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''Brettanomyces'' strains may possess both alpha and beta-glucosidases. These enzymes allow ''Brettanomyces'' strains to break down a broad range of sugars, including long -chain carbohydrate molecules (polysaccharides, dextrins, and cellulose/cellobiose), and to liberate glycosidically bound sugars which are unfermentable to ''Saccharomyces'' yeasts. <ref name="Steensels"></ref><ref>[http://www.scribd.com/doc/277758178/Insight-into-the-Dekkera-anomala-YV396-genome Insight into the Dekkera anomala YV396 genome. Samuel Aeschlimann. Self-published on Eureka Brewing Blog. Spet 2015.]</ref>.

Glycosides are flavorless compounds often found in plants/fruits that are composed of a molecule (often a flavor active compound) bound to a sugar molecule. The glycosidic bond can be broken, releasing the sugar molecule and the potentially potential flavor active compound. These bonds can be broken with exposure to acid, as well as specific enzymes (beta-glucosidase) which can be added synthetically or produced naturally by some microorganisms, including some strains of ''Brettanomyces'' that have beta-glucosidase enzyme activity (mostly ''B. anomalus'' strains) <ref>[https://en.wikipedia.org/wiki/Glycoside "Glycoside." Wikipedia. Retrieved 06/27/2016.]</ref>. The release of flavor molecules from glycosides is thought to contribute to the flavor development of aging wines, as well as kriek (cherry) lambic <ref name="Daenen2">[http://onlinelibrary.wiley.com/doi/10.1111/j.1567-1364.2008.00421.x/pdf Evaluation of the glycoside hydrolase activity of a Brettanomyces strain on glycosides from sour cherry (Prunus cerasus L.) used in the production of special fruit beers. Luk Daenen, Femke Sterckx, Freddy R. Delvaux, Hubert Verachtert & Guy Derdelinckx. 2007.]</ref>. It is speculated that flavor compounds from hops can also be released from glycosides <ref name="Daenen1">[http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2007.03566.x/full Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts. L. Daenen, D. Saison, F. Sterckx, F.R. Delvaux, H. Verachtert, G. Derdelinckx. 2007.]</ref>, however, at least one study has shown no significant difference in a blind taste test between hopped beer exposed to the beta-glucosidase enzyme enzymes and hopped beer that was not exposed to the enzyme <ref name="Vervoort">[http://onlinelibrary.wiley.com/wol1/doi/10.1111/jam.13200/abstract Characterization of the recombinant Brettanomyces anomalus β-glucosidase and its potential for bioflavoring. Yannick Vervoort, Beatriz Herrera-Malaver, Stijn Mertens, Victor Guadalupe Medina, Jorge Duitama, Lotte Michiels, Guy Derdelinckx, Karin Voordeckers, and Kevin J. Verstrepen. 2016.]</ref>.

Other than sugars, nitrogen is an essential nutrient for yeast, and generally occurs in wort in the form of amino acids <ref>Principles of Brewing Science, A Study of Serious Brewing Issues. George Fix. Brewers Publications. 1999. Pg 40.</ref>. ''Brettanomyces'' can survive in environments that are very low in nitrogen. While nitrogen usage for ''S. cerevisiae'' is well understood, the general utilization of nitrogen by ''Brettanomyces'' and its preferred sources for nitrogen under the stressful conditions of fully fermented beer and wine are not yet well known. However, it is known that ''Brettanomyces'' can use a wide range of sources for nitrogen, and its requirements for nitrogen as a nutrient are extremely low when oxygen is available. When oxygen is not present, nitrogen is required for the survival and growth of ''Brettanomyces''. Sources of nitrogen include amino acids such as lysine, histidine, arginine, asparagine, aspartic cid, glutamic acid, and alanine <ref name="smith_divol_2016" />. Some strains of ''Brettanomyces'' can metabolize other nitrogen sources, such as the amino acids proline and arginine <ref name="Crauwels1"></ref>. Ammonium nitrates may also be utilized by some strains of ''B. bruxellensis''. Although studies have been contradictory and some have not documented whether conditions were aerobic or anaerobic (these contradictions might also be due to strain differences between the ''B. bruxellensis'' strains that were used in different studies), it appears as though some strains of ''B. bruxellensis'' might be able to take advantage of trace amount of amino acids that ''S. cerevisiae'' does not use during fermentation, and nitrates and nitrites that ''S. cerevisiae'' is not able to consume, as well as amino acids from yeast autolysis (proline, leucine, tryptophan, and gamma aminobutyric acid) <ref name="smith_divol_2016"></ref>. Other compounds from ''Saccharomyces'' autolysis may also be used by ''Brettanomyces'', such as glucose, fatty acids, nucleotides, polysaccharides, polypeptides, and other proteins <ref>Private correspondence with Richard Preiss by Dan Pixley. 08/23/2016.]</ref><ref>[http://oeno-one.eu/article/view/1701 Influence of yeast autolysis after alcoholic fermentation on the development of Brettanomyces/Dekkera in wine. Michèle Guilloux-Benatier, D. Chassagne, Hervé Alexandre, Claudine Charpentier, Michel Feuillat. 2001.]</ref>. The role that oxygen plays in the ability of ''B. bruxellensis'' to uptake nitrogen from various sources might be an important one, and something that should be examined in science going forward <ref name="smith_divol_2016"></ref>.

''Secondary metabolites'' are compounds that are not essential to the life of an organism <ref>[http://en.wikipedia.org/wiki/Secondary_metabolite Wikipedia. Secondary Metabolite. Retrieved 6/2/2015.]</ref>. ''Brettanomyces'' will use a range of secondary metabolites to produce many of the fruity and funky esters, phenols, and acids that this genus of yeast has become known for. ''Brettanomyces'' has also been observed anecdotally to produce thin beer when fermented on it's its own, and this has at least partially been attributed to the lack of glycerol production by ''Brettanomyces''. The lack of glycerol production has been attributed to a genetic predisposal to prefer pyruvate production over glycerol production during fermentation, and it has been speculated that this gives ''Brettanomyces'' an adaptive advantage <ref>[http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0155140 Independent Evolution of Winner Traits without Whole Genome Duplication in Dekkera Yeasts. Yi-Cheng Guo, Lin Zhang, Shao-Xing Dai, Wen-Xing Li, Jun-Juan Zheng, Gong-Hua Li, Jing-Fei Huang. 2016.]</ref><ref name="yakobson_introduction"></ref>. The major secondary metabolites of ''B. bruxellensis'' fermentation have been identified in one study as the ethyl phenols (4EP and 4EG), the alcohols isoamyl alcohol, 2-methyl-butanol, 2-ethylhexanol, phenethyl alcohol, and an ester ethyl 2-methyl butyrate. Many other compounds are considered minor secondary metabolites and are produced in varying degrees or not at all based on the strain of ''Brettanomyces'', but may still be produced in high enough concentrations to contribute to the flavor and/or aroma in beers fermented with ''Brettanomyces''. The types and amounts of flavor compounds produced by ''Brettanomyces'' cover a wide spectrum, and many factors such as species/strain, amino acid precursors, the presence of oxygen, and other nutrients, play a large role in the production of these compounds. In one study on ''Brettanomyces'' in wine, some strains rated as being perceived positively if the strains metabolized certain compounds slower and produced other compounds slower, indicating that the age of the fermented beverage also plays a large role in how beverages fermented with ''Brettanomyces'' are perceived <ref name="Lucy_2015" />. Some strains of ''B. bruxellensis also produce the amines cadaverine, hexylamine, phenylethylamine, putrescine and spermidine, under wine-model conditions <ref name="Agnolucci_2017" />.

''Brettanomyces'' is capable of synthesizing several ethyl esters from ethanol and fatty acids. Among the most prolific of these are ethyl acetate, ethyl lactate, phenethyl acetate, ethyl caproate, ethyl caprylate, ethyl deconoate <ref name="Tyrawa_2017" />, along with the hydrolysis (breakdown) of isoamyl acetate. During non-mixed fermentations where lactic acid is minimal to none, insignificant amounts of ethyl lactate ester is esters are produced, whereas ethyl caprylate and ethyl caproate have a general increase. With the addition of lactic acid, ethyl lactate levels are greatly increased although may still not reach the flavor threshold level of 250 mg/L (strain dependent), and ethyl acetate is generally slightly increased. The amounts of esters produced varies vary widely based on species and strain <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>. A similar but slower evolution of esters has been seen in a long -term study on examining how Belgian lambic from Cantillon ages in bottles. The study found that lactic acid (produced by lactic acid bacteria) and ethyl lactate increased as bottles aged, while ethyl decanoate and isoamyl acetate decreased, all presumably from ''Brettanomyces'' metabolism over time <ref>[http://horscategoriebrewing.blogspot.com/2016/02/thoughts-on-spitaels-and-van.html "Thoughts on Spitaels and Van Kerrebroeck et al, 2015." Dave Janssen. Hors Catégorie Blog. 02/20/2016. Retrieved 03/15/2016.]</ref>.

Ester production peaks towards the end of growth, and is influenced by temperature, aeration/agitation, and pH. Spaepen and Verachtert found in one study that the optimal temperature for growth and thus ester production was 28°C (77°F), although they did not test higher temperatures. This study also found that continuously shaken samples produced relatively less fewer 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, no agitation, 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>. Another study by Tyrawa et al. found that all strains of ''B. bruxellensis'' tested produced above threshold levels of ethyl caproate, ethyl caprylate, and ethyl deconoate esters at 15°C versus 22.5°C, but for some strains the higher fermentation temperature of 22.5°C produced significantly more of these esters than the lower 15°C temperature (other strains produced similar levels of esters at both temperatures, although they fermented slower at 15°C) <ref name="Tyrawa_2017" />.

Pitching rate of ''Brettanomyces'' may have a slight effect on ester production levels, but the differences caused by pitching rate probably do not have a significant impact on the sensory character of the beer <ref name="MTF_Brett_Secondary"></ref>. ''Brettanomyces'' produces higher levels of esters when fermented without competition from ''S. cerevisiae'', and this correlates with higher ''Brettanomyces'' cell growth when not in competition with ''S. cerevisiae'' (see [[100%25_Brettanomyces_Fermentation#Are_100.25_Brett_Beers_Really_Cleaner.3F|100% ''Brettanomyces'' Fermentation]]) <ref name="Hubbe">[https://lookaside.fbsbx.com/file/Final%20work%202%20-%20Thomas%20H%C3%BCbbe.pdf?token=AWyH17JH23uJ-wby5L7bZBZ-_G9EbxFbtNZhoHdq9nFQXDyOlNW66kYos4cpt_oOzIGzmllGYexkcE6o3bESICERaG8rSM4SruxzJVAaDb7UaoeAfVvLY_7uNezyeiynjnVG1T1zYyf-Zl4f2E6NwyOIX0y9hlh78XXVWFGHZySDEA Effect of mixed cultures on microbiological development in Berliner Weisse (master thesis). Thomas Hübbe. 2016.]</ref>. The aromatic amino acids phenylalanine, tryptophan, and tyrosine have been associated with higher ester formation <ref name="Lucy_2015" />.

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 are 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 sometime 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 and reaches an equilibrium. 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 an 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 and 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>. Pitching rate of ''Brettanomyces'' has an effect on the breakdown of isoamyl acetate with higher pitching rates breaking down this ester at a faster rate <ref name="MTF_Brett_Secondary"></ref>.

In the presence of oxygen, ''Brettanomyces'' strains produce acetic acid as a byproduct of glucose fermentation. This is thought to be a defensive tactic against competing microorganisms (e.g. ''Brettanomyces'' has been shown to produce more acetic acid when co-fermented with ''S. cerevisiae'', and ''S. cerevisiae'' has been shown to have less viability over time in the presence of acetic acid and ethanol) <ref name="Hubbe"></ref>. Depending on the brewer's palate and the degree of acetic production, this can be a desirable or undesirable trait. The degree of acetic acid production varies among different ''Brettanomyces'' strains, and is limited by limiting oxygen exposure. Acetic acid produced by ''Brettanomyces'' is also used in the synthesis of [[Secondary metabolites|acetate esters]] such as ethyl acetate, perhaps as a mechanism to protect itself after hindering other microbes via the acetic acid precursor. ''Brettanomyces'' has been shown to produce enough fatty acids in anaerobic fermentation to drop the pH to 4.0, which can also be esterified (see the ester table above) <ref name="yakobson1"></ref>. Many of these acids can have an unpleasant rancid odor and/or taste, which may be noticeable in young ''Brettanomyces'' beers before these acids are esterified.

Michael Lentz and Chad Harris tested whether or not the hydroxycinnamic acids (HCAs) inhibit the growth of ''Brettanomyces''. They found that high levels of hydroxycinnamic acids (HCAs), which includes ferulic acid, p-coumaric acid, and caffeic acid, do inhibit the growth of ''Brettanomyces''. Ferulic acid is the strongest inhibitor of these three HCAs with most strains tested not being able to grow in wort that contained 12 mM (millimolar) of ferulic acid. Caffeic acid was generally shown to be the weakest inhibitor of the three HCAs tested. Levels of 25 mM p-courmaric coumaric acid inhibited the growth of all strains tested, and levels of 30 mM of caffeic acid inhibited all strains tested. The ability of HCAs to inhibit growth is different from strain to strain of ''Brettanomyces''. Inhibition does not appear to be species dependent. Some strains display a lag time and grow more slowly in the presence of high amounts of HCA's, but still eventually achieve maximum growth compared to if they were grown without exposure to HCAs, while others lag and then stop growing before reaching maximum growth <ref name="Lentz"></ref>.