Development of an integrated process for continuous winemaking

Tese de Doutoramento em Engenharia Química e BiológicaMicroorganisms play an important role in the food industry, particularly in the processing of foods and beverages. In wine production, two consecutive fermentation processes occurs, involving yeasts (alcoholic fermentation) and lactic acid bacteria (malolactic fermentation), its control is being crucial to obtain high quality products. Considering that the raw material used in wine production is seasonal these two fermentations are conducted in batch mode. The advances made in cell immobilization techniques in the last decade, led to further exploration of these techniques for continuous fermentation processes. This work aims at the implementation of a continuous wine production process by the use of sequential continuous fermentations with immobilized cells, in the different stages of the fermentative process of winemaking, i.e. in alcoholic fermentation and in malolactic fermentation. For this purpose, separate immobilization of commercial strains of Saccharomyces cerevisiae and Oenococcus oeni were carried out by adsorption on different natural waste materials such as: grape skins, grape stems, grape seeds and corn cobs. Following preliminary batch trials the best immobilized cell systems were studied in the corresponding packed bed reactors operated in continuous mode. The influence of the operating parameters of the system over the cell physiology and quality of the final product - physicochemical and sensory characteristics (colour, aroma and taste) - was evaluated. The operational stability of the immobilized systems was also characterized. Fermentations with immobilized cells were much faster than fermentations with free cells and the quality of the product was maintained, making the process more advantageous and economical. Also, the uses of natural supports that are agro-industrial residues make the process more environmentally friendly. The quality of the wines produced using immobilized cells was confirmed by sensory evaluation as it was difficult to distinguish between wines produced by the different methods. Volatile aroma compounds evaluation confirmed the good quality of wines produced with immobilized cells. Initially, wines produced with immobilized cells presented a darker colour compared with wines produced by the traditional free cells method. However, with time and with the reuse of the immobilized cell system the colour stabilized and wines became similar to the wines produced with free cells. The used immobilized cell systems were found to have strong operational and mechanical stabilities. Moreover it was found that immobilized cells can be stored for at least 1 month at 4 °C and used again in fermentation processes of winemaking, as confirmed by its successful application in continuous alcoholic and malolactic fermentation processes. Overall, the integrated continuous process of winemaking demonstrated to have good operational stability making it a valuable alternative for the production of good quality wines.Os microrganismos desempenham um papel relevante na indústria alimentar, nomeadamente no processamento de alimentos e de bebidas. No caso particular da produção de vinho, ocorrem dois processos fermentativos, onde intervêm leveduras (fermentação alcoólica) e bactérias lácticas (fermentação maloláctica), cujo controlo é crucial para obtenção de produtos de qualidade. Devido à sazonalidade da matéria-prima, estas duas fermentações são tradicionalmente conduzidas em descontínuo. Nos últimos, os avanços conseguidos nas técnicas de imobilização celular e o uso de suportes passíveis de contacto com produtos alimentares, conduziu a uma maior exploração de processos fermentativos em contínuo. Este trabalho teve por finalidade a implementação, em contínuo, com células imobilizadas, das etapas fermentativas de um processo de vinificação, i.e. a fermentação alcoólica e a fermentação maloláctica. Para o efeito foi ensaiada a imobilização de culturas comerciais de Saccharomyces cerevisiae e de Oenococcus oeni, por adsorção natural em suportes resultantes de resíduos da indústria agroalimentar: películas de uva, bagaço de uva e carolo de milho. Após operação em modo descontínuo as melhores combinações célula-suporte foram estudadas em reator empacotado. A qualidade final do produto, quer em termos físico-químicos quer em termos organolépticos (aspeto, gosto e aroma), depende não só da qualidade da uva utilizada, que depende da casta, do terroir e do ano de vindima, mas também dos procedimentos adotados durante todo o processo de vinificação. Sendo assim foi avaliada a influência dos parâmetros de operação do sistema sobre a fisiologia celular e a qualidade do produto final. Foi também avaliada a estabilidade operacional dos sistemas imobilizados. As fermentações conduzidas com células imobilizadas foram muito mais rápidas do que fermentações com células livres, tornando o processo mais vantajoso e económico. Assim também o uso dos resíduos agroindustriais torna o processo mais ambientalmente amigável. A avaliação sensorial dos vinhos produzidos com células imobilizadas e com células livres demonstrou que é difícil distingui-los. A avaliação aromática dos vinhos elaborados com células imobilizadas demonstrou que estes vinhos são de boa qualidade. Inicialmente os vinhos produzidos com células imobilizadas apresentaram uma cor mais escura comparados com os vinhos produzidos pelo método tradicional com células livres. No entanto, com o tempo e com a reutilização dos sistemas imobilizadas a cor tende a estabilizar e torna-se semelhante aos dos vinhos produzidos com células livres. Os sistemas de células imobilizadas evidenciaram estabilidades operacionais e mecânicas. Além disso, verificou-se que as células imobilizadas podem ser armazenadas durante pelo menos 1 mês a 4 °C e utilizadas novamente nos processos fermentativos de vinificação. Os processos de fermentação em contínuo (alcoólica e maloláctica) com células imobilizadas, foram bem sucedidos e bem conduzidos. O processo integrado de vinificação em contínuo demonstrou uma estabilidade operacional eficaz. Os resultados obtidos foram considerados promissores para fomentar estudos futuros

Caracterização aromática varietal das castas brancas recomendadas para a produção de vinho verde

Dissertação de mestrado em Engenharia BiológicaPortugal é um país de longa tradição vinícola. A região demarcada dos Vinhos Verdes é uma região que pelas suas características naturais de solo e clima, pelo aspecto sócio-económico e agrotécnico da sua agricultura e pelas pressões demográficas que tem sofrido ao longo dos tempos produz um vinho ímpar no mundo – o Vinho Verde. O Vinho Verde é um vinho com características únicas, devidas essencialmente ao meio geográfico, tendo em conta os factores naturais e humanos que estão na sua origem. Podem-se classificar os aromas do vinho em três grupos: o primário é, o secundário e o terciário. Os aromas primários ou varietais são os aromas da uva, que se encontram no mosto e passam para os vinhos. Estes aromas são muito frágeis e manifestam-se principalmente em vinhos brancos muito jovens e frescos. No caso dos Vinhos Verdes, uma vez que normalmente se pretende realçar as qualidades da casta de origem, é a componente varietal que assume maior importância com vista à sua elaboração. Neste trabalho pretende-se estudar a composição aromática varietal, das sete castas recomendadas para a Região Demarcada dos Vinhos Verdes, que são as seguintes: Alvarinho, Arinto, Avesso, Azal, Batoca, Loureiro e Trajadura. Este estudo assume importância, tanto no que se refere à sua caracterização e diferenciação como no que diz respeito ao conhecimento mais aprofundado da matéria-prima, com vista ao melhor aproveitamento tecnológico, além disso, o facto de poderem existir compostos voláteis capazes de funcionar como marcadores da variedade, a sua caracterização pode assumir papel relevante na autenticação das uvas e dos vinhos. Numa segunda tarefa, semelhante à anterior, pretende-se estudar a possibilidade de discriminar os vinhos obtidos a partir das 7 variedades com base na quantificação de dois álcoois com 6 átomos de carbono ((E)-3-hexenol e (Z)-3-hexenol). O estudo realizou-se para as sete castas da mesma vindima (2005) e uma vez que se trata de uma região Demarcada bastante ampla, torna-se importante fazer este estudo em mais do que um local, neste caso em Arcos de Valdevez e Felgueiras. Ao nível do aroma, todas as variedades apresentam compostos semelhantes, embora os perfils aromáticos baseados nos compostos monoterpénicos, óxidos e diois monoterpénicos e norisoprenódes em C13 sejam diferentes. Os resultados demonstraram que as castas Loureiro, Alvarinho e Arinto podem ser diferenciadas. A casta Loureiro diferencia-se facilmente das outras 6, no que respeita à fracção livre do aroma das uvas, pois o composto mais abundante é o linalol. Assim também foi possível diferenciar a casta Loureiro por razão (E)-3-hexenol/(Z)-3-hexenol.Portugal is a country with a long tradition in winemaking. The demarcated region of Vinhos Verdes with its natural resources sociological and economic aspects, agriculture and demographic peculiarities is producing a wine like no other in the world- the Vinho Verde. The Vinho Verde is a wine with unique characteristics, due to geographic area, together with the natural and human factors that are in his origin. Wine aromas are generically classified into: Primary aromas (deriving from the grape), Secondary aromas (deriving from the fermentation processes) and Tertiary aromas (deriving from the ageing process). Primary aromas are derived from the grape and persist through the winemaking process. Aromas of this type are normally those described as floral or fruity and will gradually decrease with extended ageing of a wine, because of that they are typical characteristic of the young and fresh wines. The aim of this work is to study the aromatic varietal composition of the seven grapes recommended in the Demarcated Region for the production of Vinhos Verdes that are the following: Alvarinho, Arinto, Avesso, Azal, Batoca, Loureiro and Trajadura. The study assume the importance, as much on what refers its characterization and differentiation as on more profound knowledge of the prime material, with the view of a better technological use. Some volatile compounds can have the function of marks for the type of grape variety so the characterization of the aromatic varietal composition can have an important part in the authentication of the grape variety and the wine. The study was made for the seven grape varieties for the year of 2005, from two places Arcos de Valdevez and Felgueiras. In a second part of this work was studied the possibility of differenciation of the wines prodused by this grape varieties on the base of quantification of two alcohols with 6 carbon atoms ((E)-3-hexenol and (Z)-3-hexenol). All the varieties showed similar aromatic compounds, although the aromatic profiles based on the alcohols, monoterpenic alcohols and C13 norisoprenoids were different. By an Analyse of the Principal Components was made the conclusion that the grape varieties Alvarinho, Arinto and Loureiro can be discriminate from the others verieties. The Loureiro variaety was descriminated easily due to its aromatic profile from the free fraction, of the grapes, where the most abundant compound was linalool. The results showed that (E)-3- hexenol/(Z)-3-hexenol ratio clearly discriminate the Loureiro wines

Characterization and differentiation of seven vinhos verdes grape varieties on the basis of monoterpenic and C13-norisoprenoid compounds

The aromatic profiles of the 7 recommended cultivars for Vinho Verde wine production have been established with respect to monoterpenic and C13-norisoprenoids compounds, present either in free or in glycosidically bound fractions. Eighteen monoterpenic compounds have been identified and quantified in the free form and 22 in the glycosidically bound form. Fifteen C13-norisoprenoids compounds have been identified and quantified in the glycosidically bound form. Loureiro variety can be easily differentiating from the 6 other varieties by important levels of linalool in the free fraction, above the odour perception threshold; in contrast, Alvarinho, Avesso, Arinto, Azal and Trajadura, geraniol prevails. Batoca variety showed a very poor monoterpenic profile in the free form. In the glycosidically bound form Alvarinho is the richest in total concentration of monoterpenic compounds, followed by Loureiro.Les profils aromatiques des 7 cépages recommandés pour la production de Vinho Verde ont été établis en ce qui concerne les C13-norisoprenoïdes et les composés monoterpéniques, trouvés dans les deux fractions de l´arôme, livre et glycosillée. Dix-huit monoterpènes ont été identifiés et quantifiées, dans la fraction libre, et d’autres 22 attachés à des glycosides. Quinze C13-norisoprenoïdes ont été identifiés et quantifiés dans la fraction glycosillée. Le cépage Loureiro peut facilement être différencié des autres 6 variétés par les niveaux importants de linalol dans la fraction libre, au-dessus du seuil de perception olfactive; par contre, le geraniol prédomine pour Alvarinho, Avesso, Arinto, Azal et Trajadura. Le cépage Batoca a montré un très pauvre profil monoterpénique dans la fraction libre. Pour la fraction glycosillée, le cépage Alvarinho est le plus riche, en ce qui concerne la concentration totale en composés monoterpéniques ; le Loureiro est le deuxième cépage plus riche.Estação Vitivinícola Amândio GalhanoDivisão de Vitivinicultura e Fruticultura da Direcção Regional de Agricultura de Entre Douro e Minh

First study on volatile composition of three Vitis vinifera cultivars from Betanzos (NW Spain) : blanco lexítimo, agudelo and serradelo

Wine aroma is formed by volatile compounds of different chemical nature and origins. Compounds arising from grape metabolism vary as a function not only of the variety but also of climatic conditions and others. Betanzos is the most northern viticole geographic area from Galicia (NW Spain). Wines produced from Vitis vinifera cv. Blanco lexítimo, Agudelo and Serradelo from Betanzos (NW Spain), harvest in 2006 and 2007, were submitted to gas chromatography (GC/FID). A total of 35 varietal and fermentative aroma compounds were identified and quantified which include terpenes and C13-norisoprenoids, alcohols, acids, esters, phenol volatiles and C6 compounds. The results for quantification of the aroma compounds in the three varieties from Betanzos shows that the mean concentration between years was 25039.42ug/L for Blanco lexítimo 21913.2ug/L for Agudelo and 19804.6μg/L for Serradelo variety. Alcohols, acids and esters were the most important volatile compounds in the varieties from Betanzos.El aroma del vino está formado por compuestos volátiles de diferente naturaleza y origen. Estos compuestos varian no solo con la variedad de uva sino también con las condiciones climáticas y otros factores. Betanzos es la zona vitícola situada más al norte de Galicia (Noroeste de España). Los vinos producidos con tres variedades de Betanzos, Blanco lexítimo, Agudelo y Serradelo cultivadas en las cosechas 2006 y 2007, fueron analizados mediante GC/FID. En los vinos fueron identificados y cuantificados un total de 35 compuestos varietales y fermentativos, en cuales se incluyen C13-norisoprenoides, alcoholes, acidos, esters, fenoles volatiles y otros. Los resultados de la cuantificación de los compuestos del aroma en los vinos de las tres variedades de Betanzos muestra que la concentración mediaentre los años estudiados fue de 25039.42ug/L para Blanco lexítimo 21913.2ug/L para Agudelo y 19804.6μg/L para Serradelo. Alcoholes, acidos and esteres fueron los compuestos más importantes en los tres vinos elaborados con las variedades de Betanzos, siendo el más aromático el elaborado con la variedad Blanco lexítimo, caracterizado por aromas frutales.Xunta de Galicia (Spain) and Adega Lorenzo Bescansa S.U.L

Monoterpenic characterization of white cultivars from Vinhos Verdes appellation of origin (North Portugal)

The monoterpenic composition of Alvarinho, Arinto, Avesso, Azal, Batoca, Loureiro and Trajadura Vitis vinifera cultivars from the Vinhos Verdes region (Portugal), in respect to free volatile compounds as well as glycosidically bound aroma precursors, was determined by GC-MS after adsorption on XAD-2 resin. Eighteen monoterpenic compounds were identified and quantified in the free form and twenty-two compounds in the glycosidically bound form. In the free fraction, the Loureiro variety could be easily differentiated from the other six varieties by the important levels of linalool, above the odor perception threshold. This was in contrast to Alvarinho, Avesso, Arinto, Aza and Trajadura, where geraniol prevailed. The Batoca variety showed a very poor monoterpenic profile in the free form. On the other hand, Alvarinho was the richest variety in respect to the glycosidically bound form, followed by Loureiro. In this fraction, linalool and 3,7-dimethylocta-1,5-dien-3,7-diol were the most abundant compounds in the Loureiro grape cultivar; additionally, the isomer (E)-8-hydroxylinalool was in a higher concentration than (Z)-8-hydroxylinalool (sub-region of Sousa),while for the rest of the varieties the (Z) isomer prevailed.The authors thank the Comissao de Viticultura da Regiao dos Vinhos Verdes - EVAG. Divisao de Vitivinicultura e Fruticultura da Direccao Regional de Agricultura de Entre Douro e Minho - DVF-DRAEDM for providing the grape samples for this study. Thanks to Dr. Vilanova (Mision Biologica de Galicia - CSIC, Spain) and Dr. Solange I. Mussatto (University of Minho) for their help in the revision of the manuscript

Utilização da extração em fase sólida seguida de análise por GC-MS na caracterização aromática de uvas da casta Alvarinho

Uma das características mais significativas dos vinhos é o aroma[1]. De acordo com a sequência biotecnológica empregue na elaboração do vinho, o aroma está geralmente dividido em três grupos: aroma primário, que engloba o varietal e o préfermentativo, aroma secundário ou fermentativo e aroma terciário ou pós-fermentativo[1]. Embora seja elevado o número de constituintes voláteis de um vinho passíveis de contribuir para o seu aroma, a tipicidade aromática é principalmente devida aos compostos que procedem das uvas, isto é os compostos de origem varietal. O aroma varietal é constituído por substâncias voláteis presentes numa forma livre, odorífera, e na forma de precursores glicoconjugados, inodoros; estes precursores são suscetíveis de ser hidrolisados durante todas as etapas, desde a produção à abertura da garrafa, originando compostos voláteis. Vários estudos têm sido desenvolvidos no sentido de verificar quais os compostos voláteis presentes em diversas variedades de uva e de que modo estes compostos permanecem, desaparecem ou se transformam durante a vinificação, a conservação e o envelhecimento dos vinhos. O presente trabalho, inserido no âmbito do projeto AGROCONTROL, co-financiado pelo “ON.2 – O Novo Norte” e QREN através do Fundo Europeu de Desenvolvimento Regional (FEDER), teve como objetivo a identificação e quantificação dos compostos voláteis de amostras de uvas da casta Alvarinho da colheita de 2011[2-4]. As amostras de uvas deram origem a sumos por trituração num liquidificador, dos quais foram extraídos os compostos voláteis na forma livre e na forma de glicoconjugados, por extração em fase sólida (SPE – Solid Phase Extraction), obtendo-se extratos que foram analisados por GC-MS (GasChromatography-Mass Spectrometry). A extração foi realizada pela passagem de 100 mL de sumo de uva por cartuxos de Lichrolute® EN (Merck), sendo, posteriormente, os compostos aromáticos da fração livre eluídos com 5 mL de mistura azeotrópica pentano-diclorometano (2:1) e os precursores glicoconjugados extraídos com 7 mL de acetato de etilo. Os compostos glicoconjugados foram depois hidrolisados enzimaticamente, com um preparado enzimático comercial (AR 2000), de modo a ser libertados as agliconas aromáticas. A determinação, semi-quantitativa, foi efetuada pelo método do padrão interno, usando 4-nonanol para o efeito. . A identificação dos compostos voláteis foi realizada por comparação dos índices de retenção e dos espectros de massas dos compostos com os de substâncias de referência puras. Os ensaios foram realizados em triplicado. Os resultados obtidos foram comparados com os resultados anteriormente descritos para a mesma casta[5]. Verificou-se que, para os compostos terpénicos analisados, quer na fração livre, quer na fração glicoconjugada, o perfil de compostos terpénicos foi semelhante ao descrito[5]. Os resultados obtidos mostraram uma boa repetibilidade do método utilizado. Este trabalho demonstra a grande aplicabilidade das técnicas analíticas como SPE e GC-MS em áreas como a vinicultura e a enologia

Fermented beverages : process technology and management, volatile compounds, instrumental methods of analysis

The authors have been developed their main efforts studying fermented beverages, being wine the most important product [1]. Recently, other fermented products, obtained from various fruits, namely tropical fruits, and even from cheese whey have been studied [2]. The main goal is to study the technological parameters which may influence the quality of the final products, in order to manage the process, accurately. As aroma is one of the most important attributes of fermented beverages, the development of analytical methodologies to identify and quantify key volatile compounds, was also an objective. Volatile compounds related to different origins (varietal, fermentative, post-fermentative) and belonging to various chemical families (terpenes, norisoprenoids, alcohols, esters, volatile fatty acids, volatile phenols, …) were studied in final products as well as in raw-materials, namely grapes. Correlation between chemical and sensory analyses has been also considered. Methodologies used to extract volatile compounds from beverages include liquid-liquid micro-extraction, solid-phase extraction and solid-phase micro-extraction. Gas chromatography with flame ionisation, pulsed photometric and mass detectors was used. The main results may be summarized as followed: characterization and discrimination of grape varieties based on varietal volatile composition, from Vinhos Verdes region and from Galicia (Spain); influence of viticultural and some technological parameters and storage conditions on the volatile composition of grapes and wines; correlation between sensory (aroma) and chemical analysis (volatile compounds); implementation of simple and accurate analytical methodologies to quantify volatile compounds of fermented beverages; characterisation, respecting volatile compounds, of diverse beverages, namely obtained from tropical fruits and from cheese whey

Correlation between instrumental and sensory analysis for the characterization of Vitis vinifera wines

In this work, sensory analysis of was used to evaluate the wine aroma character with different aroma attributes according to Norm ISO 11035.1 In parallel wine volatiles were identified and quantified by gas chromatography according the methodology proposed by Oliveira et al. (2006).2 The objective of this work was to study the correlation between instrumental analysis and sensory perception of wine constituents. Thirty-five Albariño white young wines from 2006 vintage were evaluated by GC-FID and sensory analysis by trained and native panel composed by 10 wine tasters from Appellation of Origin Rias Baixas (Galicia, Spain). Forty volatile compounds were identified and quantified by Gas Chromatography and forty-seven aroma descriptors were developed using quantitative descriptive analysis and evaluated with line scaling method. Principal component analysis showed the distribution of the wines in basis to chemical and sensory characteristics. The correlation coefficients of the flavour scores between sensory evaluation and instrumental analysis (R2 > 0.50) were found for methanollacticvanilla, guaiacolhoney, 3-methyl-1-propanolgreen-pepper, 1-butanolliquorice, ethyl lactategreenpepper liquorice. These correlations linking chemical compounds and sensory descriptors may be attributed to possible relations between them, to the presence of other produced compounds which were not analysed or, to some associations among the analysed compounds

Easy and accurate methodology to quantify volatile compounds in fermented beverages

Volatile compounds of fermented beverages may derive from raw matter, but the majority are formed during fermentative processes. In the some specific cases, e. g. wines, they may also arise during conservation and ageing. These volatiles belong to a diversity of chemical families, including alcohols, esters, fatty acids, volatile phenols, terpenes and norisoprenoids. Levels of individual compounds may range from few ng/L to hundreds of mg/L. Although major volatiles may be analysed directly by gas chromatography, most of them needs a previous step of concentration. Concentration was traditionally achieved by solvent extraction, liquid-liquid mixture, soxhlet and SPE, followed by evaporation; more recently solvent-free extractive techniques SPME and SBSE, became usual due to their detection limits and selectivity. However, these techniques have some drawbacks: environmental aspects when using large quantities of solvents, price, needs of specialized equipment and workmanship. Furthermore, chromatographic detectors may be selective, e. g. pulse flame photometric detector (PFPD) for sulphurous compounds or nitrogenous compounds, or somewhat universal as mass spectrometer (MS). However, although MS permits the quantification of a wide range of volatiles, the equipment is very expensive and cost-maintenance. Another detector largely used by research centres and institutions linked to beverages sector, the flame ionisation detector, acquire attractiveness due to its good response to the methyl groups of organic compounds and its appellative price. These constraints makes difficult to achieve a universal methodology to analyse, in a single and quick approach, all the constituents of a fermented beverage like wine, beer or other products obtained by the fermentation of fruit juices. Moreover, when analysts of research centres and tutelary institutions need to compare samples, to quantify some few key volatile compounds or even to confirm its authenticity, they aspire for an easy and accurate methodology, which additionally uses inexpensive equipment. This work refers to the implementation of a quick methodology to quantify volatile compounds of fermented beverages, carried out by liquid-liquid micro-extraction followed by chromatographic analysis. The optimized methodology, reached to maximize the number of extractions per day, only uses 8 mL of sample and 400 L of dichloromethane. Each battery of extracts, ready for chromatographic analysis by GC-FID, may be obtained after 30 min, and only uses ordinary labware. Although the proposed methodology may permit the identification and quantification of a large number of compounds, it was implemented for 40, belonging to the following families: alcohols, esters, terpenes, volatile fatty acids, volatile phenols and norisoprenoids. Validation was performed in terms of linearity, limits of detection and quantification, repeatability, reproducibility and recovery