Towards the understanding of the impact of nitrogen on wine fermentation: from classical to genomic approaches
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2014
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Hoje em dia, a procura de leveduras com elevada capacidade fermentativae que
tenham impacto favorável sobre a qualidade do vinho, é um dos principais objetivos na
indústria enológica.Nesta linha, o estudo do impacto do azoto na qualidade do vinho é de
grande relevância, uma vez que é importante não só para assegurar o crescimento de
leveduras e cinética fermentação adequados, mas também por poder afetar a produção
dos principais metabolitos resultantes da fermentação dos açúcares.Neste trabalho,
explorámos primeiro a diversidade fenotípica de 20 estirpes de Saccharomyces
cerevisiae de origem geográfica diferente em termos de resistência a várias condições de
“stress” normalmente encontradas durante a fermentação alcoólica, bem como do seu
potencial para a produção de compostos aromáticos. Com o objetivo de avaliar como o azoto afeta, não apenas o crescimento e atividade fermentativa das leveduras, mas
também a qualidade do produto final, oito estirpes selecionadas foram usados numa
abordagem experimental fatorial (concentração de azoto x fonte de azoto x estirpe de levedura). Os resultados sugerem que o uso de diferentes fontes e concentrações de azoto resulta na produção de vinhos com perfis aromáticos divergentes, e altamente
dependentes da estirpe utilizada. A determinação de metionina e a sua administração
antes da fermentação são cruciais para suprimir a formação de H2S e permitir a
obtenção de vinhos com diferentes perfis sensoriais. A diversidade exo‐metabólica
encontrada entre as estirpes de levedura mostrou que o “pré‐screening” utilizado pode ser empregue na selecção da estirpe de levedura mais adequada a ser utilizada,
dependendo tanto da composição em azoto do kosto, bem como do estilo de vinho
pretendido. Com base nos dados obtidos relativamente aos requisitos em azoto, cinética de crescimento, desempenho fermentativo e perfis metabólicos, três estirpes distintas de S. cerevisiae foram selecionadas para análise funcional comparativa em duas
condições de fermentação divergentes no que respeita à disponibilidade em azoto. A
correlação entre expressão genéticae parâmetros fisiológicos encontrada neste estudo revelou que a maior expressão de genes de resposta ao “stress” está associada a uma alta capacidade fermentativa. Além disso, a descoberta de genes comuns correlacionados,
tanto com a atividade de fermentação bem como com o consumo de azoto, sublinham o
papel deste nutriente na capacidade fermentativa das leveduras. As enormes diferenças encontradas na expressão genéticadas três estirpes de leveduras ressaltam a importância de se considerarem leveduras com diferente “background” genético na procura de biomarcadores de deficiência de azoto. No entanto, parte dos resultados apresentados são uma previsão da classificação de resposta comum à limitação de azoto,
o que facilitará o diagnóstico de deficiência deste nutriente nos mostos e no desenvolvimento de estratégias para otimizar o desempenho fermentativo de leveduras em fermentações industriais.
A introdução de culturas “starter” de leveduras, consistindo numa mistura de
estirpes de leveduras de S. cerevisiae e não‐Saccharomyces, está a emergir como uma
opção interessante na indústria enológica mundial, com vista a satisfazer as exigências dos consumidores de vinhos com alta complexidade de sabor e distinção estilística. No entanto, não há dados sobre o impacto desta estratégia no metabolismo do azoto da principal levedura de fermentação, S. cerevisiae. Neste trabalho, pretendeu avaliar‐se o
efeito do nível de azotodos mostos no comportamento fermentativo e metabólico de S. cerevisiae, tanto em cultura pura como em cultura mista com uma estirpe de
Hanseniaspora guiliermondii. Os resultados mostraram claramente que a sua presença
compromete o crescimento e desempenho fermentativo da estirpe de S. cerevisiae.
Considerou‐seque a potencial interação levedura‐levedura poderia também ser revelada pela expressão diferencial de genes em ambos os tipos de fermentação. Várias alterações detectadas na expressão genéticade S. cerevisiae parecem responder a mudanças na disponibilidade de nutrientes no mosto, principalmente azoto e vitaminas, e que estão
potencialmente associados à atividade metabólica de H. guilliermondii. Observou‐se que as diferenças na expressão de genes de S. cerevisiae envolvidos na formação de
compostos aromáticos poderiam explicar os diferentes perfis de aroma obtidos em
culturapura e mista. Estes resultados levantam a questão de perceber se o impacto de estirpes de não‐Saccharomycesno perfil aromático são resultado de uma contribuição direta com produção dos compostos e/ou de influência no comportamento metabólico da levedura S. cerevisiae.
Em suma, e abrindo perspectivas interessantes para futuras pesquisas na biologia de levedura, os dados apresentados nesta tese fornecem informações importantes para
uma compreensão mais profunda do papel do azoto na fermentação alcoólica, que
podem ajudar os enólogos na obtenção de vinhos de alta qualidade.
Nowadays, the pursuit for yeast strains displaying both high potential fitness to conduct alcoholic fermentations and favourable influence on the wine quality is one of the major goals in wine making industry.In this line, the study of the impact of nitrogen on wine quality is of major relevance since it is not only important for ensuring adequate yeast growth and fermentation kinetics, but also can affect the production of the major metabolites arising from sugar fermentation. In this work, we first explored the phenotypic diversity among 20 Saccharomyces cerevisiae strains from different geographic origin in terms of their resistance to several stress conditions usually found during alcoholic fermentation, as well as of their potential to produce aroma compounds. Aiming to assess how nitrogen affects not only yeast cells growth and fermentative activity, but also the quality of the final product, eight selected strains were used in a factorial design experimental approach (N concentration x N source x Yeast strain). Our results suggest that the use of different nitrogen sources and different nitrogen concentrations result in the production of wines with divergent aroma profiles. We found that methionine determination and its management prior to fermentation are crucial for suppressing H2S and for endowing beverages with diverse sensory traits. The exo‐metabolic diversity found among the yeast strains showed that the prescreening used can be employed inthe selection of the most suitable yeaststrainto be used depending on both grape‐juice nitrogen composition and wine style envisioned. Based on the data obtained regarding nitrogen requirements, growth kinetics, fermentative fitness, and metabolic profiles, three contrastingS. cerevisiae strains were selected for comparative functional analysis throughout two divergent fermentation conditions of nitrogen availability. The correlations of gene expression with the physiological parameters found in our study revealed that higher expression of stress responsive genes is associated with a high fermentative capacity. Also the finding of common genes correlated with both fermentation activity and nitrogen up‐takeunderlie the role of nitrogen on yeast fermentative fitness. The huge dissimilarities found in gene expression among the three yeast strains underscore the importance of considering different yeast strain backgrounds in the search for biomarkers of nitrogen deficiency. Nevertheless, the results presented are a preview on classification of common nitrogen limitation response, which will facilitate diagnosis of deficiency of this nutrient in the musts and the development of strategies to optimize yeast performance in industrial fermentations. The introduction of yeast starter cultures consisting in a blend of S. cerevisiae and non‐Saccharomyces yeast strains is emerging as an interesting option for production of wines complexity of flavour and stylistic distinction. However, there is no data regarding the impact of this strategy on nitrogen metabolism of the main fermenting yeast, S. cerevisiae. In this work, we aimed at evaluate the effect of nitrogen level of grape‐musts on fermentative and metabolic behaviour of S. cerevisiae in mixed culture with a non‐ Sacharomyces strain of Hanseniaspora guiliermondii. The results have clearly shown that its presence compromise the growth and fermentation fitness of S. cerevisiae. We reasoned that potential yeast‐yeast interaction would also be revealed by the differential expression of genes in both fermentations. Several changes in S. cerevisiae gene expression detected appear to respond to changes in nutrient availability, mainly nitrogen and vitamins, in the fermenting must that are potentially linked to Hanseniaspora guilliermondii metabolic activity. We found that differences in the expression of S. cerevisiae genes involved in aroma compounds formation could explain the differences in the aroma profiles obtained in single and mixed‐culture. These results raises the question whether the impact of non‐Saccharomyces in the sensorial profile in co‐culture fermentations results from a direct contribution with the production of aromatic compounds and/or from influencing the metabolic behaviour of the fermentative yeast S. cerevisiae. In sum, while opening interesting perspectives for future research in yeast biology, the data presented in this thesis clearly provides important information towards the deeper understanding of the role of nitrogen in alcoholic fermentation that can assist winemakers in the pursuit of high quality wines.
Nowadays, the pursuit for yeast strains displaying both high potential fitness to conduct alcoholic fermentations and favourable influence on the wine quality is one of the major goals in wine making industry.In this line, the study of the impact of nitrogen on wine quality is of major relevance since it is not only important for ensuring adequate yeast growth and fermentation kinetics, but also can affect the production of the major metabolites arising from sugar fermentation. In this work, we first explored the phenotypic diversity among 20 Saccharomyces cerevisiae strains from different geographic origin in terms of their resistance to several stress conditions usually found during alcoholic fermentation, as well as of their potential to produce aroma compounds. Aiming to assess how nitrogen affects not only yeast cells growth and fermentative activity, but also the quality of the final product, eight selected strains were used in a factorial design experimental approach (N concentration x N source x Yeast strain). Our results suggest that the use of different nitrogen sources and different nitrogen concentrations result in the production of wines with divergent aroma profiles. We found that methionine determination and its management prior to fermentation are crucial for suppressing H2S and for endowing beverages with diverse sensory traits. The exo‐metabolic diversity found among the yeast strains showed that the prescreening used can be employed inthe selection of the most suitable yeaststrainto be used depending on both grape‐juice nitrogen composition and wine style envisioned. Based on the data obtained regarding nitrogen requirements, growth kinetics, fermentative fitness, and metabolic profiles, three contrastingS. cerevisiae strains were selected for comparative functional analysis throughout two divergent fermentation conditions of nitrogen availability. The correlations of gene expression with the physiological parameters found in our study revealed that higher expression of stress responsive genes is associated with a high fermentative capacity. Also the finding of common genes correlated with both fermentation activity and nitrogen up‐takeunderlie the role of nitrogen on yeast fermentative fitness. The huge dissimilarities found in gene expression among the three yeast strains underscore the importance of considering different yeast strain backgrounds in the search for biomarkers of nitrogen deficiency. Nevertheless, the results presented are a preview on classification of common nitrogen limitation response, which will facilitate diagnosis of deficiency of this nutrient in the musts and the development of strategies to optimize yeast performance in industrial fermentations. The introduction of yeast starter cultures consisting in a blend of S. cerevisiae and non‐Saccharomyces yeast strains is emerging as an interesting option for production of wines complexity of flavour and stylistic distinction. However, there is no data regarding the impact of this strategy on nitrogen metabolism of the main fermenting yeast, S. cerevisiae. In this work, we aimed at evaluate the effect of nitrogen level of grape‐musts on fermentative and metabolic behaviour of S. cerevisiae in mixed culture with a non‐ Sacharomyces strain of Hanseniaspora guiliermondii. The results have clearly shown that its presence compromise the growth and fermentation fitness of S. cerevisiae. We reasoned that potential yeast‐yeast interaction would also be revealed by the differential expression of genes in both fermentations. Several changes in S. cerevisiae gene expression detected appear to respond to changes in nutrient availability, mainly nitrogen and vitamins, in the fermenting must that are potentially linked to Hanseniaspora guilliermondii metabolic activity. We found that differences in the expression of S. cerevisiae genes involved in aroma compounds formation could explain the differences in the aroma profiles obtained in single and mixed‐culture. These results raises the question whether the impact of non‐Saccharomyces in the sensorial profile in co‐culture fermentations results from a direct contribution with the production of aromatic compounds and/or from influencing the metabolic behaviour of the fermentative yeast S. cerevisiae. In sum, while opening interesting perspectives for future research in yeast biology, the data presented in this thesis clearly provides important information towards the deeper understanding of the role of nitrogen in alcoholic fermentation that can assist winemakers in the pursuit of high quality wines.
Descrição
Tese de Doutoramento em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Levedura de vinho , Saccharomyces cerevisiae , Fermentação alcoólica , Azoto , Expressão genética , Biologia das leveduras