Abstract
Beer is an alcoholic drink obtained from the controlled fermentation of wort by yeast. Demand for distinctly flavoured beer is increasing and researchers are seeking reliable, cost-efficient methods to enhance the aroma and flavour of beer. During fermentation, brewing yeast is responsible for the production of volatile organic compounds (VOCs), which are important contributors to the sensory profile in beer. Factors that affect yeast growth are known to be capable of influencing VOCs synthesized. Previous studies have reported that a wide range of audible sound frequency and intensity combinations can impact on yeast growth and the production of metabolites, including VOCs. The effects of sound treatment on yeast growth, rate of fermentation and the generation of VOCs in beer is not understood. Moreover, sound delivery in previous studies have been poorly controlled or ill-defined due to ineffective delivery of audible sound via a loudspeaker through air into liquid/solid cultures without accounting for the transmission losses through the medium and the hard vessel walls and the effect of reflection within the vessel. As the method of sound delivery in these previous studies has been poorly controlled, there is a need to better control sound treatment so that the impact of sound stimulation on fermentation can be more accurately assessed and optimized. Such careful and accurate studies will enable the industrial relevance of the application of sound on beer production to be more confidently assessed.
Thus, this dissertation aimed to understand the effects of different frequencies and intensities of audible sound on fermentation by yeast in beer as measured by number of yeast cells in suspension, changes in wort gravity (as a measure of carbohydrate metabolism) and the production of VOCs. To achieve this, two methods of controlled audible sound delivery systems were used. The first method was an underwater sound delivery system where reinforced laminated barrier film bags (2 L) containing pitched wort were partially submerged in a tank filled with water and the audible sound delivered via underwater speakers. The second method of delivery of audible sound to ferments was using an oscillating linear actuators (LAs) acting directly on the flexible wall of the laminate bags containing pitched wort. The use of LAs were intended to increase the particle motion component of the sound in relation to the pressure component. Sound files were generated at different frequency ranges with bespoke MATLAB® scripts and played continuously.
Spray-dried malt extract was used to prepare a standardized wort (12 oBrix), bulk pitched with yeast (~10 x 10^6 cell/mL) and subjected to sound stimulation at a selected frequency range and intensity. A handheld density meter or a Hanna digital refractometer was used to assess gravity during the fermentations. The number of yeast cells in suspension were determined using an Oculyze BB cell counter with methylene blue as a stain. Headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HSPM-GCMS) was used to identify and measure the VOCs. A Gompertz sigmoid function was used to model and predict the terminal gravity of the ferment, which signalled the end of fermentation. Methyl chloroformate derivatisation was used to extract and measure the yeast extracellular metabolites in ferments.
The underwater application of audible sound to beer fermentations elicited limited changes in the fermentation performance (changes in wort gravities, and yeast cells in suspension) and VOCs. The ferments treated with different audible sound frequencies and intensity levels fermented slower and had fewer yeast cells in suspension compared to the control ferments without added sound. There were no significant differences in most VOCs measured between ferments exposed to sound compared to the control, however the control ferments had small but significantly higher abundance of higher alcohols (i.e., 2-methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 1-hexanol, and phenylethyl alcohol) compared to the treated samples. In contrast, the application of audible sound at either 800-2000 Hz, 138.95±0.12 dB @ 20 µPa or 4000-10000 Hz, 138.82±0.23 dB @ 20 µPa to ferments using LA significantly increased the fermentation rate and the number of yeast cells in suspension. Statistical modelling predicted that ferments treated with LA reached terminal gravity 20-40 h ahead of ferments without sound exposure. Most VOCs in ferments exposed to audible sound using LA were not significantly different to the control, although some small effects were observed at certain points during fermentation (i.e., ethyl esters, HAs). In contrast, audible sound at 4000-1000 Hz, 140 dB @ 20 µPa and 800-2000 Hz, 140 dB @ 20 µPa significantly increased (p<0.05) the abundance of 39/68 targeted metabolites compared to ferments without sound. This observation indicates that different audible sound frequency and intensity influenced some metabolic pathways via which these metabolites are synthesised. In conclusion, for the fist-time two novel methods of control audible sound delivery to ferments were successfully designed and applied during beer fermentation. Sound delivery using oscillating linear actuators can significantly increase the fermentation rate without altering flavour compounds and could enhance production throughput. Sound delivery using LA showed significant effects on fermentation parameters (gravity, yeast in suspension) compared to underwater sound treatments owing to the enhanced particle motion in relation to pressure component of sound.