Changes

After primary fermentation, the mostly attenuated beer is sometimes moved to a secondary fermentation vessel (and sometimes not; read below). In commercial production secondary fermentation is often conducted in wine barrels (mostly because it is messy to conduct primary fermentation in barrels), however, home brewers can accomplish this phase in glass or plastic carboys with low oxygen permeability. A mixed culture of ''Brettanomyces'', ''Lactobacillus'' and ''Pediococcus'' is then introduced to the beer. If barrels are being used then these microbes may simply come from the walls of the barrel, originating from a previous batch. Alternatively, the brewer might inoculate the wort with a mixed culture directly, either with a house culture or by introducing the dregs of bottled sour beer. Upon their introduction, these new microorganisms begin converting the longer chain sugars left over from the primary fermentation. These sugars are primarily converted into alcohol and lactic acid, increasing the degree of attenuation and lowering the pH of the beer. This also corresponds with a decrease in ''S. cerevisiae'' cell counts, and the release of amino acids and vitamins from yeast autolysis helps feed lactic acid bacteria and ''Brettanomyces'' <ref name="Hubbe">[https://www.facebook.com/groups/MilkTheFunk/1407620505932826/ Effect of mixed cultures on microbiological development in Berliner Weisse (master thesis). Thomas Hübbe. 2016.]</ref>. Other flavor-impacting secondary metabolites are also produced, depending on the strains used. For example, if the beer contains ''Brettanomyces'' this often results in the production of a high amount of fruity esters such as ethyl acetate and ethyl lactate, as well as "funky" phenols and other flavor compounds specific to ''Brettanomyces'' (see [[Brettanomyces#Secondary_Metabolites|''Brettanomyces'' secondary metabolites)]]. In the presence of oxygen, acetic acid is also produced by ''Brettanomyces'' (and acetic acid bacteria if they are present) which in low amounts can be complementary, adding to the complexity of the beer. These flavor compounds are essential to the flavor profile of mixed fermentation sour beer. For example, traditional Berliner Weisse was fermented with a mixed culture containing ''Brettanomyces'', and this was considered the most important aspect of achieving the fruity ester character of that beer style historically (see [https://docs.google.com/spreadsheets/d/1CNrO46TPSFpjhO3HX1-CbKK5rhFd7uGWdpONf7AJAlU/edit#gid=0 Benedikt Koch's table comparing esters of traditional Berliner Weisse versus kettle soured Kindl Weisse and Belgian gueuze]).

Co-pitching all of the microbes to begin with, including the primary ''Saccharomyces'' culture, can produce different results than staggering the pitches of individual species over time. For example, many brewers pitch a single mixed culture that contains ale yeast, ''Brettanomyces'', and lactic acid bacteria. Other brewers, such as Vinnie Cilurzo at Russian River, prefer to pitch their ale yeast first, and then pitch ''Brettanomyces'' and/or lactic acid bacteria after the primary fermentation <ref>Tonsmeire, Michael. "American Sour Beers: InnovativeTechniques for Mixed Fermentations". Brewers Publications. 2014. Pgs 100-101.</ref>. See this [http://brulosophy.com/2018/05/14/mixed-fermentation-combined-vs-staggered-microbe-pitch-exbeeriment-results/ Brulosophy experiment comparing co-pitching versus staggered pitching] (note that oxygen exposure during the staggered pitch and other variables in this experiment could account for some of the differences between the two beers) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2093241697370700/?comment_id=2093637253997811&comment_tracking=%7B%22tn%22%3A%22R%22%7D Dan Pixley and Zach Taggart. Milk The Funk Facebook thread on the "MIXED FERMENTATION: COMBINED VS. STAGGERED MICROBE PITCH" blog post. 05/14/2018.]</ref>.