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Kiyomichi et al. (2023) developed a simple method to accurately detect ATHP, ETHP, and APY using gas chromatography-mass spectrometry with stir bar sorptive extraction (SBSE-GC–MS instrumentation). Thermal desorption and injection were performed using a Twister thermal desorption unit (TDU) and a Gerstel CIS 4 cooled injection system with 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 a constant pressure of 70 kPa, corresponding to an initial flow of 1.3 mL.min−1 <ref>[https://www.sciencedirect.com/science/article/abs/pii/S0308814623000705 Daiki Kiyomichi, Céline Franc, Pierre Moulis, Laurent Riquier, Patricia Ballestra, Stéphanie Marchand, Sophie Tempère, Gilles de Revel. Investigation into mousy off-flavor in wine using gas chromatography-mass spectrometry with stir bar sorptive extraction. Food Chemistry, Volume 411, 2023, 135454, ISSN 0308-8146, https://doi.org/10.1016/j.foodchem.2023.135454.]</ref>. See also [https://ives-technicalreviews.eu/article/view/7867 "Simultaneous assay of mousy off-flavor markers in wine," 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>.
The effectiveness of these methods in beer has not been reported to our knowledge (please report any research or anecdotes in the [https://www.facebook.com/groups/MilkTheFunk/ MTF Facebook group]).
==Forms of THP==
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" />.
===Lactic Acid Bacteria===
===Acetic Acid Bacteria and Mould===
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>.
===Maillard Reactions===
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>.
===Oxygen Reactions===
===MTF Threads and Other Forum Posts===
Below is a list of discussions on internet forum threads that may shed light on specific strains and individual experiences. Keep in mind that many of the opinions and experiences are anecdotal, although commonalities and shared experiences may prove to be useful and accurate.
* [https://www.masterbrewerspodcast.com/326 MBAA podcast episode 326, "THP" with Paulina Streimikyte and Glen Fox.]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1766965029998370/ Joe Idnoni from House Cat Brewing and Brian Ogden from Attaboy Beer report that Caseinate de Potassium (a fining agent used in wine-making) was used to significantly reduce THP off-flavors in a kettle sour beer.] Note that casein is a milk allergen, and might require declaration as a milk allergen (refer to your local government requirements) <ref>[https://www.fda.gov/Safety/Recalls/ucm456836.htm "UPDATED - Allergy Alert on Undeclared Milk in Nutrition Resource Services, Inc.'s Whey, Casein, and Colostrum Protein Products". FDA website. 08/03/2015. Retrieved 12/21/2017.]</ref>.
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1852891514739054/ Tariq Ahmed of Revel Cider discusses their protocol for reducing THP in spontaneously fermented ciders.]