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==Brewing Methods==
<!--Removing graph for now due to out of date information
[[File:Funky_Ferm.jpg|thumb|upright=2.5|Conceptual graph of traditional microbe and wort dynamics|Conceptual graph of dynamics of funk expression and inoculation timing of Brettanomyces. Y-axis for each microbe group depicts relative activity which combines in a conceptual sense: growth, attenuation, and production of flavor compounds. Plot was drawn by Drew Wham based on concepts discussed in American Sour Beer <ref> Tonsmeire, M. (2014). American Sour Beers. Brewers Publications </ref>. See the table to the left for updated information on where this graph may no longer be accurate. ]]
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! Technique !! More Funk !! Less Funk !! Note
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| Inoculation timing || After ''Saccharomyces '' has finished fermentation || At the start of Fermentation || The timing of the ''Brettanomyces'' pitch may not have a significant effect a lot of the time; pending data from George Van Der Merwe or another source of scientific data <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2103639509664252/?comment_id=2117621214932748&comment_tracking=%7B%22tn%22%3A%22R%22%7D Richard Preiss. Milk The Funk Facebook thread on co-pitching versus subsequent pitching of ''Brettanomyces''. 06/04/2018.]</ref>.
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| ''Brettanomyces'' Inoculation cell count || Lower cell count or higher cell count || Higher cell count or lower cell count|| Although more data is needed, pitching rate of ''Brettanomyces'' may not have a measured impact on beer flavor. See [[Brettanomyces secondary fermentation experiment]].
===Dosing Clean Beer with ''Brettanomyces'' At Bottling===
One method that some brewers attempt is adding a small pitch of ''Brettanomyces'' to a clean beer at bottling time. This can be done either in the bottling bucket/tank, or added to each bottle individually. If adding ''Brettanomyces'' to each bottle individually, a 1 mL dosage of ''Brettanomyces'' from a starter should be enough since pitching rate seems to have little impact on the beer <ref>[[Brettanomyces_secondary_fermentation_experiment]]</ref>. Some brewers believe that adding the ''Brettanomyces'' at bottling time results in a more complex beer. It is speculated that the extra stress of pressure within the bottles helps to create this complexity, although evidence for this is lacking. Other stressors such as lack of oxygen are also speculated to have an impact.
One challenge with this approach is that it is difficult to predict how much ''Brettanomyces'' will further attenuate the beer once in the bottle. Over-carbonation and bottle bombs can easily be an issue with this method if the brewer is not careful. Each degree Plato adds ~2 volumes of CO2 <ref>[http://braukaiser.com/wiki/index.php/Accurately_Calculating_Sugar_Additions_for_Carbonation#Remaining_or_Residual_Extract "Accurately Calculating Sugar Additions for Carbonation." Kai Troester. Braukaiser.com. Retrieved 08/07/2016.]</ref>. Since different species and strains of ''Brettanomyces'' ferment different types of sugars, some strains might be safer for dosing at bottling time. For example, most strains of ''B. anomulus'' do not ferment maltose, which is around 50% of sugar in wort, so this makes it a good choice for adding to the beer at bottling. However, if the amount of additional attenuation is already known for a particular beer and a particular strain of ''Brettanomyces'', than any strain can be used.
See also:
* [https://www.masterbrewerspodcast.com/062 Interview with Nick Mader on the MBAA podcast.]
* [[Brettanomyces_secondary_fermentation_experiment|''Brettanomyces'' secondary pitch rate experiment by Lance Shaner and Richard Preiss.]]
* [[Mixed_Fermentation#Staggered_Versus_Co-Pitching|Mixed Fermentation Staggered vs Co-pitching]].
* [[Lactobacillus#Effects_on_Mixed_Fermentation|Effects of ''Lactobacillus'' on cofermentationco-fermentation]]. ==Effects of ''Saccharomyces'' Strain Selection and Staggered vs Co-Pitch==Anecdotally, brewers often claim that the primary strain of ''S. cerevisiae'' makes a difference when co-fermented with ''Brettanomyces''. However, until recently, there has been limited scientific analysis to back this claim, and some brewers might disagree with this claim. Similar differences in anecdotal experiences have been shared by brewers in regards to co-pitching ''Saccharomyces'' and ''Brettanomyces'' at the same time, or adding the ''Brettanomyces'' pitch after the ''Saccharomyces'' fermentation has finished (often called a "staggered pitch"). ===Review of Scientific Analysis===[[File:Tyrawa Fig 15.JPG|thumbnail|250px|right|Figure 15 from [https://atrium.lib.uoguelph.ca/xmlui/handle/10214/14757 Caroline Tyrawa's Masters thesis]. Uploaded with permission from Caroline Tyrawa and Dr. G. van der Merwe's laboratory at University of Guelph Department of Molecular and Cellular Biology.]][[File:Tyrawa Fig 19.JPG|thumbnail|250px|right|Figure 19 from [https://atrium.lib.uoguelph.ca/xmlui/handle/10214/14757 Caroline Tyrawa's Masters thesis]. Uploaded with permission from Caroline Tyrawa and Dr. G. van der Merwe's laboratory at University of Guelph Department of Molecular and Cellular Biology.]] According to [https://atrium.lib.uoguelph.ca/xmlui/handle/10214/14757 Caroline Tyrawa's masters thesis], some of the flavor impacts of the ''Saccharomyces cerevisiae'' fermentation can remain intact despite the flavor impacts of a subsequent ''Brettanomyces bruxellensis'' fermentation. This indicates that the strain selection for ''S. cerevisiae'' in a co-fermentation with ''B. bruxellensis'' can make a difference to the final product. Tyrawa tested the fermentation of three different strains of ''S. cerevisiae'' and three different strains of ''B. bruxellensis''. The strains of ''S. cerevisiae'' were the Cal Ale strain (neutral), the Vermont Ale strain (fruity), and the St. Remy Belgian strain (phenolic). The three ''B. bruxellensis'' strains were the BSI Drei strain, a strain isolated from a winery, and a strain isolated from a brewery. The ''B. bruxellensis'' strains were selected for their desirable flavor impact in beer as well as their ability to ferment the sugars present in wort. Each of the 6 strains were given a primary-only fermentation. In addition, for each of the ''S. cerevisiae'' strains, they were also co-fermented in combination with each of the ''B. bruxellensis'' strains pitched at one time (a total of 9 combinations). Another set of co-fermentations were done by allowing the ''S. cerevisiae'' strains to ferment for 7 days before adding the ''B. bruxellensis'' strains (staggered pitches). The fermentations were analyzed during a total of 21 days at 22°C. The rate of sugar fermentation was measured, as well as analysis of key flavor compounds <ref name="Tyrawa_Masters">[https://atrium.lib.uoguelph.ca/xmlui/handle/10214/14757 Demystifying Brettanomyces bruxellensis: Fermentation kinetics, flavour compound production, and nutrient requirements during wort fermentation. University of Guelph, Masters Thesis. Department of Molecular and Cellular Biology. 2020.]</ref>. The primary fermentations consumed the wort sugars as would be predicted, with the ''S. cerevisiae'' strains completing fermentation in 5 days, and the ''Brettanomyces'' strains reaching a similar level of attenuation after a 1-2 day lag phase but at a slower rate which continued during all 21 days and probably continuing past 21 days if they were allowed to continue fermentation. For the staggered pitches, the ''S. cerevisiae'' strains appeared to be finished with their fermentation by the time the ''B. bruxellensis'' strains were pitched on day 7. After a day or two of lag, the ''B. bruxellensis'' strains slowly continued to attenuate the wort over the next 14 days and did not taper off at the end of the additional 14 days (21 days total counting the initial ''S. cerevisiae'' fermentation), indicating that attenuation by the ''B. bruxellensis'' strains may not have been finished. For the staggered co-pitches, the highest fermentation rate was achieved with the all three of the B. bruxellensis strains that were co-fermented with the St. Remy Belgian strain and the lowest fermentation rate was with the Cal Ale strain, indicating that the strain choice of ''S. cerevisiae'' affects the fermentation rate over time in combination with the strain of ''B. bruxellensis''. The specific combination of ''B. bruxellensis'' strain and ''S. cerevisiae'' strain can have different effects on fermentation rate as well. For the Cal Ale and Vermont Ale strains, the wine strain of ''B. bruxellensis'' fermented the most efficiently, while the BSI Drei strain fermented most efficiently in combination with the St. Remy Belgian strain. The author did not speculate on why this might be the case. Interestingly, the set of fermentations where both the ''S. cerevisiae'' and ''B. bruxellensis'' were inoculated at the same time did not have this effect. These co-fermentations where the strains were pitched at the same time there looked much like the primary fermentation of the ''S. cerevisiae'' strains where the fermentations were mostly done after 5-6 days with no noticeable attenuation after this short time. This data indicates that higher and slower attenuation occurs when ''Brettanomyces'' is inoculated after the primary ''S. cerevisiae'' fermentation has finished, but not when ''S. cerevisiae'' and ''B. bruxellensis'' are inoculated at the same time <ref name="Tyrawa_Masters" />. Key flavor compounds were analyzed for the different fermentations. Overall, the results showed that some flavor compounds produced by ''S. cerevisiae'' remained even when co-fermented with ''Brettanomyces'' (either co-pitched, or staggered pitch), indicating that the strain selection for ''S. cerevisiae'' for a co-fermentation remains important for the final flavor profile of the beer (see Figure 15). It is also possible that since this experiment was only conducted for 21 days that the ''Brettanomyces'' did not have enough time to have its full flavor impact. In general, there were no significant flavor differences between the co-pitched fermentations versus the staggered pitch fermentations (despite there being a very significant attenuation difference as previously mentioned) <ref name="Tyrawa_Masters" />. The ester profiles of the co-fermentations (both the staggered and co-pitch) were a little bit subdued compared to the primary fermentations with ''Brettanomyces'', indicating that primary fermentations with ''Brettanomyces'' produces higher amounts of esters versus co-fermentation of ''Brettanomyces'' with ''S. cerevisiae''. For example, lower levels of ethyl caproate, ethyl butyrate, ethyl caprylate, ethyl decanoate, ethyl nonanoate, and ethyl lactate, were seen in the co-fermentations versus the ''Brettanomyces'' primary fermentations with some of these compounds dropping below flavor threshold levels (ethyl caproate, for example). The ester production of ''Brettanomyces'' peaked at 14 days, and then esters slowly degraded. In the co-fermentations, the ''Brettanomyces'' appeared to be degrading acetate esters produced by the ''S. cerevisiae'', such as phenyl ethyl acetate, and producing higher amounts of of ethyl acetate. Phenol production began as soon as ''Brettanomyces'' was pitched, and this has been hypothesized to play a large role in replenishing NAD<sup>+</sup> to alleviate the initial lag growth phase in ''Brettanomyces''. Interestingly, levels of 4-ethylphenol were produced at a faster rate in the 100% ''Brettanomyces'' fermentations, but by the end of the 21 day trial period the the 100% ''Brettanomyces'' ferments had slightly lower concentrations of 4-EP versus the co-fermentations with ''S. cerevisiae'', indicating that perhaps 100% ''Brettanomyces'' fermentations produce more phenols up front, but after some time of aging co-fermentations can produce slightly higher levels of phenols (see Figure 19) <ref name="Tyrawa_Masters" />. See also:* [https://www.youtube.com/watch?v=IudVmYyWXss Presentation from Caroline Tyrawa via Escarpment Laboratories Crowdcast video.] ===''Brettanomyces'' Strain Selection===[https://ir.library.oregonstate.edu/downloads/gh93h631p Riley Humbert's Bachelors thesis] reported that the ability to hyper attenuate beer in secondary fermentation of ''B. bruxellensis'' is strain dependent with some strains fermenting at a slower rate than others (this was also the case for these strains when primary fermenting wort). The fermentation rate did not correlate to phenol production; for example, one strain that produced the most 4-vinyl phenol and 4 ethyl phenol was one of the slowest fermenters. Humbert also reported that strains of ''B. bruxellensis'' isolated from beer performed better than those isolated from wine, which is in agreement with previous experiments <ref>[https://ir.library.oregonstate.edu/downloads/gh93h631p Riley Humbert for the degree of Honors Baccalaureate of Science in Chemical Engineering presented on May 21, 2021. Title: Performance of Brettanomyces Yeast Strains in Primary and Secondary Beer Fermentations.]</ref>.
==See Also==
===External Resources===
* [https://www.whitelabs.com/sites/default/files/belgianchart_0.pdf White Labs list of Belgian yeast strains and characteristics, including phenols/spiciness.]
* [https://beerandbrewing.com/podcast-episode-253-matt-van-wyk-and-brian-coombs-of-alesong-brewing-and-blending/ Craft Beer & Brewing Podcast Episode 253: Matt Van Wyk and Brian Coombs of Alesong (52 mins in).]
==References==