Changes

THP in wine ("mouse taint") was first described in wine by Müller-Thurgau and Osterwalder in 1913, although mention of an off-flavor in wine that 'is closely resembling to the smell of a residence of mice' dates to 1894 in "A Treatise on wines" by J.L.W. Thudichum. They Müller-Thurgau and Osterwalder attributed it to being produced by bacteria. They also established that the ability to detect THP varies from person to person In the 1940's, some scientists proposed that THP was purely a chemical reaction, however their evidence was inconclusive. In 1977, Tucknott et al. was able to identify that THP production was attributed to both some ''Brettanomyces'' yeast and lactic acid bacteria, and that ethanol and L-lysine played a role in its production, and this was confirmed by Heresztyn et al. in 1986 <ref name="Grbin_1996">[http://iwrdb.org/cgi-bin/koha/opac-detail.pl?biblionumber=27016&shelfbrowse_itemnumber=19456 Developments in the sensory, chemical and microbiological basis of mousy taint in wine. Grbin, P.R, Costello, P.J, Herderich, M. 1996.]</ref>.

The chemical analysis of THP has proven to be a difficult endeavor in science. In 1995, Herderich et al. out of Australia developed a method for chemically analyzing THP for the first time. It was until this time that all three forms of THP (ETHP, ATHP, and APY) could be identified consistently in contaminated wine. It was also during this time that the three forms of THP contributed to the flavor of various foods, such as tortilla chips and taco shells. For example, APY was found in the fermentation of cocoa in 1995. In 1995, Grbin et al. described ''Brettanomyces'' yeast as the yeast that produces THP, while wine strains of ''S. cerevisiae'' did not <ref name="Grbin_1996" />.  In 2000, the Australians Grbin and Henschke showed that some strains but not all of ''B. bruxellensis'', ''B. anomalus'', ''B. nardenensis'', and ''B custersianus'' produce THP, and that THP production was influenced by the carbon source, but not dependent on it (THP was still produced in dry wines with little available nutrients, and fermentation rate was not always related to THP levels) <ref name="Grbin_2000">[http://onlinelibrary.wiley.com/doi/10.1111/j.1755-0238.2000.tb00186.x/abstract PAUL R. GRBIN and PAUL A. HENSCHKE. 2000.]</ref>.  In 2007, the Australian team of Grbin et al. developed a complex method of analyzing forms of THP using a Finnigan TSQ 70 mass spectrometer directly coupled to a Varian 3400 gas chromatograph. The chromatograph was equipped with a 30 m J&W Carbowax 20 CAM fused silica column, 0.25 mm i.d, and 0.25μm film thickness. They were able to confirm that higher lysine levels increased the amount of ATHP produced (but not ETHP), although with diminishing increases of THP as the lysine level was increased. They also discovered that L-ornithine also functioned as a precursor for THP production in ''Brettanomyces''. The group proposed a biochemical pathway for the different forms of THP in ''Brettanomyces' <ref name="Grbin_2007" />.

All species of [[Brettanomyces]] can produce forms of Tetrahydropyridine tetrahydropyridine in varying amounts, although some below threshold. Additionally, Lactic Acid Bacteria (LAB) including [[Lactobacillus]] and [[Pediococcus]] can produce forms of THP. Acetic Acid Bactera (AAB) has also been demonstrated to produce forms of THP <ref name="Snowdon"></ref><ref name="Grbin_2000" />.

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-lysineor D-lysine <ref name="Grbin_2007" />, 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 throughout fermentation, but are generally high throughout a beer's lifetime (including after fermentation) <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>. Wheat generally has a slightly lower amount of lysine than barley, and oats have a slightly higher amount of lysine than barley <ref>[http://www.aaccnet.org/publications/cc/backissues/1983/Documents/chem60_461.pdf Amino Acid Composition of Six Grains and Winter Wheat Forage. Morey, D.D. 1983.]</ref><ref>[https://diy.soylent.com/ingredients/oats "Oats". DIY Soylent website. Retrieved 02/07/2017.]</ref><ref>[https://diy.soylent.com/ingredients/barley-malt-flour "Barley malt flour". DIY Soylent website. Retrieved 02/07/2017.]</ref><ref>[https://diy.soylent.com/ingredients/wheat-flour-wholegrain "Wheat flour, whole-grain". DIY Soylent website. Retrieved 02/07/2017.]</ref>.

Oxygen plays a key role and 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 ''Brettanomyces'' growth, and ''Brettanomyces'' 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><refname="Grbin_2007">[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 ''Brettanomyces'' growth <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 (although the exact threshold of how much oxygen is required has not been determined). For example, the purity of the CO² supply should thus be taken into consideration when force carbonating. At 0.5% impurity (the impurity is air, 1/5 of which is oxygen) and at 2 volumes of CO², ~1,420 ppb of O² would be added to the packaged beer, which is an exceedingly high amount of oxygen. The CO² supply should ideally be 99.990% pure or better (this would introduce 46 ppb of oxygen at 2 volumes of CO²). The method that the CO² is added can also determine how much oxygen is introduced into the packaged beer. Sparging CO² (bubbling it through the beer) dissolves significantly less oxygen due to Henry's Law (see reference), while injecting (flushing) dissolves significantly more oxygen <ref>[https://tapintohach.com/2014/01/27/how-the-purity-of-sparged-carbon-dioxide-affects-the-oxygen-concentration-of-beer/ How the Purity of Sparged Carbon Dioxide Affects the Oxygen Concentration of Beer. Tap Into Hach blog. 01/24/2014. Retrieved 06/29/2017.]</ref><ref>[https://tapintohach.com/2013/12/02/how-the-purity-of-injected-carbon-dioxide-affects-the-oxygen-concentration-of-beer/ How the Purity of Injected Carbon Dioxide Affects the Oxygen Concentration of Beer. Tap Into Hach blog. 12/02/2013. Retrieved 06/29/2017.]</ref>. Vessel purging methods with CO² are also less efficient than some might expect, and might still leave enough oxygen behind to stimulate THP production (see [http://www.homebrewtalk.com/showpost.php?p=8004741&postcount=3 this HomebrewTalk thread]). Pitching fresh ''Saccharomyces'' at bottling/kegging time and naturally carbonating the beer with sugar has reportedly reduced mousy off-flavor detection, perhaps because ''Saccharomyces'' metabolizes both the oxygen and sugar faster than ''Brettanomyces''. Different strains of ''S. cereivisaecerevisiae'' might be more efficient than others at helping reduce THP. For example, Mitch Ermatinger from Speciation Artisan Ales anecdotally observed that switching from CBC1 conditioning yeast to EC1118 reduced THP off-flavors from 1 month to two weeks <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1839193092775563/?comment_id=1839266972768175&reply_comment_id=1839402709421268&comment_tracking=%7B%22tn%22%3A%22R7%22%7D Mitch Ermatinger. Milk The Funk Facebook group post on THP reduction using CBC1 and EC1118. 10/03/2017.]</ref> (see [[Packaging#Re-yeasting|Packaging]] for details on re-yeasting at packaging time).

The level of ATHP production varies widely between species and strains of ''Brettanomyces'', with some strains producing insignificant amounts and others producing very high amounts above taste threshold<ref name="Grbin_2000" />. Additionally, ATHP production 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 ''Brettanomyces'' 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 ''Brettanomyces'' is capable of producing APY from L-ornithine<ref name="Grbin_2007" />, the amount produced is much less than that of LABand high amounts of L-ornithine are required. In wine, there isn't enough L-ornithine present to production significant amounts of APY from L-ornithine. Therefore, the presence of APY (which is much easier to detect aromatically than ATHP) indicates a bacterial contamination in wine (it is unknown if this applies to beer) <ref name="Snowdon"></ref>.

Although research is limited, acetic acid bacteria (''Gluconobacter'' sp.and many strains of ''Acetobacter aceti'') have been shown to occasionally produce forms of THP <ref name="Snowdon"></ref>.