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Vicinal diketones monitoring during lager beer fermentation: the importance of nitrogenous compounds
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Abstract(s)
As dicetonas vicinais, o diacetil e a 2,3-pentanediona, são um sub-resultado da
biossíntese de valina, leucina e isoleucina por parte da levedura. Estes compostos conferem um
aroma desagradável à cerveja e por isso a monitorização dos mesmos é imprescindível durante
a fermentação.
Uma nova metodologia para a determinação de compostos orgânicos voláteis em
amostras de fermentação de cerveja por GC-MS foi desenvolvida, após a optimização da
extração destes compostos por HS-SPME. As condições óptimas consistiram numa extração a
40ºC durante 20 minutos, através da exposição de uma fibra Car-PDMS num vial de 20 mL,
contendo 10 mL de amostra e 3,3 g de NaCl. Adicionalmente, desenvolveu-se uma metodologia
para a determinação de aminoácidos por HPLC-fluorescência.
As metodologias desenvolvidas apresentam boa linearidade nas gamas de concentração
em estudo e valores de recuperação entre os 89,88-133,80% (voláteis) e os 77,60-112,39%
(aminoácidos). A precisão, avaliada em termos de repetibilidade e reprodutibilidade,
caracteriza-se por desvios inferiores a 10 e 15%, para a determinação dos compostos orgânicos
voláteis e dos aminoácidos, respectivamente. Os valores de LOD e LOQ obtidos confirmam a alta
sensibilidade dos métodos desenvolvidos.
As metodologias acima referidas, bem como a metodologia standard para a
determinação do FAN, foram aplicadas para monitorizar 21 aminoácidos e 22 compostos
orgânicos voláteis em três cubas de fermentação lager. Diferentes métodos de conservação
foram testados e a adição de cloreto de cálcio associada ao armazenamento a -26°C por 2 dias
foi o tratamento escolhido para evitar a evolução das amostras até à análise das mesmas.
Identicamente ao que se verificou em relação às substâncias nitrogenadas, as principais
variações a nível dos compostos orgânicos voláteis ocorrem também nas primeiras 150 horas
de fermentação. A análise das amostras em estudo permitiu relacionar a abundância dos
compostos nitrogenados com as baixas taxas de fermentação observadas, justificando assim a
formação/redução tardia das dicetonas vicinais. Efectivamente, associou-se o aparecimento de
um segundo pico de diacetil à exaustão dos aminoácidos do grupo A, da leucina e da isoleucina.
Vicinal diketones, diacetyl and 2,3-pentanedione, impart unpleasant aromas to beer, when their concentrations are higher than their odour threshold limits. The formation of these off-flavours is related to valine, leucine and isoleucine biosynthesis. Therefore, vicinal diketones as well as amino acids determination is a major concern during beer fermentation. A new methodology for the determination of volatile organic compounds in fermentation samples by GC-MS, including a design of experiments for compounds extraction optimization by HS-SPME, was successfully developed. A 20-minute extraction at 40°C, by exposing a Car-PDMS fibre in a 20 mL vial containing 10 mL of sample and 3.3 g of NaCl was adopted. A methodology for the determination of amino acids by HPLC-fluorescence, including on-line derivatization was also developed. The developed methodologies present a good linearity in the concentration ranges in study. Recovery mean values ranged from 89.88 to 133.80% and from 77.60 to 112.39%, while methods precision, evaluated in terms of repeatability and reproducibility, showed variations lower than, respectively, 10% and 15% for volatile organic compounds and amino acids determination. Also, LOD and LOQ values confirm the high sensitivity of these methodologies. The methodologies mentioned above and standard FAN methodology were applied to monitor 21 amino acids and 22 volatile organic compounds in three batches during the fermentation process. Conservation tests were performed and chloride salts addition and storage at -26°C for two days was the most suitable treatment for sample preservation. Volatile organic compounds evolution during the analysed lager fermentations showed that, similarly to nitrogenous compounds, major changes occur in the first 150 hours of fermentation. Low free amino nitrogen content may explain low fermentation rates and be the cause of delayed vicinal diketones formation/reduction. In addition to group A amino acids complete assimilation, leucine and isoleucine exhaustion leads to the occurrence of two diacetyl peaks and to increased fermentation time.
Vicinal diketones, diacetyl and 2,3-pentanedione, impart unpleasant aromas to beer, when their concentrations are higher than their odour threshold limits. The formation of these off-flavours is related to valine, leucine and isoleucine biosynthesis. Therefore, vicinal diketones as well as amino acids determination is a major concern during beer fermentation. A new methodology for the determination of volatile organic compounds in fermentation samples by GC-MS, including a design of experiments for compounds extraction optimization by HS-SPME, was successfully developed. A 20-minute extraction at 40°C, by exposing a Car-PDMS fibre in a 20 mL vial containing 10 mL of sample and 3.3 g of NaCl was adopted. A methodology for the determination of amino acids by HPLC-fluorescence, including on-line derivatization was also developed. The developed methodologies present a good linearity in the concentration ranges in study. Recovery mean values ranged from 89.88 to 133.80% and from 77.60 to 112.39%, while methods precision, evaluated in terms of repeatability and reproducibility, showed variations lower than, respectively, 10% and 15% for volatile organic compounds and amino acids determination. Also, LOD and LOQ values confirm the high sensitivity of these methodologies. The methodologies mentioned above and standard FAN methodology were applied to monitor 21 amino acids and 22 volatile organic compounds in three batches during the fermentation process. Conservation tests were performed and chloride salts addition and storage at -26°C for two days was the most suitable treatment for sample preservation. Volatile organic compounds evolution during the analysed lager fermentations showed that, similarly to nitrogenous compounds, major changes occur in the first 150 hours of fermentation. Low free amino nitrogen content may explain low fermentation rates and be the cause of delayed vicinal diketones formation/reduction. In addition to group A amino acids complete assimilation, leucine and isoleucine exhaustion leads to the occurrence of two diacetyl peaks and to increased fermentation time.
Description
Keywords
Cerveja lager Fermentação Compostos orgânicos voláteis Dicetonas vicinais Aminoácidos Lager beer Fermentation Volatile organic compounds Vicinal diketones Amino acids Applied Biochemistry . Faculdade de Ciências Exatas e da Engenharia