odorants with a particular emphasis on the oak lactones. Previous sensory studies and synthetic work are discussed. Of great importance to this work are the recent advancements in 1,2-dioxine chemistry, highlighted in this chapter. Chapter 3 details the synthetic work completed for the preparation of all four possible oak lactone stereoisomers. A suitably substituted racemic 1,2-dioxine featured as the common intermediate and enabled preparation of the gamma-lactone moiety upon reaction with a chiral malonate diester and separation of the diastereomers by column chromatography. A key step involved the decarboxylation of the ester cleaved gamma-lactone diastereomers, which could be directed to give either the cis- or trans-products. Standard chemical transformations were then utilised to produce the desired stereoisomers of oak lactone. Chapter 4 describes the results from the sensory studies that were completed on the synthetic oak lactone samples. Odour detection thresholds were measured in both a white and a red wine. The thresholds in the former medium were calculated to be 24 ug/L, 172 ug/L, 132 ug/L and 305 ug/L, while in the latter medium the thresholds were calculated to be 57 ug/L, 380 ug/L, 175 ug/L and 285 ug/L, for (4S,5S)-cis-, (4S,5R)-trans-, (4R,5R)-cis- and (4R,5S)-trans-oak lactone, respectively. Difference testings were completed on the pairs of enantiomers and also on mixtures of the nature-identical isomers: between the cis-enantiomers a significant difference was found at the 99% confidence level, while between the trans-enantiomers and also the mixtures of cis- and trans-isomers little difference was observed. Chapter 5 contains the experimental procedures for Part A. Part B: Chapter 6 discusses the sensory properties of some gamma- and delta-lactones, with the focus on a series of five-alkyl substituted gamma-lactones: gamma-octalactone, gamma-nonalactone, gamma-decalactone and gamma-dodecalactone. Topics covered in this chapter include chirality, biosynthetic pathways and quantification results in wine from previous studies for these gamma-lactones. Chapter 7 concerns the method development for the quantification of gamma-lactones in wine using a stable isotope dilution assay (SIDA). Deuterated analogues were prepared from commercially available racemic gamma-lactones for use as internal standards. Initially a head space solid-phase microextraction (HS SPME) method was developed using d5-standards; however, analysis of bottled wine samples revealed the presence of co-eluting compounds that contained several of the selected ions. Thus an alternative method was developed using d7-standards, with a specific focus on sample clean-up, via solid-phase extraction (SPE). Using this procedure, 44 white and 120 red wines were analysed for their gamma-lactone content. The lactones were found to be significantly more common in the red wines, with gamma-nonalactone the most abundant lactone in this series. Chapter 8 deals with the extension of the SIDA method, as developed in Chapter 7, for use with a chiral gas chromatography column. Optically pure standards were prepared, from either L- or D-glutamic acid, and used to determine the order of elution of the enantiomers. A method was developed for the quantification of the individual enantiomers of gamma-octalactone, gamma-nonalactone, gamma-decalactone and gamma-dodecalactone. The enantiomeric distribution of gamma-nonalactone was investigated in 34 red wines; the (R)-stereoisomer was found to be dominant with an average of 59%, although there were wines analysed that did contain the (S)-stereoisomer in greater amounts. Chapter 9 describes the results from the sensory studies that were completed on the individual enantiomers of the gamma-lactones. Odour detection thresholds were measured in a red wine. The thresholds were calculated to be 238 ug/L, 285 ug/L, 34 ug/L and 8 ug/L for the (R)-enantiomers, while the thresholds were calculated to be 135 ug/L, 91 ug/L, 47 ug/L and 39 ug/L for the (S)-enantiomers, of gamma-octalactone, gamma-nonalactone, gamma-decalactone and gamma-dodecalactone, respectively. Chapter 10 contains the experimental procedures for Part B. Chapter 11 contains the appendices, followed by the references in Chapter 12.