BIOCHEMISTRY ●

The British fungus The brewing science of Brettanomyces

By Ed Wray, Campden-BRI sugars without the need for a second- ary fermentation by Brettanomyces. However, adding Brettanomyces to Brettanomyces is a genus of bring about a secondary fermentation non-Saccharomyces yeast was used in Courage Imperial Russian of great importance to the Stout – and in Belgium the method continues to be used in the production brewing industry. It was of the Trappist beer Orval. It also plays named by N H Claussen, an important role in Belgian sour beer production and recently interest in us- Director of the Carlsberg ing this yeast has grown to the extent Laboratory, in a paper that some breweries now use it for primary fermentation. published in the Journal of the 1 Institute of Brewing in 1904 . Current classifi cation Spore forming D. bruxellensis As with most micro-organisms the Brettanomyces species have been laussen had identifi ed from English reclassifi ed as scientifi c understanding Cstock ales the organism respon- has advanced. Brettanomyces had only sible for “both the condition of these been known to reproduce asexually, beers and their fl avour.” He named but the discovery of ascospore forma- it Brettanomyces (British fungus) due tion in Brettanomyces by Van der Walt to its close connection to the British and Kerken2 led to its reclassifi cation brewing industry. of the genus as Dekkera. Species have Stock ales underwent a long also been reclassifi ed which adds to maturation and for them to condition the diffi culty of keeping up with cur- a ‘true’ secondary fermentation, by rent nomenclature. At the present time secondary yeast, was necessary. Using there are four species3 in the genus, Hansen’s pure yeast culture method two of which have been seen to form for producing stock ales obtained poor spores. results as the pure cultures were free The spore formers are Dekkera Vegetative Brettanomyces cells of Brettanomyces. Demand for stock anomala (into which the species claus- tanomcyes4 and, as it is the most com- ales was, however, in terminal decline senii has been merged) and Dekkera monly used term by those that brew by the time of Claussen’s paper. Mild bruxellensis (into which the species with these organisms, I shall continue or running beers, served soon after lambicus has been merged) and the to use it in this article. brewing, replaced them, and like the non-spore-formers are Brettanomy- Genetically Brettanomyces shows a cask beers of today they came into ces custersianus and Brettanomyces marked degree of diversity, which will condition though the action of Saccha- naardenensis. As it is likely that the no doubt keep the taxonomists busy. romyces on residual or added priming genus will be reclassifi ed back to Bret- Researchers looking at B. bruxellensis

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yeast by utilising dextrins that they A similar situation is found in beers cannot ferment. Andrews and Gilliland that have a secondary Brettanomyces of the Guinness laboratory described fermentation after primary fermenta- how a secondary attenuation limit tion with Saccharomyces. The ester was determined after fermentation profile of the beer is changed, some- with Saccharomyces by using a culture thing that will not happen if Saccharo- of Brettanomyces9. It can produce myces is used for conditioning. Other both an intra- and extra-cellular esters, such as ethyl caproate (pineap- α-glucosidase, both of which can po- ple) and ethyl caprylate (fruity, winey, tentially hydrolyse dextrins with >9-12 waxy) are also associated with the glucose unit, producing glucose and flavour of lambic beers and are likely the next lower dextrin10. In a research to be produced by Brettanomyces15. project carried out at Heriot-Watt University, Chad Yakobson found that Phenolic compounds glucose levels could even increase Some of the characteristic flavour during fermentation as this dextrin compounds produced by Brettano- breakdown occurs. myces are phenols16. Flavour-active Sugar utilisation is highly vari- compounds produced include 4-vinyl- able within the genus9,11. The slow guiacol (clove flavour), 4-vinylphenol growth of Brettanomyces means that (barnyard, medicinal, plastic) and in mixed fermentations it is out- 4-vinylcatechol (plastic, bitter, smoky). competed by Saccharomyces and Unlike Saccharomyces, Brettanomyces With the head brewer Anne-Françoise Pypaert in the Orval cannot take full advantage of the has the enzyme vinylphenol reductase laboratory wort glucose and maltose. In pure which will reduce these compounds to have found large variation in chromo- culture Brettanomyces fermentations their corresponding ethyl derivatives3 some size and number4 and that it has however, it is found that most strains : 4-ethylguiacol (spicy, clove), 4-ethyl- a core diploid genome however triploid can utilise both these sugars, though phenol (medicinal) and 4-ethylcatechol strains are common5. this does to some extent repress (medicinal, barnyard). In beer the dextrin utilisation and limit super- concentration of 4-ethylphenol is lower Non-Saccharomyces yeast attenuation12. Some strains produce than 4-ethylguaicol, though in wine the As a non-Saccharomyces yeast, Bret- a β-glucosidase which allows them situation is reversed17. tanomyces metabolism differs from to ferment the wood sugar cellobi- that of normal brewing yeast strains. It ose. β-glucosidase is also involved Other flavour compounds is able to use a wider range of nitrogen in the bio-transformation of hop Mousy off-flavours can be caused by sources, which gives it an advantage compounds leading to the release of Brettanomyces due to production of over Saccharomyces species in nutri- glycosidically-bound flavour active ETHP (2-ethyltetrahydropyridine) and tionally depleted environments such volatile compounds13. ATHP (2-acetlytetrahydropyridine). as beer4. This has also been used as The amount produced is strain spe- the basis for selective media such as Esters and esterases cific, though the presence of oxygen lysine agar6. Researchers looking into the complex stimulates their production4. During Brettanomyces will ferment glucose world of lambic fermentations have anaerobic growth, Brettanomyces will faster in the presence of oxygen than found that all the Brettanomyces iso- produce a number of fatty acids, some it will anaerobically. This was named lates they examined showed esterase of which have cheesy or goaty flavours the Custers effect after the researcher activity not found in Saccharomyces. such as isovaleric acid, caproic acid that discovered this in 19407. It will The esterases found in Brettanomyces and caprylic acid3,11,15. also produce acetic acid at the same do not only break down esters, they So not all the flavours produced by time – one of the reasons that Bret- also have ester-synthesising activity14. Brettanomyces are desirable. A num- tanomyces is often associated with In lambic beers after Brettanomyces ber of factors influence which flavours sour beer. However, in an anaerobic growth, high levels of ethyl acetate are produced, these include the strain, environment this does not occur so it (fruity, solvent flavour) and ethyl pitching rate, wort composition and is perfectly possible to make beers us- lactate (fruity, creamy) were found, fermentation conditions11,12. Flavours ing Brettanomyces that are not sour. As but very low levels of iso-amyl acetate can also change over time, as for ex- many beers fermented using Brettano- (banana). ample fatty acids are esterified. When myces are aged in wooden barrels or Brettanomyces beers are matured for vats, it is worth noting that the larger a long time they will require regular the vessel, the less oxygen ingress sampling to determine if the desired there is over time per unit volume flavour profile has developed. Some of beer8, which perhaps explains the breweries will then use pasteurisation fondness the old porter brewers had when packaging to prevent further for giant vats. flavour changes12.

Carbon source Fermentation One of Brettanomyces best known Brettanomyces can be used to carry out characteristics is its ability to attenu- fermentations in a number of different ate beer further than normal brewing Orval Brett cultures awaiting use ways. When using B.clausenii (WLP645)

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Journal. Vol 26, Issue 1, 292-296. 3. Crauwels, S. et al. (2015). Brettanomy- ces Bruxellensis, Essential Contributor in Spontaneous Beer Fermentation Providing Novel Opportunities for the Brewing Indus- try. Brewing Science. Vol 68, 110-121. 4. Steensels, J. et al. (2015). Brettanomyces yeasts – From spoilage organisms to valua- ble contributers to industrial fermentations. International Journal of Food Microbiology, issue 206, 24-38 5. Borneman, A.R. et al. (2014). Insights into the Dekkera bruxellensis Genomic Landscape: Comparative Genomics Reveals Variations in Ploidy and Nutrient Utilisa- tion Potential amongst Wine Isolates. PLOS genetics. Vol 10, Issue 2, 1-11. 6. Morris, E.O. and Eddy, A. A. (1957). Method for the measurement of wild yeast infection in pitching yeast. Journal of the Institute of Brewing. Vol 63, Issue 1, 34–35. 7. Custers, M.J.T. (1940). Onderzoekingen over het Gistgeslacht Brettanomyces. PhD thesis, University of Delft. 8. Sparrow, J. (2005). Wild Brews: beer The Orval brewhouse beyond the influence of brewer’s yeast. for secondary fermentations in a way Brewers Publications, Colorado. p197. similar to that first described by Claus- 9. Andrews, J. and Gilliland, R.B. (1952). sen, I found that the gravity of the beer Super-attenuation of beer: a study of three would drop by another two degrees organisms capable of causing abnormal at- Sacch (0.5 degree Plato) and the fer- tenuation. Journal of the Institute of Brewing. mentation would be complete after two Vol 58, Issue 3, 189-196. months at room temperature. As many 10. Shantha Kumara, H.M.C. et al. (1993). Localisation and characterisation of brewers will be aware, a very small α-glucosidase activity in Brettanomyces amount of Brettanomyces can have lambicus. Applied and Environmental Micro- a large effect and the recommended biology. Vol 59, no. 8, 2352-2358. pitching rate for secondary fermenta- 11. http://brettanomycesproject.com tions ranges from 100 to 2,000,000 12. Tonsmeire, M. (2014). American Sour cells per ml!12 I used approximately Beer. Brewers Publications, Colorado. 500,000 cells per ml. 13. Daenen, L. et al. (2007). Screen and Pure Brettanomyces fermentations evaluation of the glucoside hydrolase acitiv- are slow to start due to its inability ity in Saccharomyces and Brettanomcyes to produce glycerol4 but will still be brewing yeasts. Journal of Applied Microbiol- ogy, Vol 104, 478-488. completed within weeks with most 14. Spaepen, M. and Verachtert, H. (1982). Tzar Top brewed by Kent’s Old Dairy Brewery strains giving an apparent attenuation Esterase activity in the genus Brettanom- of 80-90%. The lack of glycerol produc- can be eight months before the Bret- cyes. Journal of the Institute of Brewing. Vol tion can also make pure Brettanomyces tanomyces starts to out-compete the 88, issue 1, 11-17. beers taste thinner than normal. As Saccharomyces, but once established, 15. Spaepen, M. et al. (1978). Fatty acids and the Brettanomyces has easier access it can continue to grow for years18. esters produced during the spontaneous to nutrients and is less stressed when Growing in mixed cultures with lactic fermentation of lambic and gueuze. Journal no competing organisms are present, acid bacteria leads to increased at- of the Institute of Brewing. Vol 84, issue 5, the flavour development has been re- tenuation9,19, as does adding lactic acid 278-282. ported as more muted than that found to the wort11. 16. Licker, J.L. et al. (1999). What is “Brett” (Brettanomcyes) flavor?: a preliminary when it is used for secondary fermen- Despite its long history in brewing, investigation. In Chemistry of Wine Flavor; tations – though in my experience this the full potential of making beer with Waterhouse, A. et al. ACS Symposium Se- is not always the case. Brettanomyces is only now being found ries. Washington. Pitching rates similar to that of and there is still much to learn about 17. Schifferdecker, A.J. et al (2014). The Saccharomyces are recommended this interesting organism. wine and beer yeast Dekkera bruxellensis. for primary fermentation and some Yeast, 31: 323-332. brewers oxygenate as normal whereas References 18. Van Oevelen, D. et al. (1977). Microbio- others to restrict oxygenation to stress 1. Claussen, NH. (1904). On a Method for logical aspects of spontaneous wort fer- the yeast and increase production of the Application of Hansen’s Pure Yeast mentation in the production of lambic and System in the Manufacturing of Well-Con- flavour compounds. Some brewers gueuze. Journal of the Institute of Brewing, ditioned English Stock Beers. Journal of the Vol 83, issue 6, 356-360. ferment at around 20°C, though others Institute of Brewing, Vol 10, Issue 4, 308-311. 19. Martens, H. et al. (1997). Microbiological will allow it to rise as high as 27-28°C 2. Van der Walt, JP and Kerken, AE. (1960). aspects of a mixed yeast-bacterial fermen- 12 to promote ester formation . The Wine yeasts of the Cape part IV – As- tation in the production of special Belgian In the “spontaneous” fermenta- cospore formation in the genus Brettanomy- acid ale. Journal of the Institute of Brewing, tions of lambic and similar beers it ces. Antonie van Leeuwenhoek International Vol 103, issue 2, 85-91. www.ibd.org.uk Brewer and Distiller International December 2016 z 17