Abstract
Hops (Humulus lupulus L.) are responsible for imparting bitterness and aroma to beer and contain both terpenoids and terpenoid glycosides as part of their composition. Terpenoids from the essential oil are well known for the distinctive hop aromas they impart to beer. In contrast, terpenoid glycosides are non-volatile and have no aroma, but they can be hydrolysed by yeasts during fermentation or isolated enzymes resulting in the release of a free terpenoid. Yeasts can also biotransform terpenoids via isomerisation, reduction or esterification reactions, modifying the chemical composition and aroma profile of beer. The diversity of reactions makes it difficult to predict the impact of hop addition on the aroma of beer. The goal of the research presented in this thesis was to gain a better understanding of the biotransformation of terpenoids and terpenoid glycosides during beer fermentation.
Terpenoid glycosides were isolated from hops using solid phase extraction (SPE). The ability of either Saccharomyces cerevisiae SafAle US-05 yeast or added β-glucosidase to subsequently hydrolyse the terpenoid glycoside during fermentation was estimated by measuring the volatile organic compounds (VOCs) produced using headspace solid-phase microextraction gas chromatography/mass spectrometry (HS SPME-GC/MS). The addition of yeast resulted in the detection of caryophyllene and citronellol during fermentation. The addition of β-glucosidase resulted in the detection of caryophyllene oxide, citronellol, and α-terpineol. However, the amount of terpenoids released from hop-derived terpenoid glycosides was small, and these reactions were therefore considered to be likely to have far less of an effect on beer aroma than the biotransformation of free terpenoids, which consequently became the focus of the remainder of the thesis.
To investigate the biotransformation of terpenoids by SafAle US-05, one of eleven terpenoid (geraniol, nerol, citronellol, citral, α-terpineol, citronellyl acetate, caryophyllene, linalool, limonene, β-pinene and myrcene) was individually added to a model beer system at a concentration of 10 ppm and the VOCs present in the headspace of the samples before and after fermentation (at 20°C) measured using SPME-GC/MS. The detection of terpenoids which differed from the terpenoid added was evidence of biotransformation, as in the absence of yeast only the terpenoid added was detected. For example, when geraniol (10 ppm) was added, geraniol, citronellol acetate and citronellol were detected at the end of fermentation.
To determine the impact of yeast species and strain on terpenoid biotransformation during fermentation, geraniol (5 ppm) was added to a model beer system containing either a single S. cerevisiae (SafAle US-05 or SafAle WB-06) or a S. pastorianus (SafLager W-34/70 and SafLager S-23) strain. VOC in the ferments headspace were measured ever 6 hours over a 5 day fermentation (at 20°C) using proton transfer reaction-time of flight-mass spectrometry (PTR-ToF-MS). During fermentation, the depletion of geraniol was closely followed by the detection of citronellol, citronellol acetate and geraniol acetate. The S. pastorianus strains produced a higher concentration of terpenoids than the S. cerevisiae strains.
Higher alcohols and esters are important VOCs that characterize the quality and palatability of beer. The effect of yeast species and strains (SafAle US-05, SafAle WB-06, SafLager W-34/70 or SafLager S-23) on the formation of flavour-relevant esters (ethyl acetate, isoamyl acetate, ethyl hexanoate and ethyl octanoate) and higher alcohols (isoamyl alcohol, isobutyl alcohol and phenylethyl alcohol) were monitored over a 5 day fermentation (at 20°C) using PTR-ToF-MS. The two S. pastorianus strains produced comparable concentrations of the key higher alcohols and esters. However, the key higher alcohol and ester concentrations varied greatly between the two S. cerevisiae strains.
To investigate the dynamics of the biotransformation reactions over time, citronellol, citral, and geraniol (10 ppm) were individually added to a model beer system, along with either SafAle US-05 or SafAle WB-06. The VOCs in the headspace were measured every 6 hours over a 4 day fermentation (at 20°C) using fast-gas chromatography proton transfer reaction-time of flight-mass spectrometry (Fast-GC PTR-ToF-MS). Fast-GC PTR-ToF-MS enabled isobaric and isomeric compounds, such as geranial and neral, to be to differentiated from each other. In the samples spiked with either geraniol and citral (geranial/neral), both citronellol and citronellol acetate were detected throughout beer fermentation by both yeast strains. In the samples spiked with citronellol, citronellol acetate was detected throughout beer fermentation by both yeast strains, with SafAle WB-06 initially producing about double the mean peak area compared to SafAle US-05. However towards the end of fermentation the abundance became similar for both yeast strains. During fermentation, acetate esters are produced through enzyme-catalysed condensation reactions, where alcohol transferase enzyme (AFT) facilitates the combination of an acetyl group from acetyl coenzyme A (acetyl-CoA) with a higher alcohol. At the beginning of fermentation, when the concentration of higher alcohols are low, acetyl-CoA and AFT are available for the esterification of citronellol to citronellol acetate. However, as fermentation progresses and the concentration of higher alcohols increases and the rate of fermentation declines, the availability of acetyl-CoA and AFT for esterification of citronellol is reduced. Therefore, the observed decrease in citronellol acetate production after day 1 in the current research is likely due to the increase in higher alcohol concentration.
The use PTR-ToF-MS and Fast GC PTR-ToF-MS has been instrumental in enhancing our understanding of the intricate complexities of dynamic VOC production and biotransformation reactions by yeast. This research has advanced the current understanding of terpenoid biotransformation during beer fermentation, providing valuable information that can support brewers to control and optimise beer aroma and quality.