Esters and higher alcohols (fusel alcohols) are important group of metabolites that make a substantial contribution to the development of the flavour profile of the final product. The aim of this research was to develop tools that would permit analysis of flavour volatiles formed during fermentation and use them to determine the impact of initial dissolved oxygen (DO) levels on key fermentation performance indicators. Analysis of solvent extracts and headspace of fermented samples by gas chromatography-mass spectrometry (GC MS) was optimised to permit the quantification of higher alcohols and esters and compared to the data generated by a novel direct mass spectroscopy technique known as atmospheric pressure chemical ionisation-mass spectrometry (APCI-MS). Using these analytical techniques the impact of key process conditions, particularly the effect of initial dissolved oxygen, on fermentation performance and formation of flavour compounds were investigated. It was observed that reductions in the initial DO, increases cell growth rates and utilisation of wort metabolites during the first few hours fermentation. However, as the fermentation continued the final yeast biomass yield was adversely affected. The flavour metabolite concentrations during fermentation were not found to be significantly affected, except in a limited number of scenarios where the changes observed would be unlikely to be perceived as they were well below the sensory threshold. Although reducing initial DO yielded lower peak values of VDK, this apparent benefit was mitigated by the observation that diacetyl uptake was slower in fermentation, where reduced oxygen had been made available to the yeast. The net impact of reducing initial DO was therefore limited in the scenarios examined, however it is anticipated that some negative impacts may be observed if cropped yeast from reduced DO fermentations were repeatedly recycled. Thus future work would involve a serial re-pitching experiment, preferably in a pilot scale, to ascertain the impact of reduced DO using serially re-pitched yeast cells. 2

Premature yeast flocculation (PYF) is a sporadic problem encountered during industrial brewing fermentations. Current hypothesis states that factors, thought to arise from fungal infection of the barley in the field and/or the malt in the maltings cause yeast to flocculate prematurely and/or heavily before the depletion of the sugars in the wort. This results in poorly attenuated worts, with higher residual extract and lower ABV, flavor abnormalities (i.e. diacetyl, SO2), lower carbonation levels, disruption of process cycle times and potential issues with the re-use of the yeast in subsequent fermentations. Consequently, PYF generates significant financial and logistical problems both to the brewer and the maltster. In the current study a small-scale fermentation assay was developed and optimized to predict the PYF potential of malts, as well as to investigate the importance of yeast strain in the incidence and severity of the phenomenon. Furthermore, the impacts of the PYF factor(s) (i.e. arabinoxylans, antimicrobial peptides) on yeast fermentation performance and metabolite uptake were also studied, whilst the Biolog detection system was investigated as a potential rapid tool which to detect PYF. The results obtained suggested that our in-house assay can be successfully used to predict the PYF potential of malts 69 or 40 h post-pitching depending upon the yeast strain used. Whilst ale yeasts were not found susceptible to PYF, lager yeasts exhibited different degrees of susceptibility even to the same PYF factor(s). More specifically, the more flocculent lager yeast SMA was found to be more susceptible than the medium flocculent lager yeast W34/70. However, interestingly, the fermentation performance of a PYF+ wort could be significantly improved by using a non-flocculent and relatively insensitive to PYF lager yeast. It was also shown that worts with lower amount of glucose and maltose could be responsible for poor fermentation profiles and/or heavy PYF as well as elevated residual sugars and lower fermentability. The observation that linoleic acid (6 mg.l-1) exacerbated PYF (P = 0.047) and made its detection more rapid was found to be contrary to the “titration hypothesis” (Axcell et al., 2000) which hypothesized that the addition of fatty acids might “titrate” out antimicrobial peptides so that they can no longer bind to the yeast cells. High gravity fermentations with worts inducing PYF did not have a significant effect (P > 0.05) on yeast physiological characteristics or fermentation performance suggesting that the PYF+ sample used in this study was inducing PYF though the ‘bridging’ polysaccharide mechanism rather than through the antimicrobial peptides. The Biolog system can be used for the metabolic characterization of different flocculence lager yeasts incubated in different fermentation media, whilst wort composition had a significant effect in redox reduction reactions.

- / -