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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'' 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. Samples for flavor metabolite analysis were filtered with a 0.2µM syringe filter and stored in 15mL conical tubes and stored at 4ºC prior to flavor metabolite analysis. Metabolite analysis was performed via HS-SPME-GC-MS using the methods outlined in Rodriguez-Bencomo et al. (2012).<ref>[http://link.springer.com/article/10.1007%2Fs12161-012-9390-x Optimization of a HS-SPME-GC-MS Procedure for Beer Volatile Profiling Using Response Surface Methodology: Application to Follow Aroma Stability of Beers Under Different Storage Conditions Rodriguez-Bencomo, J. J., Muñoz-González, C., Martín-Álvarez, P. J., Lázaro, E., Mancebo, R., Castañé, X., & Pozo-Bayón, M. A. 2012.]</ref>
The beers will also be analyzed at a 3 month time point.
File:Orgacids-omegaesc.png|'''Figure 5''' Measurement of volatile organic acids at 3 weeks.
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===== Sensory Analysis =====
At the tasting two weeks after bottling, all three participants correctly identified the Control. The participants noted that the Control had more clove aroma and fuller mouthfeel compared to the two Brett samples. The Brett samples were described as fruity and slightly acidic. No taster was able to discern a significant difference between the Low and High samples.
At the tasting four weeks after bottling, all three participants again correctly identified the Control due to the clove aroma and fuller mouthfeel. The Brett samples were described as fruity but with a stronger medicinal flavor than at two weeks. There was still no discernible difference between the Low and High Brett samples.
===== Metabolite Analysis =====
At 3 weeks, conversion of 4-vinylguiacol (4-VG) to 4-ethylguiacol (4-EG) was observed in all samples dosed with ''Brettanomyces'' (Figure 1). This indicates that regardless of pitch rate, conversion of above-threshold amounts of 4-VG (clove) produced by ''Saccharomyces'' was metabolized to above-threshold levels of 4-EG (spicy, smoky). Additionally, ''de novo'' production of 4-ethylphenol (4-EP, barnyard/medicinal) was observed at around sensory threshold levels regardless of ''Brettanomyces'' pitch rate when compared to the ''Saccharomyces'' control. Taken together, these results indicate that volatile phenol production does not correlate to pitch rate in the range tested, as early as 3 weeks into secondary fermentation.
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 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.