Yeast performance for beer production in very high gravity fermentations

Abstract

The main objective of this thesis was the optimization of the fermentation performance of a brewer’s yeast strain to produce beer from very high gravity (VHG) wort. In this study, two approaches were considered to overcome the limitations imposed by the stress inherent to VHG fermentations. The first approach was based on the characterization of the effect of fermentation variables on yeast performance and the second strategy consisted on the nutritional treatment of the yeast slurry before pitching the wort. The study of the effect of the environmental variables namely wort concentration, temperature, pitching rate and oxygenation was performed in EBC-2L tubes. The results obtained demonstrate that very high gravity worts at 22ºP can be fermented in the same period of time as 15ºP wort, by raising the temperature to 18ºC, the initial oxygen level to about 22 ppm and increasing the pitching rate to 22 million cells per ml. By using this strategy, a finished beer at 11.5ºP extract will increase the volumetric brewing capacity (by dilution) by 91% for the 22ºP wort, comparing to 30% for 15ºP wort. The effect of the operation variables on the most important aromatic compounds was limited. Within the range of tested conditions, the concentration of the higher alcohols did not change beyond the respective thresholds as well as the acetaldehyde concentration. However, the esters variation indicated that the concentrations obtained at the very high gravity conditions could affect the aroma profile. This suggests that beer produced with VHG worts would acquire a fruity aroma when compared with beer produced from standard gravity worts. The main objective of this thesis was the optimization of the fermentation performance of a brewer’s yeast strain to produce beer from very high gravity (VHG) wort. In this study, two approaches were considered to overcome the limitations imposed by the stress inherent to VHG fermentations. The first approach was based on the characterization of the effect of fermentation variables on yeast performance and the second strategy consisted on the nutritional treatment of the yeast slurry before pitching the wort. The study of the effect of the environmental variables namely wort concentration, temperature, pitching rate and oxygenation was performed in EBC-2L tubes. The results obtained demonstrate that very high gravity worts at 22ºP can be fermented in the same period of time as 15ºP wort, by raising the temperature to 18ºC, the initial oxygen level to about 22 ppm and increasing the pitching rate to 22 million cells per ml. By using this strategy, a finished beer at 11.5ºP extract will increase the volumetric brewing capacity (by dilution) by 91% for the 22ºP wort, comparing to 30% for 15ºP wort. The effect of the operation variables on the most important aromatic compounds was limited. Within the range of tested conditions, the concentration of the higher alcohols did not change beyond the respective thresholds as well as the acetaldehyde concentration. However, the esters variation indicated that the concentrations obtained at the very high gravity conditions could affect the aroma profile. This suggests that beer produced with VHG worts would acquire a fruity aroma when compared with beer produced from standard gravity worts. The main objective of this thesis was the optimization of the fermentation performance of a brewer’s yeast strain to produce beer from very high gravity (VHG) wort. In this study, two approaches were considered to overcome the limitations imposed by the stress inherent to VHG fermentations. The first approach was based on the characterization of the effect of fermentation variables on yeast performance and the second strategy consisted on the nutritional treatment of the yeast slurry before pitching the wort. The study of the effect of the environmental variables namely wort concentration, temperature, pitching rate and oxygenation was performed in EBC-2L tubes. The results obtained demonstrate that very high gravity worts at 22ºP can be fermented in the same period of time as 15ºP wort, by raising the temperature to 18ºC, the initial oxygen level to about 22 ppm and increasing the pitching rate to 22 million cells per ml. By using this strategy, a finished beer at 11.5ºP extract will increase the volumetric brewing capacity (by dilution) by 91% for the 22ºP wort, comparing to 30% for 15ºP wort. The effect of the operation variables on the most important aromatic compounds was limited. Within the range of tested conditions, the concentration of the higher alcohols did not change beyond the respective thresholds as well as the acetaldehyde concentration. However, the esters variation indicated that the concentrations obtained at the very high gravity conditions could affect the aroma profile. This suggests that beer produced with VHG worts would acquire a fruity aroma when compared with beer produced from standard gravity worts. The second studied approach to overcome VHG limitations corresponded to the nutritional supplementation of the yeast slurry. For this, several simple and complex nutrients reported in the literature as being relevant in the fermentation performance were tested, and the ones with higher positive effect were selected. The combination of linoleic acid (6 mg.g-1), MgCl2 (5 g.l-1) and Tween80 (6 mg.g-1) on the supplementation of the yeast slurry during 24h before pitching the wort produced higher ethanol concentration (11.7%, v/v) than the unsuplemented yeast at the same conditions (10.1%, v/v). The scale-up of the studied conditions to pilot plant fermentations indicated that although the results obtained in EBC tall tubes are extremely useful to compare brewing conditions, the absolute profile of an industrial scale fermentation would be different. In large 250 L fermentations, many physico-chemical indicators were different between a beer produced from a VHG wort at 18ºC and a beer from a standard 15ºP wort fermented at 12ºC. However, in the sensorial triangular test those beers were indistinguishable. This is an important indicator of the feasability of a VHG brewing at 18ºC with 22ºP wort to produce an identical beer as the one produced in standard conditions. The study of the cropped yeast indicated that the yeast settled on the bottom of the cylindroconical fermenter after a lager brewing fermentation can be in similar physiologic conditions. The lab-fermentation trials using samples collected along the yeast cropping presented similar profiles. However, the fractionation of the yeast slurry by density indicated that several types of cells with distinct physiological state are present in the brewer’s storage tank. This work suggests that the last formed cells at the end of the fermentation may have their physiological state compromised by the absence of nutrients. Another finding suggests that the proportion of young virgin cells in the cropped yeast may increase during brewing fermentations. This fact can have an important impact on the yeast slurry recycling procedure widely used by the brewers.

A presente tese teve como objetivo principal a otimização do desempenho fermentativo de uma estirpe de levedura cervejeira para produção de cerveja a partir de mosto de muito alto extrato. Neste estudo foram abordadas duas estratégias para ultrapassar as limitações impostas pelo stress inerente às fermentações de mosto concentrado. A primeira abordagem consistiu na otimização das variáveis ambientais influentes na atividade da levedura. A segunda estratégia baseou-se no pré-acondicionamento nutricional da levedura antes de ser usada para inocular o mosto cervejeiro. O estudo das variáveis ambientais de fermentação nomeadamente a concentração do mosto, a temperatura e as taxas de oxigenação e de inóculo foi efetuado em tubos EBC- 2L com a levedura cervejeira. Os resultados obtidos demonstraram que o mosto de alto extrato a 22ºP pode ser fermentado no mesmo período de tempo que as fermentações de mosto a 15ºP aumentando a temperatura de 12ºC para 18ºC, o oxigénio dissolvido para 22 ppm e o inóculo para 22 milhões de células por mililitro. A cerveja após diluição para um extrato de 11.5ºP aumentaria a capacidade volumétrica em 91% for 22ºP, em comparação com o aumento de 30% para o mosto a 15ºP. Foi ainda demonstrado que o efeito das condições ótimas de produtividade no perfil aromático é limitado. A variação na concentração de ésteres não ultrapassa o limite de deteção nos compostos analisados, embora o aumento na concentração de ésteres indique que a cerveja produzida nestas condições apresenta um aroma mais frutado quando comparado com a cerveja produzida nas condições padrão. A segunda alternativa estudada para ultrapassar as limitações mosto VHG correspondeu à suplementação nutricional da levedura. Para tal foi efetuado um levantamento de nutrientes simples ou compostos referenciados na literatura como influentes no desempenho fermentativo, seguido de um rastreio daqueles com maior impacto positivo na fermentação. A combinação dos nutrientes como o ácido linoleico (6 mg.g-1), MgCl2 (5 g.l-1) e Tween80 (6 mg.g-1) usada para acondicionamento da pasta de levedura durante 24h antes de inocular permitiu uma concentração final em etanol de 11.7%. A fermentação nas mesmas condições com levedura não suplementada produziu 10.1% em etanol. Os estudos de aumento de escala das fermentações cervejeiras para a escala piloto indicaram que embora os resultados obtidos em tubos EBC-2L são uteis para comparação de condições experimentais, os perfis absolutos de uma fermentação à escala industrial serão diferentes. Em fermentações com volume de 250 L, vários parâmetros físicoquímicos apresentaram diferenças entre a cerveja produzida por mosto VHG a 18ºC e a cerveja produzida a partir de mosto 15ºP a 12ºC. Contudo, na análise sensorial triangular essas cervejas foram consideradas idênticas sendo um excelente indicador da viabilidade da produção de uma cerveja por fermentação de alto extrato idêntica à produzida nas condições padrão. O estudo da levedura recolhida após a fermentação cervejeira sugere que a levedura sedimentada no cone do fermentador após a fermentação cervejeira do tipo lager pode encontrar-se em condições fisiológicas similares. Os ensaios fermentativos laboratoriais realizados com as amostras de leveduras retiradas ao longo da remoção da levedura do cone para o tanque de armazenamento apresentaram perfis similares de desempenho. Contudo, o fraccinamento de amostras de levedura homogeneizada do tanque de armazenamento de acordo com a densidade demonstrou a existência de diferentes tipos de células relativamente ao estado fisiologico e ao número da geração. Este trabalho sugere que as últimas células capazes de se dividirem durante a fermentação cervejeira possam ter baixo estado fisiológico causado pela ausência de nutrientes. Os resultados indicam ainda que a proporção de células virgens na levedura recolhida poderá aumentar ao longo da fermentação. Este facto terá um impacto importante na prática corrente da indústria cervejeira na reutilização da levedura entre ciclos fermentativos.