Optimization of mead production: design of different strategies for improvement of alcoholic fermentation
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2016-04-04
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O hidromel é uma bebida tradicional que contém 8% a 18% (v/v) de etanol e resulta da fermentação alcoólica de mel diluído, realizada por leveduras. Como o mel é uma matriz com
uma baixa concentração de nutrientes e outras condições desfavoráveis de crescimento,
normalmente são vários os problemas encontrados na produção de hidromel, nomeadamente,
paragens ou amuos de fermentação, parâmetros insatisfatórios de qualidade, bem como
propriedades sensoriais desagradáveis. Além disso, a fermentação do hidromel é um processo
moroso e a qualidade do produto final é muito variável.
Neste contexto, o objectivo global deste projecto de doutoramento foi a optimização do
crescimento e desempenho fermentativo da levedura para conduzir à maximização da
qualidade do hidromel. Para atingir esta finalidade foram seleccionadas duas estirpes da
levedura Saccharomyces cerevisiae, QA23 e ICV D47, para realizar vários ensaios de
fermentação.
Inicialmente, para melhorar a fermentação do hidromel, avaliou-se o potencial do
enriquecimento nutritivo do mosto-mel. O mosto foi suplementado com sais, vitaminas ou
uma mistura de ambos, embora essas adições não tenham tido efeitos positivos no
crescimento de leveduras, no perfil de fermentação ou nas características do hidromel.
Na tentativa de optimizar o processo fermentativo, outras estratégias foram testadas,
incluindo a utilização de elevadas densidades celulares ou a imobilização das células. O
aumento da quantidade de inóculo resultou numa redução do tempo de fermentação, embora
não se tenham verificado efeitos positivos adicionais no crescimento das leveduras. Com a
menor quantidade de inóculo, detectaram-se pequenas diferenças na cinética de crescimento
entre as duas estirpes.
Para avaliar a concentração de alginato mais eficaz para a imobilização, as duas estirpes
de levedura foram encapsuladas em esferas com 2 ou 4% (p/v) de alginato, embora nenhuma
das concentrações tenha sido capaz de evitar a saída das células das esferas para o meio.
Assim, a imobilização das células de levedura em camada simples de Ca-alginato ou em
camada dupla de alginato-quitosano foi aplicada na produção de hidromel. A imobilização
não teve qualquer efeito adverso na viabilidade celular, uma vez que foram observadas
diferenças mínimas na cinética de fermentação entre as fermentações conduzidas com células
livres ou encapsuladas. Além disso, a imobilização em camada dupla de alginato-quitosano
não apresentou nenhuma vantagem comparativamente com a camada simples de Ca-alginato, uma vez que o número de células livres no meio, resultante da saída das células das esferas,
foi semelhante.
Adicionalmente aos estudos de cinética de crescimento das leveduras e de desempenho
fermentativo, pesquisaram-se, em todas as fermentações, tanto a produção de compostos
voláteis aromáticos como as características físico-químicas dos hidroméis. No final da
fermentação alcoólica procedeu-se à identificação e quantificação dos compostos voláteis dos
hidroméis por GC-FID e GC-MS. Os resultados obtidos mostraram que a composição
aromática final do hidromel dependeu da concentração do inóculo: a formação de compostos
voláteis em concentrações acima dos seus limites de detecção foi particularmente pronunciada
com concentrações baixas de inóculo. As células imobilizadas produziram hidroméis com
concentrações mais elevadas de compostos com características frutadas e de compostos
indesejáveis. Os ésteres, acetato de isoamilo, octanoato de etilo e hexanoato de etilo e o
acetaldeído foram os principais odorantes encontrados em todos os hidroméis, embora as
concentrações tenham variado de acordo com a concentração do inóculo e a imobilização das
células. Em geral, a qualidade enológica de hidroméis, excepto a acidez volátil, não foi
influenciada pela concentração do inóculo ou imobilização. O hidromel obtido com células de
levedura imobilizadas ou com maiores concentrações de inóculo apresentou mais ácido acético.
Finalmente, as fermentações foram realizadas em volumes mais elevados para avaliar
uma possível correlação entre a formação dos compostos aromáticos e os atributos sensoriais
do hidromel. A formação dos compostos de aromáticos mais agradáveis foi detectada no
hidromel fermentado por células de levedura não imobilizadas. A análise sensorial também
corroborou esta observação, revelando que os descritores de aroma mais agradáveis estavam
correlacionados com o hidromel obtido com células de levedura livres, independentemente da
estirpe.
Resumindo, as condições que melhoraram os desempenhos fermentativo e de
crescimento não resultaram, necessariamente, em hidromel de alta qualidade.
Mead is a traditional drink that contains 8 % to 18 % (v/v) of ethanol, resulting from the alcoholic fermentation of diluted honey by yeasts. Honey is a matrix with a low nutrient concentration and other unfavourable growth conditions, so several problems are usually encountered in mead production, namely delayed or arrested fermentations, unsatisfactory quality parameters, as well as unpleasant sensory properties. Also, mead fermentation is a time-consuming process and the quality of the final product is highly variable. In this context, the global objective of this PhD research project was the optimization of growth and fermentative performance of yeast leading to the maximization of mead quality. To achieve this purpose, two wine yeast strains of Saccharomyces cerevisiae, QA23 and ICV D47 were selected to conduct several fermentation trials. Initially, it was evaluated the potential of the nutritive enrichment of honey-must to improve mead fermentation. The must was supplemented with salts, vitamins or a mixture of both, although those additions had no positive effects on yeast growth, fermentation profile or on the characteristics of the mead. In this line further strategies have been tested in an attempt to optimize the fermentative process, including the use of high cell density or cell immobilization. The increasing of inoculum size resulted in a reduction of fermentation length, although no additional positive effects where verified in yeasts net growth. At the lowest innoculum size, minor differences were detected in the growth kinetics between the two strains. In order to assess the most effective alginate concentration for immobilization, the two yeast strains were entrapped in 2 or 4% (w/v) alginate beads, although neither of the concentrations was able to prevent the cell leakage from the beads. So, the immobilization of yeast cells on single-layer Ca-alginate or double-layer alginate-chitosan was applied to mead production. Immobilization had no adverse effect on cell viability, since minor differences were found on fermentation kinetics between fermentations conducted with free or encapsulated cells. Also, the double-layer alginate-chitosan had no advantage compared with the single-layer Ca-alginate, as the number of free cells in the medium, resulting from cell leakage, was similar. In addition to the studies of yeast growth kinetics and fermentative performance, all fermentations have also been screened for the production of aroma volatile compounds and for the physicochemical characteristics of meads. Identification and quantification of volatile compounds was performed by GC-FID and GC-MS in all meads at the end of alcoholic fermentation. The results obtained showed that mead final aroma composition was dependent on the inoculum size: the formation of the volatile compounds in concentrations above their detection thresholds was particularly pronounced at low inoculum sizes. Immobilized cells produced meads with higher concentrations of compounds with fruity characteristics and of undesirable compounds. The esters isoamyl acetate, ethyl octanoate and ethyl hexanoate and acetaldehyde were the major powerful odorants found in all mead, although their concentrations varied according to the inoculum size and cell immobilization. In general, the oenological quality of meads was not influenced by the inoculum size or immobilizations, except for the volatile acidity. Mead obtained with entrapped yeast cells or with higher inoculum size presented more acetic acid. Finally, fermentations were conducted in higher volumes to evaluate a possible correlation between aroma compound formation and the sensory attributes of mead. The most pleasant aroma compounds formation was detected in mead fermented by non-immobilized yeast cells. Sensory analysis corroborates this observation, revealing that the most pleasant aroma descriptors were correlated with mead obtained with yeast free cells, independently of the strain. In sum, the conditions that improved the fermentation and growth performance were not necessarily associated with high quality mead.
Mead is a traditional drink that contains 8 % to 18 % (v/v) of ethanol, resulting from the alcoholic fermentation of diluted honey by yeasts. Honey is a matrix with a low nutrient concentration and other unfavourable growth conditions, so several problems are usually encountered in mead production, namely delayed or arrested fermentations, unsatisfactory quality parameters, as well as unpleasant sensory properties. Also, mead fermentation is a time-consuming process and the quality of the final product is highly variable. In this context, the global objective of this PhD research project was the optimization of growth and fermentative performance of yeast leading to the maximization of mead quality. To achieve this purpose, two wine yeast strains of Saccharomyces cerevisiae, QA23 and ICV D47 were selected to conduct several fermentation trials. Initially, it was evaluated the potential of the nutritive enrichment of honey-must to improve mead fermentation. The must was supplemented with salts, vitamins or a mixture of both, although those additions had no positive effects on yeast growth, fermentation profile or on the characteristics of the mead. In this line further strategies have been tested in an attempt to optimize the fermentative process, including the use of high cell density or cell immobilization. The increasing of inoculum size resulted in a reduction of fermentation length, although no additional positive effects where verified in yeasts net growth. At the lowest innoculum size, minor differences were detected in the growth kinetics between the two strains. In order to assess the most effective alginate concentration for immobilization, the two yeast strains were entrapped in 2 or 4% (w/v) alginate beads, although neither of the concentrations was able to prevent the cell leakage from the beads. So, the immobilization of yeast cells on single-layer Ca-alginate or double-layer alginate-chitosan was applied to mead production. Immobilization had no adverse effect on cell viability, since minor differences were found on fermentation kinetics between fermentations conducted with free or encapsulated cells. Also, the double-layer alginate-chitosan had no advantage compared with the single-layer Ca-alginate, as the number of free cells in the medium, resulting from cell leakage, was similar. In addition to the studies of yeast growth kinetics and fermentative performance, all fermentations have also been screened for the production of aroma volatile compounds and for the physicochemical characteristics of meads. Identification and quantification of volatile compounds was performed by GC-FID and GC-MS in all meads at the end of alcoholic fermentation. The results obtained showed that mead final aroma composition was dependent on the inoculum size: the formation of the volatile compounds in concentrations above their detection thresholds was particularly pronounced at low inoculum sizes. Immobilized cells produced meads with higher concentrations of compounds with fruity characteristics and of undesirable compounds. The esters isoamyl acetate, ethyl octanoate and ethyl hexanoate and acetaldehyde were the major powerful odorants found in all mead, although their concentrations varied according to the inoculum size and cell immobilization. In general, the oenological quality of meads was not influenced by the inoculum size or immobilizations, except for the volatile acidity. Mead obtained with entrapped yeast cells or with higher inoculum size presented more acetic acid. Finally, fermentations were conducted in higher volumes to evaluate a possible correlation between aroma compound formation and the sensory attributes of mead. The most pleasant aroma compounds formation was detected in mead fermented by non-immobilized yeast cells. Sensory analysis corroborates this observation, revealing that the most pleasant aroma descriptors were correlated with mead obtained with yeast free cells, independently of the strain. In sum, the conditions that improved the fermentation and growth performance were not necessarily associated with high quality mead.
Descrição
Tese de Doutoramento em Ciências Químicas e Biológicas
Palavras-chave
Hidromel , Análise sensorial , Composto volátil , Aroma , Leveduras , Fermentação , Concentração do inóculo