Tetrahydropyridine

Forms of Tetrahydropyridines (abbreviated and often referred to generically by brewers 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) , which are classified as a ketone and a cyclic imine , 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 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 the flavor of THP. 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 off-flavor, and the mouth's salivary pH serves that purpose when tasting beer or wine with THP. 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 in the fermenter, kegs, or bottles (although aging periods as long as possibly 8-12 months have been reported on MTF ). 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) . Many brewers have noticed that pitching rehydrated wine yeast at packaging reduces the amount/duration of this flavor in kegs and bottles .

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

ATHP

ATHP (which has two forms: 2-acetyl-3,4,5,6-tetrahydropyridine and 2-acetyl-1,4,5,6-tetrahydropyridine) has a much lower flavor threshold than ETHP (see 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 .

ETHP

ETHP (2-ethyltetrahydropyridine) 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 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 its 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 . ETHP has not been shown to break down into another byproduct.

APY

APY (2-acetylpyrroline) 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. APY is primarily produced by heterofermentative LAB. .

Unidentified "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 Brettanomyces .

Production

Proposed pathway for THP production by Brettanomyces

All species of Brettanomyces can produce forms of Tetrahydropyridine in varying amounts. 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 throughout fermentation, but are generally high throughout a beer's lifetime (including after fermentation) . Wheat generally has a slightly lower amount of lysine than barley, and oats have a slightly higher amount of lysine than barley .

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 Brettanomyces growth, and Brettanomyces 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 Brettanomyces growth . 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 exceedlingly 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 packged 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 . 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 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.

Interestingly, for unknown reasons Brettanomyces cells grown under aerobic conditions and then transferred to an anaerobic environment still produced significant amounts of ATHP in the anaerobic environment. It has been suggested that the aerobic conditions made the Brettanomyces cells predisposed to creating ATHP . Oxygen exposure during Brettanomyces starters could potentially stimulate ATHP production later on down the road, even if the beer is not exposed to oxygen, although anecdotal evidence shows that this may not be a concern for brewers. It is still advised to use an aerobic or semi-aerobic starter for Brettanomyces unless the brewer believes this might be the direct cause of ATHP problems in their beer. Any other oxygen pick up after the beer has finished fermentation is the more likely cause and the brewer's post-fermentation processes should be examined first.

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. 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 .

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 Brettanomyces is capable of producing APY from L-ornithine, the 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 is much easier to detect aromatically than ATHP) indicates a bacterial contamination in wine (it is unknown if this applies to beer) .

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. 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.

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. A strain of L. plantarum (L11a) was shown to produce relatively low amounts. 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 forms of THP, although a few species do produce relatively small amounts. 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 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 .

Acetic Acid Bacteria

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

Thresholds and Quantities Found in Mousy Wine

Editor's note: the following thresholds are from studies on wine, and may not hold true for beer. As stated above, detection is influenced by pH, and so the low pH of sour beer may have the similar effect of repressing odor (more so) and taste (less so) detection, whereas non-sour Brett beers may have a higher detection rate.

• ETHP
  • 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)
• ATHP
  • 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)
• APY
  • Odor threshold (water): 0.1 µg/L
  • Concentration reported in wines exhibiting mousy off-flavour: Trace quantities up to 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.

• 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.
• MTF thread detailing experiences with certain strains and procedures (03/24/2017).
• 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.
• Kyle Weniger from Joseph James Brewing Co noticed significantly improved THP reduction storing a split batch of beer at room temperature versus storing cold.
• 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.