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Brettanomyces

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Environment and Survival
A genetic survey of 1488 different strains of ''B. bruxellensis'' from 29 countries, 5 continents, and 9 different fermentation niches was conducted in 2018 by Avramova et al. They found that these strains formed roughly 5 genetic groups with mostly separate ancestral lineages, and 1 group with a mixed ancestral lineage: 3 wine groups, 1 beer group, 1 kombucha group (most distantly related to the beer group), as well as 1 tequila/ethanol group that has multiple ancestral lineages. This was expressed mostly in the ploidy level of each group (the number of sets of chromosomes), with 2 of the wine groups, the tequila group, and the beer group containing more sets of chromosome pairs than the other groups (diploid vs triploid; this is thought to encourage adaption and hybridization). Additionally, the triploid wine group was generally more tolerant of SO<sup>2</sup> than the diploid wine groups. The genetic differences between the fermentation substrates (beer, wine, etc.) were lower but still significant, and this was explained by the frequent cross-over of equipment such as wine barrels being used for beer fermentation. When comparing the geographic differences, they found geography contributed only 5% of genetic differences, while geography explained more than 50% of genetic differences in non-wine strains, suggesting that beer, kombucha, and tequila strains are more localized genetically than wine strains and that humans probably helped the wine strains travel across the globe. They also found that although one study reported spore-forming versions of ''B. bruxellensis'' (referred to as ''Dekkera bruxellensis''), the genetic makeup of the analyzed strains determined their ability to sporulate to be non-existent or rare (only one study that we know of by [https://link.springer.com/article/10.1007%2FBF02539015 Walt and Kerken in 1960] has reported sporulation in ''Brettanomyces'' only on specific agar types with vitamins added, indicating that sporulation in ''Brettanomyces'' is extremely rare) <ref name="Avramova_2018" />. See also [https://www.facebook.com/groups/MilkTheFunk/permalink/2022801681081369/ Richard Preiss's discussion of this study on MTF].
Sulfite and SO<sub>2</sub> inhibits the growth of ''Brettanomyces'', and is often used in the wine industry to prevent the growth of ''Brettanomyces'' (although ''Brettanomyces'' is usually seen is a contaminant in wine, some wineries have identified small amounts of flavors from ''Brettanomyces'' as being beneficial to certain wine styles, and is said to increase the complexity and impart an aged character in young wines <ref name="smith_divol_2016"></ref>) <ref>[http://onlinelibrary.wiley.com/doi/10.1111/j.1745-4549.2012.00702.x/abstract Removal of Brettanomyces Bruxellensis from Red Wine Using Membrane Filtration. Umiker, Descenzo, Lee, and Edwards. 04/24/2012.]</ref>. However, it has been shown that wine strains of ''B. bruxellensis'' could survive dosages of up to 1 mg/L of molecular SO<sub>2</sub>, and the very high dosage of 2.1 mg/L was needed to kill ''Brettanomyces'' in wine <ref name="Agnolucci_2017" />. This dosage of molecular SO<sub>2</sub> requires a total amount of SO<sub>2</sub> that is beyond legal limits (350 mg/l <ref>[https://grapesandwine.cals.cornell.edu/sites/grapesandwine.cals.cornell.edu/files/shared/documents/Research-Focus-2011-3.pdf Sulphur Dioxide Content of Wines: the Role of Winemaking and Carbonyl Compounds. Nick Jackowetz, Erhu Li, and Ramón Mira de Orduña. 2011.]</ref>; see this [https://grapesandwine.cals.cornell.edu/newsletters/appellation-cornell/2012-newsletters/issue-12/article-contains-sulfites/ Cornell University] blog post that explains the difference between ''free'' and ''molecular'' SO<sub>2</sub>) and has negative effects on wine. One study found that out of 145 strains of ''B. bruxellensis'', 107 of which were wine strains with the rest being from beer, tequila, kombucha, etc., 36% of them were either tolerant (lagged growth, but achieved full growth eventually) or resistant (no lagged growth, and achieved full growth) to 0.6 mg/L of molecular SO<sub>2</sub>. 46 of the 52 resistant/tolerant strains were wine strains, thus demonstrating that wine strains of ''B. bruxellensis'' are generally more tolerant of SO<sub>2</sub> than strains of ''B. bruxellensis'' that are found in other types of beverages. It is thought that the wine strains have adapted to the conditions of winemakers adding SO<sub>2</sub> to wine <ref>[https://www.frontiersin.org/articles/10.3389/fmicb.2018.01260/full Molecular Diagnosis of Brettanomyces bruxellensis’ Sulfur Dioxide Sensitivity Through Genotype Specific Method. Avramova M, Vallet-Courbin A, Maupeu J, Masneuf-Pomarède I, Albertin W. 2018. DOI: 10.3389/fmicb.2018.01260.]</ref>. In addition, it has been proposed that SO<sub>2</sub> can induce a so-called "viable but nonculturable" (VBNC) state in ''Brettanomyces'', which means that ''Brettanomyces'' cells in this state cannot grow or be cultured on traditional media but can remain viable and create a low amount of phenol character (see [[Quality_Assurance#VBNC_In_Yeast|VBNC in Yeast]]). Some strains of ''Candida pyralidae'', ''Wickerhamomyces anomalus'', ''Kluyveromyces wickeramii'', ''Torulaspora delbrueckii'' and ''Pichia membranifaciens'' have been found to produce toxin that inhibits ''Brettanomyces'', and these toxins have been proposed as an alternative to SO<sub>2</sub> as a way to kill ''Brettanomyces'' (killer wine strains of ''Saccharomyces cerevisiae'' do not kill ''Brettanomyces''; see [[Saccharomyces#Killer_Wine_Yeast|Killer Wine Yeast]] for more information). [http://www.laboratoriosenosan.com/en/effectiveness-of-kaolin-silver-complex/ Kaolin silver complex (KAgC)] has been found to inhibit ''Brettanomyces'' and acetic acid bacteria in wine when used in legal dosages, and has been proposed as a replacement for SO<sub>2</sub> or to minimize the use of SO<sub>2</sub> <ref>[https://www.ncbi.nlm.nih.gov/pubmed/29666535?dopt=Abstract Effect of kaolin silver complex on the control of populations of Brettanomyces and acetic acid bacteria in wine. Izquierdo-Cañas PM, López-Martín R, García-Romero E, González-Arenzana L, Mínguez-Sanz S, Chatonnet P, Palacios-García A, Puig-Pujol A. 2018. DOI: 10.1007/s13197-018-3097-y.]</ref>. Other proposed replacements for SO<sub>2</sub> as a way to inhibit ''Brettanomyces'' in wine include [https://en.wikipedia.org/wiki/Pascalization high pressure processing] and [https://www.sciencedirect.com/science/article/abs/pii/S0255270106001929 pulsed electric fields] <ref>[https://www.sciencedirect.com/science/article/pii/S1466856418302972 SO2, high pressure processing and pulsed electric field treatments of red wine: Effect on sensory, Brettanomyces inactivation and other quality parameters during one year storage. Sanelle Van Wyk, Mohammed M. Farid, Filipa V.M. Silva. 2018. DOI: https://doi.org/10.1016/j.ifset.2018.06.016.]</ref>.
The addition of vitamins can have a positive impact on ''Brettanomyces'' growth. For example, while ''Brettanomyces'' does not need riboflavin (vitamin B2) or thiamine (vitamin B1) in order to grow, the presence of either or both of these two vitamins encourages ''Brettanomyces'' growth <ref>[http://onlinelibrary.wiley.com/doi/10.1002/jib.385/full The influence of thiamine and riboflavin on various spoilage microorganisms commonly found in beer. Barry Hucker, Melinda Christophersen, Frank Vriesekoop. 2017.]</ref>. Other vitamins such as p-aminobenzoic acid (PABA), folic acid (vitamin B9), nicotinic acid (vitamin B3), pantothenic acid (vitamin B5) are also not required for most strains of ''Brettanomyces'' to grow. The presence of alcohol can increase the dependence on vitamins for some strains of ''Brettanomyces'' to grow. For example, Myo-inositol (vitamin B8) and thiamine (vitamin B1) were required by two strains of ''B. bruxellensis'' when grown in 10% ethanol but not in 0% ethanol. Biotin (vitamin B7) is the one exception, and it was found that the lack of biotin inhibited the growth of some strains of ''B. bruxellensis''. Other studies contradict these previously mentioned findings, showing that thiamine was not required by the strains of ''B. bruxellensis'' tested, that pyridoxine was required, and biotin was not required. These discrepancies between scientific studies are probably due to the genetic differences between the strains selected, the growth media chosen by the scientists, and/or the growth conditions <ref>[http://www.mdpi.com/2311-5637/2/3/17 Use of Nutritional Requirements for Brettanomyces bruxellensis to Limit Infections in Wine. Nicolas H. von Cosmos and Charles G. Edwards. 2016.]</ref>.

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