Changes

===Review of Scientific Analysis===

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" />.

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 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" />.