Orientador: Gabriela Alves Macedo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-23T15:45:42Z (GMT). No. of bitstreams: 1 Lopes_DanielleBranta_D.pdf: 3780152 bytes, checksum: ba7b4a54629ff27a24abae14e788d60c (MD5) Previous issue date: 2013 / Resumo: Os aromas despertam grande interesse entre consumidores e indústrias de alimentos, especialmente por estarem diretamente relacionados ao sabor dos alimentos. Dentre os aromas de maior importância industrial destaca-se o grupo das lactonas, presentes em uma grande variedade de produtos naturais e compostos biologicamente ativos. Em virtude do destaque apresentado por essa classe de substâncias, diversos métodos para sua produção têm sido relatados. Como alternativa aos processos químicos tradicionais, propõe-se nesta pesquisa a utilização da biotecnologia de micro-organismos e enzimas para a produção de compostos aromáticos a partir de substratos naturais, como óleos vegetais hidrolisados.A fermentação microbiana é considerada um caminho potencial para a produção de aromas naturais, sendo o grupo formado pelos fungos um dos mais utilizados para esse fim. A biocatálise também é uma alternativa muito vantajosa e capaz de catalisar um grande número de reações estéreo- e regiosseletivas, o que não é alcançado por meio de síntese química clássica. O uso de enzimas isoladas torna-se preferível em relação ao uso de micro-organismos quando existem limitações relacionadas à permeabilidade do substrato na membrana da célula ou quando ocorrem reações secundárias indesejáveis.Considerando-se os argumentos citados, neste trabalho foram estudados dois métodos para a produção de lactonas. O primeiro deles, a biotransformação, foi feita a partir do uso de micro-organismos para a produção dos compostos em questão. O segundo método, a biocatálise, foi realizado através da reação de lactonização com o uso de lipases microbianas brutas isoladas e liofilizadas. Os substratos utilizados no estudo foram os óleos vegetais de mamona e linhaça previamente hidrolisados por lipases fúngicas produzidas por Rhizopus sp. e Geotrichum sp. A produção de lactonas foi alcançada em ambos os métodos. Com o uso da biotransformação, todos os micro-organismos testados (Geotrichum sp., Rhizopus sp., Aspergillus sp. (Linhagens 1068 e 1099) e Fusarium oxysporum) foram capazes de produzir diferentes tipos de lactonas, com destaque para o fungo Fusarium oxysporum, que produziu maior variedade delas (&61543;-decalactona, 2-cumaranona, mevalonolactona e pantolactona), além de alcançar o maior rendimento na produção de &61543;-decalactona (18,93 &956;L L-1), após 48 horas de reação e empregando-se os óleos de mamona e linhaça previamente hidrolisados pela lipase produzida pelo fungo Rhizopus sp.. Através do uso da biocatálise, ótimos resultados também foram alcançados, e a produção de lactonas foi possível ao se utilizar as lipases produzidas pelos fungos Rhizopus sp., Aspergillus sp. (Linhagem 1068) e Fusarium oxysporum, sendo possível a produção de &61543;-undecalactona, &61543;-decalactona e &61543;-dodecalactona, dependendo da lipase empregada e do tempo de reação. A maior produtividade foi obtida na produção de &61543;-undecalactona ao se utilizar a lipase produzida pelo fungo Rhizopus sp. após 24 horas de reação, obtendo-se um rendimento de 21,78 &956;L L-1, também a partir do uso dos óleos de mamona e linhaça hidrolisados previamente pela lipase produzida por Rhizopus sp. As lipases produzidas pelos fungos Geotrichum sp. e Aspergillus sp. (Linhagem 1099), também estudadas neste trabalho, não foram capazes de produzir lactonas / Abstract: Aromas arouse great interest among consumers and food industry, especially because heir direct relationship with food taste. In the midst of the most important industrial flavorings stands lactones group, which are present in a wide variety of natural products and biologically active compounds. Due to the prominence of this class of substances, several methods for the production of lactones have been reported. As an alternative to traditional chemical processes, it is proposed in this research the use of microbial and enzyme biotechnology for the production of aromatic compounds natural substrates, such as hydrolyzed vegetable oils. The microbial fermentation is considered a potential pathway for the production of natural flavor, and fungi group is the most widely used for this purpose. Biocatalysis is also a very useful alternative and is able to catalyze a large number of stereo- and regioive reactions, which is not achieved by classical chemical synthesis. The use of isolated enzymes is preferable over the use of microorganisms when there are limitations concerning the permeability of the substrate in the cell membrane or when undesired side reactions occur. Considering these arguments, two methods were investigated in this work for the production of lactones. The first one, biotransformation, was made the use of microorganisms for the production of these compounds. The second method, biocatalysis, was performed by lactonization reaction using crude microbial lipases that was isolated and lyophilized. Substrates used were the vegetable castor and linseed oils previously hydrolyzed by fungal lipases produced by Rhizopus sp. and Geotrichum sp. Lactones production was achieved in both methods. Using biotransformation, all microorganisms tested (Geotrichum sp., Rhizopus sp., Aspergillus sp. (Strains 1068 and 1099) and Fusarium oxysporum) were able to produce different lactone types, especially using the fungus Fusarium oxysporum, which produced a greater variety (&61543;-decalactone, 2-cumaranone, mevalonolactone and pantolactone), achieving the best performance in y-x decalactone production (18.93 &956;L L-1), after 48 h employing castor and linseed oils hydrolyzed by lipase Rhizopus sp. Through the biocatalysis use, excellent results were also obtained, and the lactones production was possible using lipases produced by fungi Rhizopus sp., Aspergillus sp. (Strain 1068) and Fusarium oxysporum. The production of y-undecalactone, y-decalactone and y-dodecalactone was achieved depending on the lipase employed and the reaction time used. Highest yield was obtained in the &61543;-undecalactone production (21.78 &956;L L-1), using castor and linseed oils hydrolyzed by Rhizopus sp. lipase. Lipases produced by Geotrichum sp. and Aspergillus sp. (Strain 1099) was also studied in this work, but were unable to produce lactones / Doutorado / Ciência de Alimentos / Doutora em Ciência de Alimentos

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: The varietal character of Sauvignon blanc wine is mostly defined by the balance between tropical and green vegetative flavour nuances. Grape derived methoxypyrazines are the main aroma contributors towards green vegetative flavours. Methoxypyrazines are heat and light sensitive. Due to warm climatic conditions in South Africa, methoxypyrazine levels decrease during grape ripening. The addition of food flavourings to Sauvignon blanc wine, a practice known as spiking, has occurred in the past to improve the green character of the wines. Adding flavourings to wine and selling the wine as natural certified wine is illegal in South Africa. Currently, adulterated Sauvignon blanc wines are identified using gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) methods to quantify methoxypyrazines and compare levels to an established database. Although of high sensitivity, GC-MS and LCMS methods are costly and time consuming, therefore not optimal for routine screening of wines. Hence the need for the development of a fast and cost effective method for routine screening of large amounts of wines to identify adulteration. Small scale vinification practices were used to prepare experimental Sauvignon blanc wine. Flavourings were added to Sauvignon blanc grape juice before fermentation, during the preparation of experimental spiked wines. Control wines, containing no flavouring, were also prepared. Commercial wines were spiked after fermentation and bottling. Each wine was only spiked with a single flavouring. The flavourings added were the juice of homogenised fresh green peppers and commercially available flavourings for wine. The following commercial flavourings were used: green pepper, asparagus, grassy and tropical. The above mentioned wines were analyzed using Fourier transform infrared (FT-IR) spectroscopy, GC-MS, LC-MS and descriptive sensory analysis. The FT-IR techniques used were Fourier transform mid infrared (FT-MIR) transmission, FT-MIR attenuated reflection and Fourier transform near infrared (FT-NIR) reflection spectroscopy. The data was interpreted using the following multivariate statistical techniques: principal component analysis (PCA), partial least squares discrimination (PLS-D) and conformity testing. Multivariate models constructed from FT-MIR and FT-NIR data were able to discriminate between spiked and control wines. Sensory analysis results clearly showed differences between non-spiked wines and spiked wines with 3-isobutyl-2-methoxypyrazine concentrations 10 times higher than naturally occurring in wine. Differences between control and spiked wines with concentrations of 3-isobutyl-2-methoxypyrazine similar to concentrations naturally occurring in wines could not be detected to prove adulteration conducting sensory analysis. However, differences between control and spiked wines with levels of 3-isobutyl-2-methoxypyrazine similar to levels naturally occurring in wines could be detected using FT-IR data in conjunction with multivariate statistics. This study showed that, FT-IR spectroscopy in conjunction with multivariate statistical methods can be a possibility for the screening and identification of wines suspected of adulteration in terms of added flavourings. Descriptive sensory analysis also proved to be a potentially useful tool. However screening and training of potential panel members are time consuming. / AFRIKAANSE OPSOMMING: Die variëteitskarakter van Sauvignon blanc wyn word grotendeels gedefinieer deur die balans tussen tropiese en groen vegetatiewe aromas. Metoksipirasiene is die hoof aroma verbindings wat verantwoordelik is vir groen vegetatiewe aromas. Metoksipirasien is hitte- en ligsensitief. Warm klimaatsomstandighede in Suid-Afrika het tot gevolg dat metoksipirasien konsentrasies daal tydens druif rypwording. Sauvignon blanc wyne is in die verlede vervals deur middel van die byvoeging van vars groen soetrissies om die groen vegetatiewe karaktereienskappe van die wyne te bevorder. Die byvoeging van geurmiddels of plantekstrakte by wyn en verkoop van daardie wyn as gesertifiseerde natuurlike wyn is onwettig in Suid-Afrika. Tans word vervalsde wyne met behulp van gaschromatografie-massaspektrometrie (GC-MS) en vloeistofchromatografie-massaspektrometrie (LC-MS) opgespoor. Kwantifisering van metoksiepirasien konsentrasies in wyne en druiwesappe word vergelyk met konsentrasies in ‘n bestaande databasis. Alhoewel GC-MS en LC-MS hoë sensitiwiteitsmetodes is, is dit duur en tydrowende metodes, dus nie optimaal vir roetine sifting nie. Dus word ‘n koste- en tydseffektiewe roetine metode benodig om vervalsing van wyne op te spoor. Eksperimentele wyne is op klein skaal berei. Geurmiddels is voor fermentasie by die druiwesap gevoeg. Kontrole wyne waarby geen geurmiddels gevoeg is nie, is ook berei. Kommersiële wyne is gegeur na fermentasie en bottelering. Elke wyn is met ‘n enkele geurmiddel gegeur. Gehomogeniseerde vars groen soetrissie asook kommersieel beskikbare geursels vir wyn is gebruik. Die volgende kommersiële geursels is gebruik: groen soetrissie, aspersie, gras en tropiese geursel. Die volgende analitiese tegnieke is gebruik vir analisering van bogenoemde wyne: Fourier transformasie infrarooi (FT-IR) spektroskopie, GC-MS, LC-MS en beskrywende sensoriese analise. Die spesifieke FT-IR tegnieke wat gebruik is, is: Fourier transformasie mid-infrarooi (FT-MIR) transmissie, FT-MIR verswakte weerskaatsing en Fourier transformasie naby-infrarooi (FT-NIR) reflektansie. Die volgende multiveranderlike statistiese tegnieke is gebruik ter interpretasie van data: hoof komponent analise (PCA), parsiële kleinste kwadraat diskriminant analise (PLS-D) en gelykvormigheidstoetsing. Multiveranderlike modelle, bereken met behulp van FT-MIR en FT-NIR data, kon diskrimineer tussen gegeurde en kontrole wyne. Resultate wat verkry is tydens sensoriese analises het duidelike verskille uitgewys tussen gegeurde en kontrole wyne met betrekking tot 3-isobutiel-2- metoksipirasien konsetrasies waar 3-isobutiel-2-metoksipirasien konsentrasies 10 keer hoër was as wat natuurlik voorkom in wyn. Geen beduidende verskille kon waargeneem word in gevalle waar wyne vervals is met laer konsentrasies van geurmiddels deur sensoriese data te ontleed nie. Nietemin, statisitiese verskille tussen kontrole en vervalsde wyne kon waargeneem word vir lae-konsentrasie-geurmiddel vervalsde wyne deur FT-IR data met behulp van multiveranderlike statisitiese metodes te ontleed. Hierdie studie het gewys dat FT-IR in kombinasie met multiveranderlike statistiese tegnieke spesifiek hoof komponent analise (PCA) en parsiële kleinste kwadraat diskriminant analise (PLS-D) asook gelykvormigheidstoetsing bruikbare tegnieke is om te onderskei tussen kontrole (egte natuurlike) en vervalsde wyne ten opsigte van die byvoeging van geurmiddels. Beskrywende sensoriese analise kan ook nuttig gebruik word, alhoewel keuring en opleiding van paneellede tydrowend is.

Depuis quelques décennies, la communauté scientifique s'intéresse de plus en plus à la recherche de nouvelles levures pour de nouvelles applications dans le domaine des biotechnologies blanches. De l'étude de la biodiversité à l'étude des propriétés métaboliques, les recherches visant à sélectionner de nouvelles levures avec des propriétés intéressantes pour l'industrie se sont intensifiées. Localisées dans le Sud-Ouest de l'océan Indien, les îles de La Réunion et de Madagascar sont aujourd'hui considérées comme des Hot Spot de la biodiversité. La grande diversité de plantes et de fruits tropicaux fournis des habitats naturels pour la propagation de nombreux microorganismes tels que les levures. A l'instar de ce qui se passe chez les plantes endémiques, nous avons formulé l'hypothèse que certains microorganismes ne peuvent être trouvés nulle part ailleurs que dans ces îles tropicales. 101 souches de levures réparties en 26 espèces différentes ont ainsi été isolées dans cette étude à partir de 13 fruits tropicaux récoltés aléatoirement dans les régions d'Antsirabe à Madagascar et de St-Paul à La Réunion. L'identification des espèces a été réalisée par analyse des séquences des domaines D1/D2 de la grande sous-unité des gènes de l'ARN ribosomique (ARNr). Les souches caractéristiques ont ensuite fait l'objet d’une analyse systématique par micro-extraction en phase solide couplée à une analyse chromatographique en phase gazeuse et couplé à un spectromètre de masse (ET-MEPS-CPG/SM) pour déterminer leurs capacités à produire des arômes. Parmi elles, deux souches isolées sur le fruit du poc-poc (Physalis peruviana) et sur le cacao (Theobroma cacao var. Criollo) semblent appartenir à de nouvelles espèces, probablement endémiques de la zone géographique étudiée. En effet, ces souches, EGPOC17 et EB23, présentent un faible pourcentage d'identité avec les séquences de l'ARNr des levures Rhodotorula mucilaginosa (97,1% d'identité) et Candida pararugosa (97,4% d'identité). 52 composés organiques volatiles (COVs) différents, classés en cinq grandes classes de molécules, les acides, les alcools, les aldéhydes, les cétones et les esters ont ainsi été identifiés. Une analyse statistique a ensuite été réalisée pour tenter de classer ces souches par catégories en fonction de leurs caractéristiques métaboliques. Avec une production de 32 molécules différentes sur les 52 identifiées, la levure Saprochaete suaveolens (anciennement connue sous le nom de Geotrichum fragrans) est de loin la souche produisant le plus grand nombre de composés d'arômes, en particulier d'esters et de composés insaturés. D'autres souches comme Candida quercitrusa, Debaryomyces nepalensis, Pichia kluyveri et Sporidiobolus pararoseus semblent également être intéressantes pour la production d'acides, d'alcools et de cétones. Parmi les COVs identifiés, des composés comme le 2 méthylbut-2-énoate d'éthyle (tiglate d'éthyle), le 2-méthylbut-2-énoate de 3 méthylbutyle (tiglate d'isoamyle), le 2-méthylbut-2-énoate de butyle (tiglate de butyle), 3-méthylbut-2-énoate d'éthyle, 2-méthylbut-2-énoate de 2-méthylpropyle et le but-2-énoate d'éthyle ont été retrouvés spécifiquement chez la levure Saprochaete suaveolens. La présence de ces molécules, très peu décrites chez les levures dans la littérature, suggère des relations taxonomiques de type «arôme-espèce» ou «arôme-genre » chez les levures. / In recent years, there has been an increasing interest in identifying and characterizing the yeast ecosystems associated with diverse types of habitat because of the many potential desirable technological properties of these microorganisms, especially in food applications. In this study, a total of 101 yeast strains were isolated directly from the skins of tropical fruits collected in several locations in the South West Indian Ocean (in the regions of Anstirabe in Madagascar and Saint Paul in Reunion Island). Species identification was determined by sequence analysis of the D1/D2 domains of the large subunit (LSU) ribosomal RNA gene. The strains were classified into 26 different species and tested for their potential to produce aromatic flavouring compounds. Among the isolated strains, two species isolated from the skins of Cape gooseberry and Cocoa beans appeared to represent putative new yeast species. Strains EGPOC17 and EB23 showed LSU D1/D2 sequence homologies of only 97.1% and 97.4% with the yeasts Rhodotorula mucilaginosa and Candida pararugosa, respectively. In total, 52 Volatile Organic Compounds (VOCs) were detected by Head Space Solid Phase Micro Extraction coupled to Gas Chromatography and Mass Spectroscopy (HS-SPME-GC/MS) analysis and these were classified into five main groups, namely, acids, alcohols, aldehydes, ketones and esters. In order to classify and discriminate the yeast biodiversity, statistical analysis was performed which allowed the yeasts to be categorized according to their flavour production. With a production of 32 compounds among 52 VOCs, Saprochaete suaveolens (Geotrichum fragrans) seemed to be the best producer of flavour compounds, especially esters and unsaturated compounds. Other yeast species including Candida quercitrusa, Debaryomyces nepalensis, Pichia kluyveri and Sporidiobolus pararoseus also appeared of potential interest based on their abilities to produce acid, alcohol and carbonyl compounds. Among the VOCs detected, 6 uncommon compounds namely ethyl but-2-enoate, ethyl 2-methylbut-2-enoate (ethyl tiglate), ethyl 3-methylbut-2-enoate, 2-methylpropyl 2-methylbut-2-enoate, butyl 2-methylbut-2-enoate and 3 methylbutyl 2-methylbut-2-enoate were identified as possible yeast species specific flavour markers.