Changes

Although the exact pathway is not known in ''Brettanomyces'' (several are proposed), the conditions for THP production are well documented. ATHP is produced by metabolizing the amino acid L-lysine, along with ethanol and a glucose or fructose molecule. Iron is also needed for THP production, although its exact role in biosynthesis is not known <ref name="Snowdon"></ref>. As with other amino acids, lysine is taken up by ''Saccharomyces'' during fermentation, and then released after fermentation. Levels of lysine fluctuate slightly through throughout fermentation, but are generally high throughout a beer's lifetime <ref>[http://link.springer.com/article/10.1385/CBB:46:1:43 The α-aminoadipate pathway for lysine biosynthesis in fungi. Hengyu Xu, Babak Andi, Jinghua Qian, Ann H. West , Paul F. Cook. Sept 2006.]</ref><ref>[http://pubs.acs.org/doi/abs/10.1021/bi9829940 Lysine Biosynthesis in Saccharomyces cerevisiae:  Mechanism of α-Aminoadipate Reductase (Lys2) Involves Posttranslational Phosphopantetheinylation by Lys5. David E. Ehmann , Amy M. Gehring , and Christopher T. Walsh. 1999.]</ref><ref>[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.2007.tb00249.x/abstract Elucidation of the Role of Nitrogenous Wort Components in Yeast Fermentation. C. Lekkas, G.G. Stewart, A.E. Hill, B. Taidi and J. Hodgson. May 2012.]</ref><ref>[http://www.sciencedirect.com/science/article/pii/S0308814699000710 Proteins and amino acids in beers, their contents and relationships with other analytical data. S. Gorinstein, M. Zemsera, F. Vargas-Albores, J-L. Ochoa, O. Paredes-Lopez, Ch. Scheler, J. Salnikow, O. Martin-Belloso, S. Trakhtenberg. 1999.]</ref>.

Oxygen has a stimulatory effect in ATHP and ETHP production(particularly ATHP), but its exact role is not understood. It has been speculated that since ATHP production is associated with ''Brett'' growth, and ''Brett'' grows better under aerobic conditions, that this is why more ATHP is produced under aerobic conditions <ref>[http://www.brettanomycesproject.com/dissertation/introduction/ Yakobson, Chad. The Brettanomyces Project; Introduction. Retrieved 3/10/2015.]</ref><ref>[http://pubs.acs.org/doi/abs/10.1021/jf071243e The Role of Lysine Amino Nitrogen in the Biosynthesis of Mousy Off-Flavor Compounds by Dekkera anomala. Paul R. Grbin, Markus Herderich, Andrew Markides, Terry H. Lee, and Paul A. Henschke. J. Agric. Food Chem., 2007.]</ref><ref name="Oelofse">[http://scholar.sun.ac.za/handle/10019.1/8437 Significance of Brettanomyces and Dekkera during Winemaking: A Synoptic Review. A. Oelofse, I.S. Pretorius, and M. du Toit. 2008.]</ref>. It has also been hypothesized that oxygen may have a direct effect on the THP molecules themselves <ref name="Snowdon"></ref>. ATHP production was also shown to increase when anaerobically precultured cells were transferred to an aerobic environment, indicating that oxygen has a direct role on the production of ATHP, not just a byproduct of ''Brett'' growth. Interestingly, for unknown reasons ''Brett'' cells grown under aerobic conditions and then transferred to an anaerobic environment still produced significant amounts of ATHP. It has been suggested that the aerobic conditions made the ''Brett'' cells predisposed to creating ATHP <ref name="Snowdon"></ref>. Limiting oxygen exposure during kegging/force carbonating is recommended for helping to reduce ATHP production; even very small amounts can have an effect. Oxygen exposure during ''Brett'' starters could also play a role in stimulate ATHP productionlater on down the road, even if the beer is not exposed to oxygen.

The level of ATHP production varies widely between species and strains of ''Brett'', with some strains not producing it at all insignificant amounts and some others producing very high amounts above taste threshold. Additionally, ATHP production appears to require requires glucose or fructose, which explains why ATHP may be seen more often in stuck wine fermentations rather than wine that has finished fermenting. ATHP production by ''Brett'' was observed in wine with glucose or fructose added, along with synthetic growth media, suggesting that the type of growth substrate does not effect production <ref>[http://www.ncbi.nlm.nih.gov/pubmed/18194246 Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine. Romano A, Perello MC, de Revel G, Lonvaud-Funel A. J Appl Microbiol. 2008 Jun.]</ref>.

Although ''Brett'' is capable of producing APY from L-ornithine, the production rate amount produced is much less than that of LAB. In wine, there isn't enough L-ornithine present to production significant amounts of APY from L-ornithine. Therefore, the presence of APY (which can be detected is much easier to detect aromatically, while than ATHP cannot) indicates a bacterial contamination <ref name="Snowdon"></ref>.

Heterofermentative [[Lactobacillus]] spp., particularly ''L. hilgardii'' and ''L. brevis'', can also produce high levels of ATHP and APY from L-lysine/L-ornithine, ethanol, and iron. L-lysine stimulates production of ATHP, and L-ornitine stimulates the production of APY <ref name="Costello">[http://pubs.acs.org/doi/abs/10.1021/jf020341r Mousy Off-Flavor of Wine:  Precursors and Biosynthesis of the Causative N-Heterocycles 2-Ethyltetrahydropyridine, 2-Acetyltetrahydropyridine, and 2-Acetyl-1-pyrroline by Lactobacillus hilgardii DSM 20176. Peter J. Costello and Paul A. Henschke. 2002.]</ref><ref>[http://www.ajevonline.org/content/37/2/127.abstract Formation of Substituted Tetrahydropyridines by Species of Brettanomyces and Lactobacillus Isolated from Mousy Wines. Tamila Heresztyn. 1986.]</ref><ref>[http://onlinelibrary.wiley.com/doi/10.1111/j.1755-0238.2001.tb00205.x/abstract Ability of lactic acid bacteria to produce N-heterocycles causing mousy off-flavour in wine. PETER J. COSTELLO1, TERRY H. LEE1, and PAULA. HENSCHKE. 2008.]</ref><ref>Sparrows, Jeff. ''Wild Brews''. Brewers Publications. 2005. Pg. 112.</ref><ref>[https://books.google.com/books?id=tFjsAuo5WocC&pg=PA348&lpg=PA348&dq=lactobacillus+Tetrahydropyridine&source=bl&ots=QUVyoFtIwK&sig=h1cdjB0r1pIRX2Bms8wVA0UiLk4&hl=en&sa=X&ei=4DX_VPz5CsH6oQSAzoGgBA&ved=0CEwQ6AEwCQ#v=onepage&q=lactobacillus%20Tetrahydropyridine&f=false Lahtinen, Ouwehand, Salminen, von Wright. Lactic Acid Bacteria: Microbiological and Functional Aspects, Fourth Edition. Pg 348.]</ref><ref>[http://ajevonline.org/content/37/2/127.short Heresztyn, Tamila. Formation of Substituted Tetrahydropyridines by Species of Brettanomyces and Lactobacillus Isolated from Mousy Wines.]</ref>. Acetaldehyde has a stimulatory effect on ATHP and APY production, but is not required. No studies have been done to show whether or not oxygen plays a role in ATHP/APY production in LAB <ref name="Snowdon"></ref>. Most species of [[Pediococcus]] do not create forms of THP, although a few species do. In particular, these include ''P. pentosaceus'' <ref>[http://www.uniprot.org/uniprot/Q03HT0 UniProt article. Retrieved 3/10/2015.]</ref><ref>[http://www.uniprot.org/uniprot/U5ZF76 UniProt article. Retrieved 3/10/2015.]</ref>, and ''P. clausenii'' <ref>[http://www.uniprot.org/uniprot/G8PEU4 UniProt article. Retrieved 3/10/2015.]</ref> (note that commercial cultures of [[Pediococcus]] are normally ''P. damnosus''). ''Oenococcus oeni'' and ''Leuconostoc mesenteroides'' have also been associated with creating THPATHP, APY, and ETHP all above threshold amounts. Since only heterofermentative species produce significant amounts of THP, it is thought that it's production is linked to the heterolactic pathway, and thus the metabolism of sugars in LAB <ref name="Costello"></ref>.