Butyric Acid

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Butyric Acid (chemical name butanoic acid [1]) is a carboxylic acid that is produced by anaerobic bacteria such as Clostridium butyricum, C. kluyveri, and Megasphaera spp [1] during glucose fermentation, and is generally considered an off flavor in sour beer. If not done right, Sour Mashing and Wort Souring can be a big producers of butyric acid. Butyric acid is produced by anaerobic bacteria. The aroma and flavor is often described as a vomit, bile, or rancid cheese. The odor threshold is quite low (240 ppb) while the flavor threshold is much higher (2000-6800 ppb), with the flavor being more easily detected the lower the pH of the beer [2][3]. It is also present in the human gut, and is the cause of the aroma of vomit [1]. This is not to be confused with Isovaleric Acid which has a more "feety" aroma and flavor. Brettanomyces can convert butyric acid into Ethyl Butyrate at low levels, which has a pineapple, tropical fruit aroma and flavor, but at high levels not all butyric acid will be converted into ethyl butyrate. Ethyl butyrate production occurred in only half of the Brettanomyces strains studied by Yakobson, with this conversion being less with the addition of lactic acid, indicating that ethyl butyrate conversion from butyric acid is strain dependent and slows in the presence of lactic acid [4][5][6]. Butyric acid has shown to have some health benefits in medical research [7][8].

Microbes and Metabolism

Clostridium spp

Clostridium is a Gram-positive anaerobic genus of bacteria that is found in plants, wounds, soil, and feces. It has also been found in spent grains. Several species of the genus Clostridium produce butyric acid, and is produced in at least 3 days. Clostridium are generally obligatory anaerobes [9], but some can be active in aerobic conditions (aerotolerant), such as T. tertium. Species of Clostridium that can produce butyric acid from glucose include C. butyricum, C. fallax, C. paraputrificum, C. sartagoformum, C. tertium, C. thermosaccharolyticum, and to a lesser degree C. pasteurianum. [10]

Clostridium contamination in brewing methods such as Sour Mashing can be controlled by lowering the pH of the wort/mash. However, some species of Clostridium are acid tolerant. For example, C. butyricum and C. tertium were found to survive a pH as low as 4.0 [11][10]. Therefore, pre-acidifying during the Sour Mashing process to a pH of 3.9 or lower may produce the best results (brewers have long reported good results with acidifying their wort/mash to a pH of 4.4; this may be a function of greatly suppressing butyric acid bacteria, rather than eliminating it completely [12]). Fermentation by yeast also inhibits growth, probably due to out-competing the bacteria [10].

Temperature resistance varies from species to species of Clostridium. Most species have an optimal growth at around 104°F (37°C) [13]. However, C. thermosaccharolyticum can survive temperatures as hot as 149°F (65°C). Additionally, spores of some species of Clostridium (such as C. tertium) can survive boiling temperatures for longer than 1.5 hours [10].

Carbon dioxide from both fermentation and artificial introduction has been shown to have a stimulatory effect on the growth of Clostridium butyricum (as well as other bacteria such as E. coli) [14]. If sanitation issues allow for Clostridium to enter the brewery, CO2 purging may encourage butyric acid formation. Even so, many brewers recommend purging with CO2 during the souring phase of kettle souring to prevent other off-flavors [15] (~15 mins in). This has the benefit of creating positive pressure inside the souring vessel so that dust cannot be sucked into the vessel during the souring phase of kettle souring. It might also have the benefit of discouraging unidentified contaminates from creating off-flavors in the wort. The true source of butyric acid formation in kettle sour beers has not been well explored, and needs further investigation.

Although C. botulinum and other identifiably pathogenic Clostridium species do not tend to grow in Sour Mashing conditions, it is advisable to not consume wort produced from a sour mash until after it is boiled. Seek specific food and safety advice from a qualified physician.

Megasphaera spp

Megasphaera species produce large amounts of butyric acid, as well as some acetic, isovaleric, valeric, caproic acids, and hydrogen sulfide. They are considered a true beer spoilage organism in beer. M. cerevisiae is a common species found in spoiled beer, and is a Gram-negative bacteria. They are obligate anaerobes, and die in the presence of oxygen. Growth occurs at a temperature range between 59°-98.6°F (15°-37°C), with an optimal growth rate at 82.4°F (28°C) [16]. Growth of M. cerevisiae cannot occur above 5.3% abv, and slows significantly at 2.66% abv. Growth also stops at a pH of 4.0 or less [17][18].

Fusobacterium nucleatum

F. nucleatum is a Gram-negative bacteria that is commonly found in dental plaque. It is capable of fermenting glucose into butyric acid, and is tolerant of up to 6% oxygen [19]. It has a optimal growth temperature at 98.6°F [20]. F. nucleatum is sensitive to a pH of 5.0 and lower, and temperatures above 140°F (60°C) [21]. Avoid contaminating wort with saliva, and this organism shouldn't be a problem.

Lactic Acid Bacteria

Lactobacillus and other LAB produce butyric acid by breaking down lipids to fats. The low level of lipids in brewer's wort most likely limits butyric acid to insignificant levels in beer production. Butyric acid production by Lactobacillus is more significant in milk based fermented products such as yogurt and cheese [22]. For example, one study on a strain of L. plantarum that was fermented in three different substrates (oats, barley, and wheat) showed no production of butyric acid [23]. Due to the low amount of lipids in wort, compounds from the reduction of lipids in grain fermentations by lactic acid bacteria have not been studied [24].

A common brewing myth is that Lactobacillus produces butyric acid in the presence oxygen, however, there is no biological basis or evidence for this claim (see effects of oxygen on Lactobacillus). The lack of evidence for this claim is supported by Santeri Tenhovirta's masters thesis in Food Science from the University of Helsinki (2019). Tenhovirta soured wort with several species of Lactobacillus, including L. delbruekii, L. plantarum, L. alimentarius, L. brevis, L. buchneri and L. rhamnosus. During the two days of souring, the vessels were not purged of air, allowing the Lactobacillus to ferment under aerobic (containing oxygen) conditions. After two days of souring, US-05 was pitched to finish the fermentation (the soured wort was not boiled). A trained sensory panel reported low to no butyric acid flavor in the beers, except for the L. delbruekii sample which contained a moderate level of butyric acid flavor, but that sample never contained viable L. delbruekii (even at pitching time), and the butyric acid in that sample was hypothesized to be caused by some unknown contaminating microbe [25][26].

Note that much research has been done on the production of γ-amino butyric acid (also called "gamma-aminobutyric acid" or "GABA" for short) by various bacteria species due to its health benefits. Lactobacillus brevis, L. plantarum, and a few other species of bacteria are able to produce GABA from the non-protein amino acid glutamate via the enzyme glutamate decarboxylase. GABA (C4H9NO2) has a different chemical formula and is a different compound than butyric acid (C3H7COOH) [27][28][1]. GABA reportedly smells "savory" or "meaty" [29]. Therefore, the information regarding the production of GABA by Lactobacillus species should not be confused with that of butyric acid.

Other Microbes

Other microbes that are butyric acid producers, probably won't be found in the brewhouse. These include bacteria that are commonly found in the guts of animals such as Butyrivibrio fibrisolvens [30] and Eubacterium limosum [31]. A few strains of Brettanomyces bruxellensis have been shown to produce butyric acid in growth media when supplemented with the amino acids phenylalanine and tyrosine [32].

Metabolism

Butyric acid pathway [33]

Clostridium species such as C. butyicum, C. lactoacetophilum, C. pasteurianum, etc. produce butyric acid and acetic acid via the butyric acid fermentation pathway. Some species of Clostridium, such as C. acetobutylicum also produce isopropanol or acetone. Carbon dioxide and ethanol are also produced during butyric acid fermentation, as well as butunol. [34]. Butanol is a solventy smelling alcohol [35].

See Also

Additional Articles on MTF Wiki

External Resources

References

  1. ↑ 1.0 1.1 1.2 1.3 Wikipedia description of Butyric Acid production
  2. ↑ Butyric (Butyric Acid). FlavorActIV Website. Retrieved 10/24/2016.
  3. ↑ Odor Thresholds Odor & Flavor Detection Thresholds in Water (In Parts per Billion). Leffingwell & Associates website. Retrieved 10/24/2016.
  4. ↑ "Pure Culture Fermentation Discussion." The Brettanomyces Project. Chad Yakobson. 2011. Retrieved 06/06/2016.
  5. ↑ "Impact of Pitching Rate." The Brettanomyces Project. Chad Yakobson. 2011. Retrieved 06/06/2016.
  6. ↑ "Impact of the Initial Concentration of Lactic Acid on Pure Culture Fermentation." The Brettanomyces Project. Chad Yakobson. 2011. Retrieved 06/06/2016.
  7. ↑ Van Immerseel F, Ducatelle R, De Vos M, Boon N, Van De Wiele T, Verbeke K, Rutgeerts P, Sas B, Louis P, Flint HJ. Butyric acid-producing anaerobic bacteria as a novel probiotic treatment approach for inflammatory bowel disease. J Med Microbiol 2010;59:141–3.
  8. ↑ Effect of Colon Flora and Short-Chain Fatty Acids on Growth In Vitro of Pseudomonas aeruginosa and Enterobacteriaceae. MATTHEW E. LEVISON. 1973.
  9. ↑ Chlostridium. MicrobeWiki. Retrieved 09/29/2015.
  10. ↑ 10.0 10.1 10.2 10.3 Butyric Acid Off-Flavors in Beer: Origins and Control. D. B. Hawthorne, R. D. Shaw, D. F. Davine, T. E. Kavanagh, and B. J. Clarke. 1991.
  11. ↑ Growth Limiting pH, Water Activity, and Temperature for Neurotoxigenic Strains of Clostridium butyricum. Hamid B. Ghoddusi, Richard E. Sherburn, and Olusimbo O. Aboaba. 2013.
  12. ↑ Conversation with Derek Springer and Malcom Frazer on MTF. 6/22/2015.
  13. ↑ Textbook of Microbiology & Immunology. Subhash Chandra Parija. 2nd Edition. Feb 10, 2014.
  14. ↑ Gaseous CO2 signal initiates growth of butyric-acid-producing Clostridium butyricum in both pure culture and mixed cultures with Lactobacillus brevis. Hakalehto, Elias and Hänninen, Osmo. July 2012.
  15. ↑ The Sour Hour on the Brewing Network. Interview with Khris Johnson. Nov 20, 2014.
  16. ↑ Brewing Microbiology. Fergus Priest, Iain Campbell. Springer Science & Business Media, Jun 27, 2011.
  17. ↑ Brewing Microbiology: Managing Microbes, Ensuring Quality and Valorising Waste. Hill, Annie. Woodhead Publishing, May 26, 2015.
  18. ↑ Monoclonal Antibodies Binding to Lipopolysaccharide from the Beer-Spoilage Bacterium Megasphaera cerevisiae Exhibit Panreactivity with the Strictly Anaerobic Gram-Negative Brewing-Related Bacteria. Barry Ziola. 2016.
  19. ↑ Fusobacterium nucleatum. MicrobeWiki. Retrieved 6/21/2015.
  20. ↑ ATCC. Fusobacterium nucleatum subsp. nucleatum   (ATCC ® 25586™). Retrieved 6/21/2015.
  21. ↑ Ribeiro-Ribas, R.N., De Carvalho, M.A.R., Vieira, C.A., Apolônio, A.C.M., Magalhães, P.P., Mendes, E.N., Oliveira, J.S., Santoro, M.M. and De Macêdo Farias, L. (2009), Purification and partial characterization of a bacteriocin produced by an oral Fusobacterium nucleatum isolate. Journal of Applied Microbiology, 107: 699–705. doi: 10.1111/j.1365-2672.2009.04250.x
  22. ↑ Conversation with Bryan of Sui Generis Blog on MTF regarding butyric acid production by Lactobacillus. 11/23/2015.
  23. ↑ Volatile compounds produced by the probiotic strain Lactobacillus plantarum NCIMB 8826 in cereal-based substrates. Ivan Salmeron, Pablo Fuciños, Dimitris Charalampopoulos, Severino S. Pandiella. 2009.
  24. ↑ Lactic Acid Bacteria as Sensory Biomodulators for Fermented Cereal-Based Beverages. Lorenzo C. Peyer , Emanuele Zannini , Elke K. Arendt. 2016.
  25. ↑ The Effects of Lactic Acid Bacteria Species on Properties of Sour Beer. Santeri Tenhovirta; master thesis in Food Science from the University of Helsinki. 2019.
  26. ↑ Santeri Tenhovirta. Milk The Funk Facebook group thread on his masters thesis and the lack of butyric acid production under O2 conditions. 06/14/2019.
  27. ↑ "Gamma-Aminobutyric Acid". Wikipedia. Retrieved 06/11/2018.
  28. ↑ Production of gaba (γ – Aminobutyric acid) by microorganisms: a review. Radhika Dhakal, Vivek K. Bajpai, and Kwang-Hyun Baek. 2012.
  29. ↑ "gamma-aminobutyric acid". The Good Scents Company. Retrieved 06/11/2018.
  30. ↑ Cotta, M A, and R B Hespell. “Proteolytic Activity of the Ruminal Bacterium Butyrivibrio Fibrisolvens.” Applied and Environmental Microbiology 52.1 (1986): 51–58. Print.
  31. ↑ Lee, Meng-Rui et al. “Clinical and Microbiological Characteristics of Bacteremia Caused by Eggerthella, Paraeggerthella, and Eubacterium Species at a University Hospital in Taiwan from 2001 to 2010.” Journal of Clinical Microbiology 50.6 (2012): 2053–2055. PMC. Web. 22 June 2015.
  32. ↑ Brettanomyces bruxellensis Aroma-Active Compounds Determined by SPME GC-MS Olfactory Analysis. C.M. Lucy Joseph, Elizabeth A. Albino, Susan E. Ebeler, Linda F. Bisson. 2015.
  33. ↑ Fermentation: Effects on Food Properties. Bhavbhuti M. Mehta, Afaf Kamal-Eldin, Robert Z. Iwanski. CRC Press, Apr 12, 2012. Pg 76,77.
  34. ↑ Principles of Microbiology. R S. Mehrotra. ata McGraw-Hill Education, 2009. Pg 53.
  35. ↑ PubMed. 1-Butanol. Retrieved 6/21/2015.
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