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==Methods==
The base beer consisted of 12 gallons of 1.053 wort (75% Pilsner, 15% Wheat, & 10% Munich) which was fermented with saison yeast from the OYL-217 C2C American Farmhouse Blend. Final gravity was 1.008 (2ºP). Bottles and bulk aging samples were then inoculated with the ''Brettanomyces bruxellensis'' strain from the same blend. Bottle inoculation rates: 1) zero (control), 2) 50,000 cells/mL, 3) 240,000 cells/mL, 4) 1 million cells/mL, 5) 2.4 million cells/mL. Bottles were then conditioned at room temperature for 3 weeks, after which they were opened for sensory and metabolite analysis.
Bottles of the Control (no Brett added), Low (50k Brett cells/mL), and High (2.4 million cells/mL) were refrigerated for 24 hours prior to tasting. The bottles were coded so that the identities of the samples were concealed. The three participants were given a 2 oz pour of each sample and asked to identify the control, Low, and High. This analysis was performed at 2 and 4 weeks post-bottling.
In general, we observed slightly elevated levels of ethyl esters with the addition of ''Brettanomyces'' (Figure 2). However, there was a high degree of variability among the different pitch rates. Identification and confirmation of trends with regard to ethyl ester production in this experiment would require multiple replicates in order to more accurately assess the data and identify trends. The most dramatic increases in ester production were seen with ethyl nonanoate (tropical fruit) and ethyl caprylate (pineapple) production.
Interestingly, we observed a potential dose-dependent decrease in isoamyl acetate (banana) concentration at 3 weeks (Figure 3). Therefore, higher pitch rates of ''Brettanomyces'' in secondary fermentation may metabolize isoamyl acetate faster, reducing the concentration of this ester. Thus, high pitch rates of ''Brettanomyces'' could be useful to quickly reduce isoamyl acetate in beers where this flavour is undesirable. We also observed a more subtle dose-dependent decrease in concentration of two other acetate esters at high ''Brettanomyces'' pitch rate, ethyl acetate (pear, solvent) and phenethyl acetate (honey, yeasty, floral) (Figures 3, 4). Interestingly, we observed higher concentration of these esters in the low pitch rate sample. Confirming this phenomenon and its potential sensory ramifications would require an expanded experimental design including replicates. Ultimately the differences are subtle, and it is likely that these do not have significant effects on the sensory character of the resultant beers. Concomitant with the slight reduction in acetate esters by addition of ''Brettanomyces'', we also observe slight increase in volatile acetic acid concentration with pitch rate (Figure 5), but the concentration regardless is well below the sensory threshold for acetic acid. We await sampling at the 3 month timepoint to assess whether these trends continue and become more clearclearer.
We were not able to identify any clear trends in levels of volatile organic acids (Figure 5). These compounds typically cause off-flavours in immature ''Brettanomyces'' fermentations.
===== Discussion =====
There are a number of limitations to this experimental design which must be considered. First, the metabolite analysis was conducted using one bottle from each pitch rate at each time point, and therefore does not result in statistically robust data. Further experimentation can use multiple biological replicates (individual growths of the same Brett strain, dosed into bottles and tested in parallel at each time point). Furthermore, the sample size of the sensory panel (n = 3) does not allow for the use of statistics on the data. Additionally, this experiment was conduced conducted with a single ''Saccharomyces''/''Brettanomyces'' combination. The choice of strain for either organism could influence the results. For example, yeast strains that do not produce 4-VG (POF-) do not provide this precursor to ''Brettanomyces'', theoretically leading to a decrease in 4-EG concentration in the resultant ''Brettanomyces'' secondary fermentation. Furthermore, the choice of ''Brettanomyces'' strain may influence the rate and amount of flavor metabolite production in secondary fermentation. As such, it would be important in further experiments to include multiple ''Brettanomyces'' strains in order to correlate the strain-dependent and dose-dependent levels of flavor metabolite production and generate a better overall picture of the effects of ''Brettanomyces'' secondary fermentation on beer.
These results challenge the notion that "stressing" ''Brettanomyces'' by underpitching leads to more or different levels of flavor-active compounds. The results also demonstrate that exceedingly small amounts of Brett cells are needed to quickly cause changes in a beer. The low end of the pitch rates tested represents approximately 1/5 of a White Labs vial pitched into a 5 gallon batch.