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Forms of '''Tetrahydropyridines''' are a group of flavor active compounds found in some wines and sour beers. While harmless, they are often considered to be off-flavors in wine and beer. With the recent increase in spontaneously fermented wine and lower sulphite usage by certain wine producers, these flavors have become more prevalent in wine <ref name="Moulis_2023">[https://oeno-one.eu/article/view/7481 Moulis, P., Miot-Sertier, C., Cordazzo, L., Claisse, O., Franc, C., Riquier, L., Albertin, W., Marchand, S., De Revel, G., Rauhut, D., & Ballestra, P. (2023). Which microorganisms contribute to mousy off-flavour in our wines?. OENO One, 57(2), 177–187. https://doi.org/10.20870/oeno-one.2023.57.2.7481.]</ref>. Forms of tetrahydropyridines (abbreviated and often referred to generically by brewers and winemakers as '''THP'''), specifically 2-acetyl-3,4,5,6-tetrahydropyridine and 2-acetyl-1,4,5,6-tetrahydropyridine (abbreviated ATHP or ACTPY), 2-ethyltetrahydropyridine (abbreviated ETHP or ETPY), and 2-acetylpyrroline (abbreviated ACPY or APY) <ref name="Snowdon"></ref>, which are classified as a [https://en.wikipedia.org/wiki/Ketone ketone] and a cyclic [https://en.wikipedia.org/wiki/Imine imine] <ref>[https://en.wikipedia.org/wiki/6-Acetyl-2,3,4,5-tetrahydropyridine "6-Acetyl-2,3,4,5-tetrahydropyridine". Wikipedia. Retrieved 07/22/2016. ]</ref>, . They are commonly attributed to the "mousy", "urine" (in high amounts) Cheerios® or Cap'n Crunch® (in low amounts), "breakfast cereal", or more generically, "cracker biscuit" flavor in sour beersand wine. The flavor is detected towards the end of the swallow, and the aftertaste can remain for a few more than 10 minutes<ref name="Stenseels_2015_Essential">[https://www.academia.edu/19646963/Brettanomyces_Bruxellensis_Essential_Contributor_in_Spontaneous_Beer_Fermentations_Providing_Novel_Opportunities_for_the_Brewing_Industry Brettanomyces Bruxellensis, Essential Contributor in Spontaneous Beer Fermentations Providing Novel Opportunities for the Brewing Industry. Jan Steensels. BrewingScience, Sept/Oct 2015 (Vol. 68). 2015.]</ref>. 30% of winemakers are not able to detect the flavor of THP in wine (we do not know if this statistic is reflected in sour beer, but some people have reported not being able to taste THP in sour beer) <ref>[https://books.google.com/books?id=8yegCgAAQBAJ&pg=PT1784&lpg=PT1784&dq=oxford+companion+to+wine+Once+detected,+the+taint+renders+the+wine+undrinkable+and+worsens+in+the+glass,+but+as+many+as+30%25+of+winemakers+are+unable+to+detect+it.&source=bl&ots=-3o-uaZUgX&sig=ET7jldMp3Xdk61FknJ54k4P40CQ&hl=en&sa=X&ved=0ahUKEwjsiZ7bxNPaAhVO8WMKHQHaBgMQ6AEIKTAA#v=onepage&q=oxford%20companion%20to%20wine%20Once%20detected%2C%20the%20taint%20renders%20the%20wine%20undrinkable%20and%20worsens%20in%20the%20glass%2C%20but%20as%20many%20as%2030%25%20of%20winemakers%20are%20unable%20to%20detect%20it.&f=false Jancis Robinson. "The Oxford Companion to Wine". Oxford University Press. Sep 17, 2005. Pg 483.]</ref>. The low pH of sour beer or wine makes it harder to detect the flavor and often impossible to detect the aroma. An increase in pH is needed in order to detect the flavor of THP, and the mouth's salivary pH serves that purpose when tasting beer or wine with THP. For example, as the mouth's pH adjusts back up after swallowing a sip of sour beer or wine, the THP becomes detectable in the aftertaste. This effect on sensory detection by low pH might also explain why some people are better at detecting it since people have different pH's on the surface of their tongues and saliva <ref name="Snowdon">[http://pubs.acs.org/doi/abs/10.1021/jf0528613 Mousy Off-Flavor: A Review. Eleanor M. Snowdon, Michael C. Bowyer, Paul R. Grbin, and Paul K. Bowyer. 2006.]</ref>. Diacetyl is sometimes mistakenly indicated as a potential cause of "mousy" flavors in sour beers, however, tetrahydropyridines are the accepted cause. The flavor tends to age out of sour beers after 2-6 months in the fermenter, kegs, or bottles (although aging periods as long as possibly 8-12 months have been reported on MTF <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1447796001915276/?comment_id=1447826811912195&comment_tracking=%7B%22tn%22%3A%22R%22%7D MTF thread about how long THP takes to age out with comment by Mark Trent. 10/24/2016.]</ref>). The exact mechanism for how THP ages out of beer is not fully understood, and it is unknown whether cold or room temperature storage speeds up the breakdown process. It is more likely that room temperature storage will result in faster breakdown of THP, and anecdotes from MTF members seem to support this <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1451857101509166/ MTF discussion regarding THP degredation under room temperature versus refrigeration temperatures. 10/28/2016.]</ref><ref>[http://www.homebrewtalk.com/f127/cheerios-character-after-bottling-504468/#post6516169 Tonsmeire, Michael. Homebrewtalk.com post 1. 11/21/2014. Retrieved 3/10/2015.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1972251579469713/?comment_id=1972290999465771&comment_tracking=%7B%22tn%22%3A%22R%22%7D Tariq Ahmed and Shane Martin. Milk The Funk thread about THP aging out of cider at room temperature. 01/30/2018.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2069944559700414/?comment_id=2070067719688098&reply_comment_id=2070405472987656&comment_tracking=%7B%22tn%22%3A%22R3%22%7D Ryan Sandlin. Milk The Funk Facebook gropu group thread on THP aging out after 90 days of room temperature storage. 04/23/2018.]</ref>. Many brewers have noticed that pitching rehydrated wine yeast at packaging reduces the amount/duration of this flavor in kegs and bottles <ref>[http://www.homebrewtalk.com/f127/cheerios-character-after-bottling-504468/#post6522207 Tonsmeire, Michael. Homebrewtalk.com post 2. 11/21/2014. Retrieved 3/10/2015.]</ref>.

Traditionally, the mousy/Cheerios® flavor from THP is considered an off flavor in both wine and sour beer. There is some debate and differing opinions as to whether or not a small amount of THP flavor is allowable (or even enjoyable) in sour beers, however most consider any level to be an off flavor <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1864484553579750/ MTF discussion on whether or not THP should always be considered an off-flavor. 10/27/2017.]</ref>. In wine, THP is also generally considered an off-flavor, although to some people a small amount of THP is acceptable in natural wine. It has greatly been eliminated as a problem in wine making due to sulfite sulphite usage and better controlled fermentations. However, in recent years there has been an increase in the popularity of natural wine which has a higher chance of being affected by THP. There has therefore also been a recent increased attention to THP in wine due to the increased popularity of natural wine <ref>[https://www.sfchronicle.com/wine/article/There-s-a-mouse-in-your-wine-14465005.php Esther Mobley. San Francisco Chronicle. 09/24/2019. Retrieved 09/25/2019.]</ref>.

Other techniques for detected THP in food have been developed, which might be applicable to wine or beer. For example, Grimm Kiyomichi et al. (20012023) developed a technique for detecting 2simple method to accurately detect ATHP, ETHP, and APY using gas chromatography-acetyl pyrroline mass spectrometry with stir bar sorptive extraction (APY or 2APSBSE-GC–MS instrumentation) in rice. The rice samples had to be heated to 80-85°C in order to extract the volatile APY, Thermal desorption and then APY levels in the headspace of the rice container could be detected injection were performed using solid phase microextraction a Twister thermal desorption unit (SPMETDU) and a Gerstel CIS 4 cooled injection system with fibers that operate a programmable temperature vaporization (PTV) inlet, installed on an Agilent 6890 gas chromatograph combined with an Agilent 5975 Mass Selective Detector (Agilent Technologies, Massy, France), equipped with a Gerstel MPS 2 autosampler (Gerstel, Mülheim an der Ruhr, Germany). An HP-5MS fused-silica capillary column (30 m × 0.25 mm, 0.25 µm, film thickness, SGE, Courtaboeuf, France) was used, with helium as carrier gas (Messer France S.A.S, Suresnes, France) at the higher temperatures a constant pressure of 70 kPa, corresponding to an initial flow of 1.3 mL.min−1 <ref>[https://www.ncbisciencedirect.nlm.nih.govcom/science/article/abs/pubmedpii/11170584 Screening for 2-acetyl-1S0308814623000705 Daiki Kiyomichi, Céline Franc, Pierre Moulis, Laurent Riquier, Patricia Ballestra, Stéphanie Marchand, Sophie Tempère, Gilles de Revel. Investigation into mousy off-pyrroline flavor in the headspace of rice wine using SPME/GCgas chromatography-MSmass spectrometry with stir bar sorptive extraction. Grimm CCFood Chemistry, Volume 411, 2023, Bergman C135454, Delgado JTISSN 0308-8146, Bryant Rhttps://doi.org/10.1016/j.foodchem.2023. 2001135454.]</ref>. It isn't known if such methods would See also work for measuring THP compounds [https://ives-technicalreviews.eu/article/view/7867 "Simultaneous assay of mousy off-flavor markers in beer or wine, but they could provide a potential option for beer and wine researchers" Ives Technical Review].

Liesel Krout, Lucille Benedict, Meg Hausman, and Zach Bodah from University of Southern Maine and Allagash Brewing Company have begun developing a new way to produce an ATHP flavor spike and detection method. See [https://digitalcommons.usm.maine.edu/cgi/viewcontent.cgi?article=1180&context=thinking_matters the poster] for more details. This method is still in development. In 2024, UC Davis described their method for detecting THP in beer for the first time. The team used Liquid Chromatography Mass Spectrometry with Electrospray Ionization (LC-MS-ESI). Extraction of ATHP in the samples was performed using QuEChERS (quick, easy, cheap, effective, rugged, and safe) technique <ref>[https://www.sciencedirect.com/science/article/pii/S2215016124000979 Paulina Martusevice, Xueqi Li, Matt Hengel, Selina C. Wang, Glen Fox, Analysis of mousy off-flavor compound 2-Acetyl-tetrahydropyridine using Liquid Chromatography Mass Spectrometry with Electrospray Ionization in sour beer, MethodsX, Volume 12, 2024, 102643, ISSN 2215-0161, https://doi.org/10.1016/j.mex.2024.102643.]</ref>. See also [https://www.masterbrewerspodcast.com/326 MBAA podcast episode 326, "THP" with Paulina Streimikyte and Glen Fox]. Other techniques for detected THP in food have been developed, which might be applicable to wine or beer. For example, Grimm et al. (2001) developed a technique for detecting 2-acetyl pyrroline (APY or 2AP) in rice. The rice samples had to be heated to 80-85°C in order to extract the volatile APY, and then APY levels in the headspace of the rice container could be detected using solid phase microextraction (SPME) with fibers that operate at the higher temperatures <ref>[https://www.ncbi.nlm.nih.gov/pubmed/11170584 Screening for 2-acetyl-1-pyrroline in the headspace of rice using SPME/GC-MS. Grimm CC, Bergman C, Delgado JT, Bryant R. 2001.]</ref>. It isn't known if such methods would also work for measuring THP compounds in beer or wine, but they could provide a potential option for beer and wine researchers. Cider makers have used a baking soda in water solution to help detect THP. Dissolve a small amount of baking soda in water, swish the solution in your mouth for a few seconds, and then spit it out. While the pH of the saliva in your mouth is raised from the baking soda solution, taste the beer/wine/cider to more easily detect THP <ref>[https://groups.google.com/g/cider-workshop/c/a9JcCERQTYk?pli=1 "testing for mouse". Testing for Mouse. Retrieved 04/06/2021.]</ref>.

ATHP/ACTPY (which has two forms: 2-acetyl-3,4,5,6-tetrahydropyridine and 2-acetyl-1,4,5,6-tetrahydropyridine(abbreviated: ATHP or ACTPY) has a much lower flavor threshold than ETHP (see [[Tetrahydropyridine#Thresholds_and_Quantities_Found_in_Mousy_Wine|Thresholds]]), and has been historically cited as the cause of mousy off-flavors detected in wine. In wine, its aroma cannot be detected due to the low pH of wine (it can be detected if the pH is raised), only the flavor. It is easier to detect in higher pH wines. ATHP is the form of THP that is the major contributor to the aroma of freshly baked bread, corn tortilla chips, and crackers. How different foods/wines/beers interact with ATHP on the palate may explain the different flavors that are detected by people, as well differing concentrations and peoples' ability to detect ATHP <ref name="Snowdon"></ref>. In food, ATHP has a slightly different form (6-Acetyl-1,2,3,4-tetrahydropyridin). Its formation in food is due to cooking and thought to be associated with Maillard reactions. This form has a toasty flavor of crackers and popcorn, and its presence disappears quickly in food due to its extreme volatility <ref name="Adams_2005" />.

ETHP/ETPY (2-ethyl tetrahydropyridine(abbreviated: ETHP or ETPY) was first identified in wine in 1973, but until recently further studies weren't able to confirm its presence in wine. Its odor threshold is quite high (see [[Tetrahydropyridine#Thresholds_and_Quantities_Found_in_Mousy_Wine|Thresholds]]), and so it was not considered a major source of mousy off-flavors in wine for some time. Consequently, research on ETHP has been limited. More recently, it was shown that Lactic Acid Bacteria (LAB) can produce above threshold levels of ETHP, making it recently important to wine researchers <ref name="Snowdon"></ref>.

2-acetyl pyrroline (abbreviated: APY, ACPY, or AP) is a more volatile but more potent form of THP. It has a significantly stronger odor and lower odor threshold in wine than ATHP. It can also be found in damp pearl millet, toasted bread, taco shells, tortilla chips, boiled potatoes, cooked sweet corn products, roasted sesame seeds, pan-fired green teas, cured tobacco leaves, peanut and pumpkin seed oils, honey, several soy-based products, and more aromatic rice such as Indian Basmati, as well as many other foods. APY has also been detected in a pale lager beer from Bavaria <ref>[https://link.springer.com/article/10.1007/BF01190873 Primary odorants of pale lager beer. Peter Schieberle. 1991.]</ref>. APY from microbial metabolism is primarily produced by heterofermentative LAB (see [[Tetrahydropyridine#Lactic_Acid_Bacteria|below]]). In food, APY formation is due to cooking and thought to be associated with Maillard reactions, and its presence ages out quickly in food. For example, Schieberle (1989) showed that heating up yeast and sucrose produced APY, simulating how APY could be produced during baking bread <ref><[https://pubs.acs.org/doi/10.1021/bk-1989-0409.ch025 Formation of 2-Acetyl-l-pyrroline and Other Important Flavor Compounds in Wheat Bread Crust. Peter Schieberle. 1989. DOI: 10.1021/bk-1989-0409.ch025.]</ref>. It is extremely volatile; so much so that the food industry has created powdered forms of APY to increase the flavor stability of some foods associated with it. Some plants such as rice crops, Pandan leaves (''Pandanus amaryllifolius''), "bread flowers" (''Vallaris glabra''), Myabi muskmelon fruit, chempedak fruit and jackfruit contain varying levels of APY naturally <ref name="Snowdon"></ref><ref name="Grbin_1996" /><ref name="Adams_2005">[http://pubs.acs.org/doi/abs/10.1021/cr040097y Chemistry of 2-Acetyl-1-pyrroline, 6-Acetyl-1,2,3,4-tetrahydropyridine, 2-Acetyl-2-thiazoline, and 5-Acetyl-2,3-dihydro-4H-thiazine:  Extraordinary Maillard Flavor Compounds. An Adams and Norbert De Kimpe. 2005.]</ref><ref>[https://www.sciencedirect.com/science/article/pii/S1672630823001105 Abiotic and Biotic Factors Controlling Grain Aroma along the Value Chain of Fragrant Rice: A Review. Ayut Kongpun, Tonapha Pusadee, Pennapa Jaksomsak, Kawiporn chinachanta, Patcharin Tuiwong, Phukjira Chan-In, Sawika Konsaeng, Wasu Pathom-Aree, Suchila Utasee, Benjamapohn Wangkaew, Chanakan Prom-U-Thai. Rice Science. 2023. https://doi.org/10.1016/j.rsci.2023.11.004.]</ref>.

A variety of pyridine and pyrazine derived compounds are formed in malt (and other foods) during the malting process as a result of Maillard reactions and have been found to be major contributors to the "malty" flavor of beer. Examples of these compounds include 2-acetylpyridine, 3-acetylpyridine, methylpyrazine, forms of dimethylpyrazine, and trimethlpyrazine. These compounds have a range of flavor descriptors such as creamy, cardboard, grainy, and burnt sugar. For example, 2-acetylpyridine (2AP), also known as 1-pyridin-2-ylethanone, is described as having a malty-biscuity, corn-chip, corn tortilla, or popcorn flavor. These compounds (in particular 2AP) can easily be confused with forms of THP, but they are not the same as the varieties of THP explained above <ref name="Adams_2005" /><ref>[https://en.wikipedia.org/wiki/2-Acetylpyridine 2-Acetylpyridine. Wikipedia. Retrieved 09/04/2018.]</ref><ref>[http://www.thegoodscentscompany.com/data/rw1012221.html 1-pyridin-2-ylethanone. The Good Scents Company website. Retrieved 09/04/2018.]</ref><ref>[https://www.aroxa.com/beer/beer-flavour-standard/2-acetyl-pyridine/ 2-acetyl pyridine. Aroxa website. Retrieved 09/04/2018.]</ref><ref>[https://onlinelibrary.wiley.com/doi/pdf/10.1002/jsfa.2740280218 Basic compounds contributing to beer flavour. Richard J. Harding, Harry E. Nursten, John J. Wren. 1977. DOI: https://doi.org/10.1002/jsfa.2740280218].</ref>. Another form is 2-formyl-1,4,5,6-tetrahydropyridine (FTHP), however, its presence in fermented beverages has not been well studied and it is less stable than other forms of THP, making it difficult to study <ref name="Martusevice_2024">[https://pubs.acs.org/doi/abs/10.1021/acs.jafc.3c09776# A Review of N-Heterocycles: Mousy Off-Flavor in Sour Beer. Paulina Martusevice, Xueqi Li, Matt J. Hengel, Selina C. Wang, and Glen P. Fox. Journal of Agricultural and Food Chemistry 2024 72 (14), 7618-7628. DOI: 10.1021/acs.jafc.3c09776.]</ref>. One indicator that a particular flavor might be THP instead of one of these malt-derived flavors is that THP is mostly detected on the aftertaste after swallowing, whereas these compounds are often detected during the swallow (although they can sometimes also be detected during the aftertaste).

It is thought that THP in mousy wines/beers is mostly produced by microorganisms. All species of ''[[Brettanomyces]] '' can produce forms of 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" />.

Moulis et al. (2023) studied THP production by 22 strains of ''Brettanomyces bruxellensis'', 20 strains of ''Oenococcus oeni'' and 10 strains of ''Lentilactobacillus hilgardii'' (formerly classified as ''Lactobacillus hilgardii''), all of which have been reported to produce THP compounds. They found that all strains could produce ATHP, but not all strains could produce ETHP or APY. This variability was determined mostly by species, but also by strain. for example, all of the 22 ''B. bruxellensis'' strains only produced ATHP and ETHP and not APY. Variability between strains was less pronounced for the species ''L. hilgardii'' compared to the ''B. bruxellensis'' and ''O. oeni'' strains (different strains of ''B. bruxellensis'', for example, produced much different levels of ATHP/ETHP, where as every strain of ''L. hilgardii'' produced relatively the same amount of APY). The researchers also noted that repeatability of THP levels was difficult to achieve, and they owed this to unknown variables such as the physiological state of the cells at time of inoculation into the test media. Interestingly, there was no correlation between strain genealogy and how much THP they produced. The researchers also isolated other species from 25 French wines with mouse taint, including ''S. cerevisiae'', ''Pichia manshurica'', ''Priceomyces carsonii'', ''Pediococcus parvulus'', but none of these strains produced THP in the test growth media <ref name="Moulis_2023" />.  ===''Brettanomyces''===

Although ''Brettanomyces'' is capable of producing APY from L-ornithine <ref name="Grbin_2007" />, the amount produced is much less than that of LAB and 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>. Additionally, Moulis et al. (2023) found that out of 25 French wines with THP, only 20% of them had ''B. bruxellensis'' in them, indicating that THP is mostly produced by bacteria or chemically in wine <ref name="Moulis_2023" />.

The presence of the "mousy off-flavor" caused by forms of THP appears to be temporary in beer. Although not much is known about the degradation or metabolic breakdown of ATHP/ETHP, it tends to age out of beer after 2-6 months. Since the odor/taste threshold for ETHP is much higher than ATHP, and ATHP appears to be metabolized into ETHP by ''Brettanomyces'' over time, this may be one of the mechanisms by which the mousy off-flavor ages out of beer. The possibility of ETHP breakdown is not mentioned in any studies that we know of, although Moulis et al. (2023) reported that for organisms that produced ETHP, there was always a 1:10 ratio between ETHP/ATHP or ETHP/APY, suggesting that this ratio might be governed by the chemistry of the media used and/or the [https://en.wikipedia.org/wiki/Reduction_potential reduction potential] <ref name="Moulis_2023" />. This was confirmed by second study by Moulis when ''B. bruxellensis'' was co-fermented or not co-fermented with APY-producing strains of ''Pediococcus paravulus'' <ref name="Moulis_2024">[https://oeno-one.eu/article/view/8060 Moulis, P., Miot-Sertier, C., Franc, C., Riquier, L., Beisert, B., Marchand, S., … Ballestra, P. (2024). Impact of Pediococcus parvulus and Saccharomyces cerevisiae on Brettanomyces bruxellensis mousy compound production. OENO One, 58(3). https://doi.org/10.20870/oeno-one.2024.58.3.8060]</ref>. Another unknown is why does ''Brettanomyces'' produce ATHP shortly after kegging and force carbonating a beer that has reached final gravity. The most likely cause is oxygen pick up during the kegging process. 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''. ====Co-fermentation with LAB====Moulis et al (2024) published a second study where they measured ATHP, ETHP, and APY produced by three ATHP/ETHP-producing strains of ''B. bruxellensis'' and three strains of APY-producing ''Pediococcus parvulus''. They measured levels of these three THP compounds when one of each ''B. bruxellensis'' was co-fermented with one each of the strains of ''P. parvulus''. They compared these levels to when each pair (one strain of ''B. bruxellensis'' and one strain of ''P. parvulus'') were fermented on their own, and then summed the total levels of each THP compound between the two separate fermentations. They found that APY levels, which was only produced by the ''P. parvulus' strains and not the ''B. bruxellensis'' strains, were much lower if the strains were co-fermented with any of the three strains of ''B. bruxellensis'. However, for ATHP and ETHP, 2 of the 3 strains of ''B. bruxellensis'' produced different levels of ATHP or ETHP when ''P. parvulus'' was co-fermented with them. The strain of ''P. parvulus'' also had an impact on this co-fermentation; some combinations of co-ferments produced more ATHP but less ETHP. The study concluded that the impact of co-fermentation inhibits APY produced by bacteria despite the strains used, and it can have an inhibitory or stimulatory impact on ATHP/ETHP, depending on the combinations of strains. The authors suggested several possible explanations <ref name="Moulis_2024" />: <blockquote>Previous results (Strickland et al., 2016) showed that less 4-ethylphenol (EP), a compound contributing to the “Brett character” in wine, was produced when ''P. parvulus'' and ''B. bruxellensis'' were jointly inoculated rather than separately. Here, ''P. parvulu''s and ''B. bruxellensis'' produce more ATHP and no APY at all when they are together. There are several hypotheses to explain that. First, Moulis (Moulis, 2023), proposed that ATHP production could correspond to a signal or a response to a signal between the different cells of ''B. bruxellensis'', to respond to stress. Here, ''B. bruxellensis'' could reply to the stress of the presence of ''P. parvulus'' by ATHP over-production. ''P. parvulus'' could thus directly influence the metabolism of ''B. bruxellensis'', resulting in the production of more ATHP than 4-ethylphenol in its presence. A second hypothesis is that ''B. bruxellensis'' could change the balance of acylation by ''P. parvulus''. Costello and Henschke (2002) proposed a formation pathway for APY and ATHP by LAB from ornithine and lysine, respectively. This formation pathway, in both cases, ends up with acylation (of piperideine for ATHP and of pyrroline for APY). ''B. bruxellensis'' could inhibit APY production either by inhibiting the pathway or by preferentially inducing ATHP acylation by ''P. parvulus''. Moreover, one of the production routes proposed by Grbin et al. (2007) involves cadaverine as a biosynthetic intermediate. Lactic acid bacteria such as ''O. oeni'' or ''P. parvulus'' are known to produce large quantities of biogenic amines (Granchi et al., 2005; Wade et al., 2019) and they can biosynthesise cadaverine to be metabolised by ''B. bruxellensis''. It would be interesting to explore these hypotheses in a future study <ref name="Moulis_2024" />.</blockquote> Moulis et al (2024) also attempted to study the effects of co-fermenting ''B. bruxellensis'' with ''Saccharomyces cerevisiae''; however, the definitive results were not presented due to a technical issue with the equipment used to measure THP compounds. Nonetheless, the authors suggested that ATHP and ETHP appeared to be higher when ''B. bruxellensis'' was co-fermented with ''S. cerevisiae'' versus when fermented on its own. The authors hypothesized that this could be due to more acetaldehyde production by ''S. cerevisiae'', which has been identified as a precursor to ATHP production. Another possibility would be nutrients released from less produced by ''S. cerevisiae'' <ref name="Moulis_2024" />.

Heterofermentative [[Lactobacillus]] spp., particularly ''L. hilgardii'' (reclassified as ''Lentilactobacillus hilgardii'') and ''L. brevis''(reclassified as ''Levilactobacillus brevis''), as well as ''Leuconostoc oeni'' <ref name="Grbin_1996" />, can also produce high levels of ATHP, and to a lesser extent APY and ETHP from L-lysine/L-ornithine, ethanol (must be present), and iron. Although many strains of heterofermentative lactic acid bacteria can produce THP, not all do. For example, Costello et al (2008) found that all strains tested of ''L. brevis'' (3 strains tested), ''L. bucherni'' (3 strains tested), and ''L.hilgardii'' (8 strains tested) produced THP, several heterofermentative species did not produce any detectable levels of THP in a grape-based media (one strain each of ''L. fermentum'' and ''L. cellobiosus''). Some strains within a species produce high amounts while others produce low amounts, for example Costell et al. (2008) found that some strains of ''O. oeni'' produced very high amounts between 50-150 µg/L while others produced very little between 5-20 µg/L in a grape-based media <ref name="Costello_2008">[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>. A strain of ''L. plantarum'' (L11a) was shown to produce relatively low amounts. L-lysine stimulates production of ATHP, and L-ornithine 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 name="Costello_2008" /><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 produce relatively small amounts. 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>, although one study found no THP in two strains of ''P. pentocaseus'' and only transient/occasional THP production in one out of five strains of ''P. parvulus'' <ref name="Costello" />. ''Oenococcus oeni'' and ''Leuconostoc mesenteroides'' have also been associated with creating ATHP, APY, and ETHP all above threshold amounts. Since only heterofermentative species produce significant amounts of THP, it is thought that its production is linked to the heterolactic pathway, and thus the metabolism of sugars in LAB <ref name="Costello"></ref>. A pathway for APY and ATHP production in ''Lactobacillus hilgardii'' was proposed by Costello and Henschke, which involves the intake of lysine or ornithine, along with ethanol (which is broken down into acetaldehyde) to produce APY and ATHP <ref name="Costello" />. ''Lactobacillus pontis'' has been shown to break down proteins via proteolysis, yielding free amino acids such as ornithine which could serve as precursors to THP formation, and it might be [[Lactobacillus#Foam_Degradation|reasonable to presume]] that other species of ''Lactobacillus'' could also free up ornithine as a precursor to THP <ref name="Adams_2005" />.

Although research is limited, acetic acid bacteria (''Gluconobacter'' sp. and many strains of ''Acetobacter aceti'') have been shown to produce forms of THP <ref name="Snowdon"></ref>. Mediterranean dried sauses covered in [[Mold|mould]] have been characterized as having APY as a flavor contributor. The source of the APY was identified with a mould that grows on the surface called ''Peniciilium nalgiovense'' <ref name="Adams_2005" />. Moulis et al. (2023) identified several strains of acetic acid bacteria in 32% of selected French wines with mouse taint, but none of the strains produced THP when tested individually in model medium <ref name="Moulis_2023" />. ===Impact from ''Saccharomyces''===While ''S. cerevisiae'' does not produce THP compounds itself, there is some preliminary data that suggests that the strain of ''S. cerevisiae'' could impact ATHP and ETHP levels. Data from the Moulis et al. (2023) study shows that ATHP levels different slightly depending on which strain of ''S. cerevisiae'' was co-inoculated with ''Brettanomnyces''. The authors hypothesize that differing levels of acetaldehyde or build up of lees may be the reason different strains of ''S. cerevisiae'' might have an impact <ref>[https://ives-technicalreviews.eu/article/view/9206 "Does Saccharomyces cerevisiae play a supporting role in mousy off-flavours production?" Pierre Moulis, Cécile Miot-Sertier, Céline Franc, Laurent Riquier, Beata Beisert, Stéphanie Marchand, Gilles de Revel, Doris Rauhut, Patricia Ballestra. Published: 7 March 2025. DOI: https://doi.org/10.20870/IVES-TR.2025.9206.]</ref>.

It's been shown that various THP compounds can be produced from heat reactions. For example, heating proline with monosaccharides produces a small amount of APY, as does the heating of yeast and sucrose. Phosphate ions are high contributors to THP production via heat reactions, with the amino acids proline, ornithine, and citrulline being precursors (the first two of which are important amino acids in yeast), and 1-pyrroline being the intermediary step. Ornithine is the precursor to heat produced ATHP in bread making <ref>[https://link.springer.com/article/10.1007/BF01197621 The role of free amino acids present in yeast as precursors of the odorants 2-acetyl-1-pyrroline and 2-acetyltetrahydropyridine in wheat bread crust. Peter Schieberle. 1990.]</ref><ref name="Adams_2005" />. 2-Acetyl-1-pyrroline forms from Maillard reactions and is key to the aroma of cooked Lipu taro <ref>[https://www.sciencedirect.com/science/article/abs/pii/S1878450X2400101X Xiatao Zhou, Liqiong Wen, Jinshan Xiao, Xueying Mo, Peng Wan, De-Wei Chen. 2- Acetyl-1-pyrroline originated from Maillard reaction is the key odorant of cooked Lipu taro. International Journal of Gastronomy and Food Science, 2024, 100968. ISSN 1878-450X. https://doi.org/10.1016/j.ijgfs.2024.100968.]</ref>. These reactions mostly occur at a relatively basic pH (7-9) <ref>[https://pubs.acs.org/doi/full/10.1021/jf970990g 2-Oxopropanal, Hydroxy-2-propanone, and 1-PyrrolineImportant Intermediates in the Generation of the Roast-Smelling Food Flavor Compounds 2-Acetyl-1-pyrroline and 2-Acetyltetrahydropyridine. Thomas Hofmann, and Peter Schieberle. 1998. DOI: 10.1021/jf970990g.]</ref>.

# Do sulfites sulphites bind to THP molecules, rendering them unperceivable <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2069944559700414/?comment_id=2070023539692516&reply_comment_id=2074259392602264&comment_tracking=%7B%22tn%22%3A%22R%22%7D Otto Forsberg. Milk The Funk Post discussion on THP and sulfites. 04/27/2018.]</ref>?

* [https://www.facebook.com/groups/MilkTheFunk/permalink/4042481855779998/ MTF thread on removing THP from wine made with low or no sulfitesulphite.]* [https://www.masterbrewerspodcast.com/237 MBAA Podcast 237: David Fuhrer on bottle conditioning using the German speise method (similar to krausening) reduces THP.]