Tetrahydropyridine

Forms of Tetrahydropyridines (THP), specifically 6-Acetyl-2,3,4,5-tetrahydropyridine (ATHP or ACTPY), 2-ethyltetrahydropyridine (ETHP or ETPY), and 2-acetyl-1-pyrroline (ACPY or APY) , which are classified as ketones , are commonly attributed to the "mousy", "urine" (in high amounts) "cheerios" or "Captain Crunch" (in low amounts), "breakfast cereal", or more generically, "cracker biscuit" flavor in sour beers. The flavor is detected towards the end of the swallow, and the aftertaste can remain for a few minutes. Not all people are able to detect this flavor, and a low pH of sour beer or wine makes it harder to detect the flavor and often impossible to detect the aroma. 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 . Diacetyl is sometimes mistakenly indicated as a potential cause of this flavor in sour beers. However, Tetrahydropyridines are the accepted cause. The flavor tends to age out of sour beers after 2-6 months (it is unknown whether cold or room temperature storage speeds this up), although the exact mechanism for this is not fully understood . Many brewers have noticed that pitching rehydrated wine yeast at bottling reduces the amount/duration of this flavor .

In food, Tetrahydropyridines are associated with the aroma of baked goods such as white bread, popcorn, and tortillas, and is formed by Maillard reactions during heating.

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.

Forms of THP

ETHP

ETHP was first identified in wine in 1973, but until recently further studies weren't able to confirm it's presence in wine. It's odor threshold is quite high (see 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 .

It has been speculated by scientists studying mousy off-flavors in wine that it's production is the result of slow metabolism of ATHP into ETHP by Brettanomyces. ETHP was observed to form much slower than ATHP, and coincided with a decrease in ATHP. This slow production of ETHP may be another reason it has been underestimated by researchers until recently .

ATHP

ATHP has a much lower flavor threshold than ETHP (see Thresholds). 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 .

APY

APY is a more volatile form of THP, and has a significantly stronger odor and much lower odor threshold in wine than ATHP. It can also be found in damp pearl millet, bread, and more aromatic rice such as Indian Basmati .

"Transient" Forms

There have been anecdotal reports of other forms of mousy off-flavors. During growth of lactic acid bacteria (LAB), mousy off-flavor detection fluctuated with high levels detected early on, and lower levels detected towards the end of growth. This indicates that there may be a transient, strain-dependent form of THP that can occur during malolactic fermentation. There have also been sensory detection of mousy off-flavors at different levels than the documented levels of ATHP, ETHP, and APY, which were not associated with LAB or Brett .

Production

Proposed pathway for THP production by Brett

(This section is in progress)

All species of Brettanomyces can produce forms of Tetrahydropyridine. 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 .

Brettanomyces

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 . As with other amino acids, lysine is taken up by Saccharomyces during fermentation, and then released after fermentation. Levels of lysine fluctuate slightly through fermentation, but are generally high throughout a beer's lifetime .

Oxygen has a stimulatory effect in ATHP and ETHP production, 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 . It has also been hypothesized that oxygen may have a direct effect on the THP molecules themselves . 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 anaerobic environment still produced significant amounts of ATHP. It has been suggested that the aerobic conditions made the Brett cells predisposed to creating ATHP . 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 ATHP production.

The level of ATHP production varies widely between species and strains of Brett, with some strains not producing it at all and some producing very high amounts above taste threshold. Additionally, ATHP production appears to require 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 .

The production of ATHP is not efficient, meaning that the amount of ATHP produced is not proportional to the amount of L-lysine consumed. Therefore, the production of ATHP appears to be a byproduct (secondary metabolite) of L-lysine catabolism . ATHP is further metabolized into ETHP by Brettanomyces, although not much is known about this metabolic process . ETHP has a significantly higher taste threshold, and is often not detected in contaminated wine .

Although Brett is capable of producing APY from L-ornithine, the production rate 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 aromatically, while ATHP cannot) indicates a bacterial contamination .

The presence of the "mousy off-flavor" caused by THP appears to be temporary in beer. Although not much is known about the degradation or metabolic break down of ATHP/ETHP, it tends to age out of beer after 2-6 months. Another unknown is why Brett produces THP shortly after kegging and force carbonating a beer that has reached final gravity. Pitching fresh Saccharomyces for bottle conditioning a beer with Brett in it has reportedly reduced THP production, perhaps through the quicker metabolism of both the oxygen and sugar that is introduced during packaging time.

Lactic Acid Bacteria

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 . 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 . Most species of Pediococcus do not create THP, although a few species do. In particular, these include P. pentosaceus , and P. clausenii (note that commercial cultures of Pediococcus are normally P. damnosus). Oenococcus oeni and Leuconostoc mesenteroides have also been associated with creating THP. 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 .

Acetic Acid Bacteria

Although research is limited, acetic acid bacteria have been shown to occasionally produce forms of THP .

Thresholds

Editor's note: the following thresholds are from studies on wine, and may not hold true for beer. As stated above, odor detection is influenced by pH, and so the low pH of sour beer may have a similar effect.

• 2-ethyltetrahydropyridine (ETHP/ETPY)
  • Odor threshold (wine): 150 µg/L
  • Concentration reported in wines exhibiting mousy off-flavour: 2.7-18.7 µg/L (ETHP is generally not the cause of the detected mousy off-flavor)
• 2-acetyltetrahydropyridine (/ATHP/ACTPY) -
  • Odor threshold (water): 1.6 µg/L
  • Concentration reported in wines exhibiting mousy off-flavour: 4.8-106 µg/L (ATHP is generally the cause of the detected mousy off-flavor)
• 2-acetyl-1-pyrroline (ACPY)
  • Odor threshold (water): 0.1 µg/L
  • Concentration reported in wines exhibiting mousy off-flavour: Tr-7.8 µg/L

Discussions

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.

• Milk The Funk Thread on March 10, 2015.
• Milk The Funk Thread on Aug 25, 2015.
• Milk The Funk Thread on 09/17/2015.
• Homebrewtalk thread started by "ne0t0ky0".
• Homebrewtalk thread started by "loctones", comments by Michael Tonsmeire.
• Babblebelt thread with comments by Chad Yakobson.
• Michael Tonsmeire observes that adding fresh Saccharomyces at bottling time seems to reduce THP production, versus force carbonating.
• Richie Preiss suggests that there is no difference in how long it takes to age out THP between storing the beer at room temperature or refrigeration temperature based on a side by side test that he did.
• Chad Yakobson on The Sour Hour, 3/11/2015 (around 50 minute mark) - indicates that he has been researching THP for 2 years, and is continuing this research. Little else is mentioned.