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Brettanomyces

559 bytes added, 18:35, 4 May 2020
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Phenols such as 4-vinylphenol (4VP; barnyard, medicinal) and 4-vinylguaiacol (4-VG; clove) can be produced in beer through the decarboxylation of hydroxycinnamic acids (HCAs) by yeast, and also in small amounts by long boils with a portion of the wort coming from wheat (3+ hours resulted in 0.3 ppm of 4VG). HCAs, such as ferulic acid and p-coumaric acid, are found in malt and released into wort during mashing at levels that are far below their flavor thresholds (greater than 500ppm flavor threshold) <ref name="lentz_2018">[http://www.mdpi.com/2311-5637/4/1/20/html#B13-fermentation-04-00020 The Impact of Simple Phenolic Compounds on Beer Aroma and Flavor. Michael Lentz. 2018. doi: 10.3390/fermentation4010020.]</ref>. Some strains of ''Oenococcus oeni'' and ''Lactobacillus'', as well as some strains of yeast such as ''Pichia'' spp, have been found to produce HCA's via cinnamoyl esterase activity in wine, although when these strains have been used in wine to increase the HCA levels, the final phenol levels produced by ''Brettanomyces'' were the same as wine that did not have an increase in HCA levels (the precursors in wine that lead to HCA's are different than they are in beer) <ref>[http://www.ajevonline.org/content/early/2018/05/02/ajev.2018.17092 Influence of Oenococcus oeni and Brettanomyces bruxellensis on Wine Microbial Taxonomic and Functional Potential Profile. Marie Lisandra Zepeda-Mendoza, Nathalia Kruse Edwards, Mikkel Gulmann Madsen, Martin Abel-Kistrup, Lara Puetz, Thomas Sicheritz-Ponten, Jan H. Swiegers, Am J Enol Vitic. May 2018. DOI: 10.5344/ajev.2018.17092.]</ref>. The esters in grape must that contain HCA's (ethyl ferulate and ethyl coumarate) can also be formed by acidic hydrolysis which occurs at the low pH of wine, and HCA's can then be released from these esters. This formation of esters is a slow process in wine, with one study reporting ~0.03 ppm of ethyl ferulate and ~0.4 ppm of ethyl coumarate at the end of primary fermentation and ~0.09 ppm of ethyl ferulate and ~1.4 ppm of ethyl coumarate after 10 months of barrel aging <ref>[https://pubs.acs.org/doi/full/10.1021/jf204908s Hydroxycinnamic Acid Ethyl Esters as Precursors to Ethylphenols in Wine. Josh L. Hixson, Nicola R. Sleep, Dimitra L. Capone, Gordon M. Elsey, Christopher D. Curtin, Mark A. Sefton, and Dennis K. Taylor. 2012. DOI: 10.1021/jf204908s.]</ref>. We are not aware of any studies that have reported an increase in HCA's from acidic hydrolysis over time in beer; however, this is a standard laboratory technique for forcing the release of HCA's from barley (although this lab technique uses a lower pH then that of sour beer). It is therefore conceivable that HCA levels could increase in sour beer over time.
While both ''Saccharomyces'' (only by "phenolic off flavor positive/POF+" strains) and ''Brettanomyces'' strains have varying capabilities based on strain of converting hydroxycinnamic acids to their vinyl derivatives <ref name="Lentz">[http://www.mdpi.com/2304-8158/4/4/581/htm Analysis of Growth Inhibition and Metabolism of Hydroxycinnamic Acids by Brewing and Spoilage Strains of Brettanomyces Yeast. Michael Lentz and Chad Harris. 2015.]</ref>, ''Brettanomyces'' is also able to reduce these vinyl phenol derivatives to ethyl phenol derivatives. Phenolic acid decarboxylase (PAD) is the enzyme that converts the HCAs into vinyl phenols. Vinyl reductase (VA) is the enzyme that reduces vinyl phenols to ethyl phenols <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1632316743463200/ Analysis of phenolic acid decarboxylase enzyme from the wine spoilage yeast Brettanomyces bruxellensis (poster). Mike Lentz, Jamie Lynch, Pricilla Walters, Rachel Licea, Henok Daniel, Kimberly Pereira. 2017.]</ref>. Phenol production has been observed to occur shortly after inoculation of ''Brettanomyces'' and has been hypothesized to play a large role in replenishing NAD<sup>+</sup> to alleviate the initial lag growth phase in ''Brettanomyces'' <ref name="Tyrawa_Masters">[https://atrium.lib.uoguelph.ca/xmlui/handle/10214/14757 Demystifying Brettanomyces bruxellensis: Fermentation kinetics, flavour compound production, and nutrient requirements during wort fermentation. University of Guelph, Masters Thesis. Department of Molecular and Cellular Biology. 2020.]</ref>. Almost all strains of ''Brettanomyces'' produce ethyl phenols, one strain of ''Brettanomyces anomalus'' has been found that has lost the genetic capability to produce phenols <ref>[https://www.frontiersin.org/articles/10.3389/fmicb.2020.00637/full Assessing Population Diversity of Brettanomyces Yeast Species and Identification of Strains for Brewing Applications. Marc Serra Colomer, Anna Chailyan, Ross T. Fennessy, Kim Friis Olsson, Lea Johnsen, Natalia Solodovnikova and Jochen Forster. 2020. DOI: https://doi.org/10.3389/fmicb.2020.00637.]</ref>.
These vinyl derivatives have similar tastes to the ethyl derivatives but have lower flavor thresholds. Levels of all compounds produced vary depending on species and strain of ''Brettanomyces''. Although the production of ethyl phenols has been identified to occur higher in substrates with more available sugars, and this has also correlated with higher growth <ref name="Barata_2008">[http://onlinelibrary.wiley.com/doi/10.1111/j.1567-1364.2008.00415.x/full The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine. André Barata, Daniela Pagliara, Tiziana Piccininno, Francesco Tarantino, Wilma Ciardulli, Manuel Malfeito-Ferreira, Virgílio Loureiro. 2008. DOI: 10.1111/j.1567-1364.2008.00415.x]</ref>, some data supports that pitching rate does not have an effect on how much phenol content is produced by ''Brettanomyces''<ref name="MTF_Brett_Secondary">[http://www.milkthefunk.com/wiki/Brettanomyces_secondary_fermentation_experiment Brettanomyces secondary fermentation experiment. Milk The Funk Wiki. Lance Shaner and Richard Preiss. Retrieved 04/21/2016.]</ref>. Additionally, Curtin et al. (2013) showed that while both cell growth and attenuation was inhibited in anaerobic conditions in wine, phenol production was not (in fact, the phenol production was inhibited by aerobic conditions). They also showed that each of the three strains of ''B. bruxellensis'' tested all produced the same amount of phenols, while other flavor compounds such as esters were produced at different levels by each of the strains <ref>[https://www.ncbi.nlm.nih.gov/pubmed/24010603 Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma. Curtin CD, Langhans G, Henschke PA, Grbin PR. 2013]</ref>. Perhaps growth itself is not as much of a factor in producing phenols, but having sugars available for metabolism is. This contradicts the somewhat popular belief that under-pitching ''Brettanomyces'' produces more "funky" flavors. Additionally, perhaps some strains are perceived as "funkier" than others due to less ester production and more fatty acid production (isobutyric acid, for example), rather than more phenol production.

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