Changes

| 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>.

Anecdotally, brewers often claim that the primary strain of ''S. cerevisiae'' makes a difference when co-fermented with ''Brettanomyces'', however. 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").

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 1-2-3 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 affects in 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⁺ to alleviate the initial lag growth phase in ''Brettanomyces''. Interestingly, levels of 4-ethylphenol were slightly lower 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 less higher levels of phenols (see Figure 19) <ref name="Tyrawa_Masters" />.