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This page documents an ongoing collaborative experiment conducted by Lance Shaner of [[Omega Yeast Labs]] and Richard Preiss of [[Escarpment LabsLaboratories]] to investigate the effect of ''Brettanomyces'' secondary /bottle pitch rate on the sensory characteristics and flavour flavor metabolites of beer during bottle conditioning.
==Introduction==
The effect of pitch rate on flavor in ''Brettanomyces'' bottle/secondary fermentation has been a contentious point of discussion amongst brewers. The vast majority of available information is strictly anecdotal, which highlights the need to analyze a ''Brettanomyces'' secondary fermentation with different pitch rates in order to better assess the effect pitch rate may have. It has been suggested previously that stressing ''Brettanomyces'' cells by underpitching will result in higher production of funky metabolites, such as phenolic compounds and organic acids. It is known that ''Brettanomyces'' is capable of production of volatile esters, phenols and organic acids during secondary fermentation, but whether or not these metabolites are produced in a dose-dependent manner is unknown. Furthermore, the rate at which these reactions occur is also poorly characterized in the context of beer secondary fermentations. In this study, we aim to determine whether there is a dose/pitch rate-dependent effect of Brettanomyces on a secondary beer fermentation, and if so, whether this effect is reduced during prolonged beer aging.
==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. 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>
==Results==
<gallery>
File:phenols-omegaesc.png|'''Figure 1''' Measurement of phenolic compounds at 3 weeks.
File:Ethylesters-omegaesc.png|'''Figure 2''' Measurement of ethyl esters at 3 weeks.
File:Acetate-omegaesc.png|'''Figure 3''' Measurement of acetic acid and acetate esters at 3 weeks.
File:Phenethyl-omegaesc.png|'''Figure 4''' Measurement of phenethyl alcohol and phenethyl acetate at 3 weeks.
File:Orgacids-omegaesc.png|'''Figure 5''' Measurement of volatile organic acids at 3 weeks.
</gallery>
==Results and Discussion== 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. 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. Thus it is possible that acetate esters can be hydrolyzed by ''Brettanomyces'' under anaerobic conditions via an unknown mechanism. We await sampling at the 3 month timepoint to assess whether these trends continue and become more clear.
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). 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.
==See Also==
==References==
<references/>
[[Category:Yeast]]
[[Category:Experiments]]