Changes

''Brettanomyces'' is capable of forming several ethyl esters (derived from ethanol and fatty acids). Among these are ethyl acetate, ethyl lactate and phenethyl acetate, along with the hydrolysis of isoamyl acetate. During non-mixed fermentations where lactic acid and acetic acid are minimal to none, these esters are produced in smaller quantities <ref>[http://www.brettanomycesproject.com/dissertation/introduction/ Yakobson, Chad]. Pure Culture Fermentation Characteristics of Brettanomyces Yeast Species and Their Use in the Brewing Industry. Production of Secondary Metabolites. 2011.</ref>.  Ester production peaks towards the end of growth, and is influenced by temperature, aeration/agitation, and pH. One study found that the optimal temperature for growth and thus ester production was 28°C (77°F), although it did not test higher temperatures. This study also found that continuously shaken samples produced relatively less esters, as well as samples that were not exposed to oxygen at all. The highest ester production was found under conditions of limited oxygen supply, not agitated, held at a temperature of 28°C (77°F), and young cells produced more esters than older cells. It also found that esterase activity (esterase is the enzyme that facilitates ester production and destruction) increases as pH rises until a pH of 7.6 is reached, after which it begins to decline again. It was shown that the ester formation/degradation was indeed caused by enzymatic activity of any ''Brettanomyces'' species/strain, and not caused by chemical reactions or from ''Saccharomyces'' or ''Kloeckera'' activity <ref name="Spaepen"></ref>.  Esters are also broken down via a process called hydrolysis. Hydrolysis breaks the esters down using the same esterase enzyme within the ''Brettanomyces'' cells that is used to create esters. In general, all acetate based esters, except for phenethyl acetate and methyl acetate, are broken down faster than non-acetate esters by ''Brettanomyces''. In lambic brewing, some time after the primary fermentation finishes, ''Pediococcus'' begins to produce lactic acid. The formation of lactic acid by ''Pediococcus'' coincides with the appearance and growth of ''Brettanomyces'', which produces more acetic acid. After another 2-3 months, the ester content of the lambic beer changes. Ethyl acetate and ethyl lactate are greatly increased, while isoamyl acetate is greatly decreased, reaching an equilibrium of these esters. Given a static amount of acetic acid, ''Brettanomyces'' reaches equilibrium of ethyl acetate within 24 hours, while ethyl lactate equilibrium takes longer and is much more complex. In lambic, the majority of ester production versus breakdown occurs within 1-3 months after lactic acid production by ''Pediococcus'' begins, and at a pH of around 3.5 and a temperature of around 15°C or less <ref name="Spaepen"></ref>.