Changes

Recently a new species of ''Brettanomyces'' has been proposed, although classification has not been fully established. The proposed name is ''Brettanomyces acidodurans'' sp. nov. Two strains of ''B. acidodurans'' were isolated from olive oil from Spain and Israel, ; however, its presence in olive oil has been described as "rare" because only two strains were found after searching dozens of olive oils. Its closest relation is to ''B. naardenesis'' by 73% of its genetic makeup. No teleomorph form was observed. This species is a strong acetic acid producer, and it is very tolerant of acetic acid in its environment. It can consume lactose and cellobiose but does not consume maltose. it is unknown but a possibility that this species contributes to the vinegary taste of spoiled olive oils, although this has generally been attributed to acetic acid bacteria <ref>[https://www.ncbi.nlm.nih.gov/pubmed/28160110 Brettanomyces acidodurans sp. nov., a new acetic acid producing yeast species from olive oil. Péter G, Dlauchy D, Tóbiás A, Fülöp L, Podgoršek M, Čadež N. 2017.]</ref>.

The ability of a given ''Brettanomyces'' strain to ferment different types of sugars might be at least partially linked to its source of isolation. For example, a strain of ''B. bruxellensis'' isolated from a soft drink could not ferment the disaccharides maltose, turanose, or the trisaccharide melezitose, whereas all of the other ''B. bruxellensis'' strains isolated from beer and wine could ferment these disaccharides/trisaccharide. The beer strains, ; however, were unable to ferment cellobiose or gentiobiose, as well as arbutin and methyl-glucoside. The wine strains were able to ferment these disaccharides, perhaps because they were adapted to the environment in which they were isolated (wine barrels). Further studies are needed to see if this is a trend throughout the species <ref name="Crauwels1"></ref>. Daenen et al. (2007) found that none of the ''B. bruxellensis'' strains isolated from lambic that they tested could utilize cellobiose (see [[Brettanomyces#Glycosides_and_Beta-Glucosidase_Activity|glycosides]] below). This data point challenges the belief that ''Brettanomyces'' lives in wooden barrels because it is able to consume the cellobiose of the wood. A study by Tyrawa et al. from [[Escarpment Laboratories]] agreed that wine isolated strains were generally better at fermenting cellobiose than strains isolated from beer at 15°C (59°F), however at 22.5°C (72.5°F) some of the beer strains started to utilize cellobiose, indicating that temperature plays a role in whether ''Brettanomyces'' can ferment certain sugars <ref name="Tyrawa_2017">[https://onlinelibrary.wiley.com/doi/abs/10.1002/jib.565 The temperature dependent functionality of Brettanomyces bruxellensis strains in wort fermentations. Caroline Tyrawa Richard Preiss Meagan Armstrong George van der Merwe. 2019. DOI: https://doi.org/10.1002/jib.565.]

Currently, research into how well ''Brettanomyces'' strains ferment the trisaccharide maltotriose has not been explored much by science, however. However, one study found that ''B. custersianus'' can ferment maltotriose. Another study found that all 7 strains of ''B. bruxellensis'' tested could ferment maltotriose, but not the trisaccharide raffinose. More investigation into this possibility is needed <ref>[http://www.asbcnet.org/events/archives/2015Meeting/proceedings/Pages/54.aspx Determination of sugar metabolism profiles of non-traditional yeasts in the Saccharomyces and Brettanomyces families. J. D. Cook, W. A. DEUTSCHMAN. ASBC Proceeding. 2015.]</ref><ref name="Crauwels1"></ref>.

Just like in other yeast species, the temperature has a direct effect on the rate of fermentation for ''Brettanomyces''. The optimal fermentation rate temperature range for ''Brettanomyces'' is between 22-32°C (77-90°F), however. However, one study by Tyrawa et al. found that several strains of ''B. bruxellensis'' fermented at 30°C "smelled terrible" <ref name=" Tyrawa_2017" />. At 20°C (68°F) fermentation rate is about half as slow. ''Brettanomyces'' will still grow at temperatures as low as 15°C (59°F) with about a third of strains being able to grow as low as 10°C (50°F) <ref name="Conterno_2006">[http://www.ajevonline.org/content/57/2/139 Genetic and Physiological Characterization of Brettanomyces bruxellensis Strains Isolated from Wines. Lorenza Conterno, C.M. Lucy Joseph, Torey J. Arvik, Thomas Henick-Kling, Linda F. Bisson. 2006.]</ref><ref>[https://www.ncbi.nlm.nih.gov/pubmed/24290676 Impact of sulfur dioxide and temperature on culturability and viability of Brettanomyces bruxellensis in Wine. Zuehlke JM, Edwards CG. 2013. DOI: 10.4315/0362-028X.JFP-13-243R.]</ref> but growth will be much slower. However, one study showed a slightly higher viability during the full-time period of fermentation at 15°C as opposed to the optimal growth and fermentation temperature range of 20-32°C. The growth rate at 15°C, while still slowly active, varies from strain to strain with some strains growing very poorly. Carbohydrates are consumed much slower, with cellobiose metabolizing ceasing for some strains (although phenol production stayed the same between 15°C and 22.5°C) <ref name="Tyrawa_2017" />. At a temperature of 35°C (95°F), fermentation is greatly inhibited due to cell death for most strains, with about a third of strains able to grow as high as 37°C (98.6°F) <ref name="Conterno_2006" />. The primary byproducts of ''Brettanomyces'' fermentation, which are ethanol, acetic acid, and CO2 are produced both during growth but also during fermentation after growth has stopped. At the more optimal fermentation temperatures of 25-32°C, ethanol and acetic acid are produced faster from fermentation, but the amounts of ethanol and acetic acid produced from fermentation are not affected by temperature (i.e. higher temperatures do not produce more ethanol and acetic acid from the same amount of sugar, they are just produced faster at warmer temperatures because fermentation is faster) <ref name="Brandam_2008" />. The warmer temperature ranges that are ideal for ''Brettanomyces'' fermentation rates and growth rates may still produce unfavorable flavors such as higher alcohols, ; however, this has not been analyzed as far as we know <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1555689637792578/ MTF conversation with Richard Preiss of Escarpment Labs. 01/20/2017.]</ref>. For more information on how fermentation temperature affects the flavor compounds of 100% ''Brettanomyces'' fermentation, see [[100%25_Brettanomyces_Fermentation#Impact_of_Fermentation_Temperature|Impact of Fermentation Temperature]].

Glycosides are flavorless compounds often found in plants/fruits that are composed of a molecule (often a flavor active compound) bound to a sugar molecule. The glycosidic bond can be broken, releasing the sugar molecule and the potential flavor active compound. These bonds can be broken with exposure to acid, as well as specific enzymes (beta-glucosidase) which can be added synthetically or produced naturally by some microorganisms, including some strains of ''Brettanomyces'' that have beta-glucosidase enzyme activity (mostly ''B. anomalus'' strains) <ref>[https://en.wikipedia.org/wiki/Glycoside "Glycoside." Wikipedia. Retrieved 06/27/2016.]</ref>. The release of flavor molecules from glycosides is thought to contribute to the flavor development of aging wines, as well as kriek (cherry) lambic <ref name="Daenen2">[http://onlinelibrary.wiley.com/doi/10.1111/j.1567-1364.2008.00421.x/pdf Evaluation of the glycoside hydrolase activity of a Brettanomyces strain on glycosides from sour cherry (Prunus cerasus L.) used in the production of special fruit beers. Luk Daenen, Femke Sterckx, Freddy R. Delvaux, Hubert Verachtert & Guy Derdelinckx. 2007.]</ref>. It is speculated that flavor compounds from hops can also be released from glycosides <ref name="Daenen1">[http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2007.03566.x/full Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts. L. Daenen, D. Saison, F. Sterckx, F.R. Delvaux, H. Verachtert, G. Derdelinckx. 2007.]</ref>, ; however, at least one study has shown no significant difference in a blind taste test between hopped beer exposed to the beta-glucosidase enzymes and hopped beer that was not exposed to the enzyme <ref name="Vervoort">[http://onlinelibrary.wiley.com/wol1/doi/10.1111/jam.13200/abstract Characterization of the recombinant Brettanomyces anomalus β-glucosidase and its potential for bioflavoring. Yannick Vervoort, Beatriz Herrera-Malaver, Stijn Mertens, Victor Guadalupe Medina, Jorge Duitama, Lotte Michiels, Guy Derdelinckx, Karin Voordeckers, and Kevin J. Verstrepen. 2016.]</ref>. Beta-glucosidase also allows the breakdown of cellobiose and cellotriose <ref>[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC241500/ Fermentation of Cellodextrins by Different Yeast Strains. Pierre Gondé, Bruno Blondin, Marc Leclerc, Robert Ratomahenina, Alain Arnaud, and Pierre Galzy. 1984.]</ref><ref name="Roos_2018">[https://www.ncbi.nlm.nih.gov/pubmed/30246252?dopt=Abstract Jonas De Roos and Luc De Vuyst. 2018. DOI: 10.1002/jsfa.9291.]</ref>. This has been believed to be a mechanism in which ''Brettanomyces'' can survive in barrels, ; however, most strains of ''Brettanomyces'' found in lambic do not seem to have the ability to produce beta-glucosidase nor utilize cellobiose. Daenen et al. (2007) found that none of the ''B. bruxellensis'' strains isolated from lambic could utilize cellobiose, but strains of ''B. anomalus'' and ''B. custersianus'' isolated from lambic could utilize cellobiose <ref name="Daenen1" /><ref name="Roos_2018" />. Additionally, a study by Tyrawa et al. from [[Escarpment Laboratories]] agreed that wine isolated strains were generally better at fermenting cellobiose than strains isoalted from beer at 15°C (59°F), however. However, at 22.5°C (72.5°F) most of the beer strains started to utilize cellobiose after a few days of incubation (they preferred other food sources such as glucose and maltose), indicating that temperature plays a role in whether ''Brettanomyces'' can ferment certain sugars <ref name="Tyrawa_2017" />, and the [[Brettanomyces#Carbohydrate_Metabolism_and_Fermentation_Temperature|table from the NCYC ''Brettanomyces'' strains]] suggests that fermenting cellobiose is generally rare for ''B. bruxellensis''. This suggests that not only is ''B. bruxellensis'' strains that are isolated from beer are generally unable to break down glycosides, but they are probably also unable to utilize the cellobiose in wooden barrels as a food source (although higher temperature might allow some beer strains to start fermenting cellobiose).

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.

It has been hypothesized that the production and destruction of various phenols by ''Brettanomyces'' is connected with the [https://en.wikipedia.org/wiki/Redox redox balance], ; however, this has not been demonstrated. Ethyl phenols have been shown to be highly attractive to fruit flies, and it has also been proposed that these aromatics allow ''Brettanomyces'' to travel from food source to food source and by doing so increasing its chances of survival in the wild <ref>[http://www.sciencedirect.com/science/article/pii/S0960982214016558 Olfaction: Smells Like Fly Food. Geraldine A. Wright. 2015.]</ref><ref name="smith_divol_2016"></ref>. Phenols have been shown to have positive effects on decreasing protein glycation, a complication associated with type 2 diabetes <ref>[http://www.asbcnet.org/publications/journal/vol/2017/Pages/ASBCJ-2017-1323-01.aspx High Phenolic Beer Inhibits Protein Glycation In Vitro. Susan M. Elrod, Phillip Greenspan, Erik H. Hofmeister. 2017. ]</ref>.

| Bruxellensis||''Dekkera bruxellensis''||''Brettanomyces bruxellensis''||Brettanomyces bruxellensis||Medium intensity Brett character. Classic strain used in secondary fermentation for Belgian style beers and lambics. || Thought to be the same as White Labs WLP648, ; however, side by side sensory comparisons seem to indicate that they might not be the same <ref name="coffey_drei">[http://www.alesoftheriverwards.com/2015/12/brettanomyces-drei-vs-brettanomyces-vrai.html Ed Coffey. "Brettanomyces Drei vs. Brettanomyces Vrai". Ale of the Riverwards blog. 12/07/2015. Retrieved 11/13/2018.]</ref>. '''Commercial pitches only.'''

Major yeast labs will often store yeast in a -80°C laboratory freezer in a media/glycerol solution, however, although this option is generally not practical for brewers <ref>[https://en.wikipedia.org/wiki/Cryopreservation Cryopreservation. Wikipedia. Retrieved 07/07/2016.]</ref>. The next best option for long-term storage of ''Brettanomyces'' is freezing with 10% glycerol in a home freezer, however. However, the effects of storing yeast at such a high and often variable temperature have not been evaluated scientifically. Traditionally ''Saccharomyces'' yeast has been stored on slants held in a refrigerator and can provide storage for a few months up to 2+ years, depending on the type of slant used (using mineral oil in slants has been shown to extend the life of stored ''Saccharomyces''). Homebrewers, however, have reported poor survival of ''Brettanomyces'' on slants. Data from a MTF member showed promising results by buffering the slant media. In this data, Brettanomyces has stored well for up to 100 days on the buffered media. It is not known for how long viability will remain high on buffered slants. For instructions on how to make slants at home capable of storing any microbe for potentially 2+ years, [http://suigenerisbrewing.blogspot.com/2015/11/easy-home-yeast-banking-and-video.html see Bryan's video on Sui Generis Brewing (requires a pressure cooker)]. Agar plates are the least effective solution and have been observed anecdotally to reduce the viability of ''Brettanomyces'' over a few months <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1083075848387295/?comment_id=1083272091701004&offset=0&total_comments=13&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Conversation with Matt Humbard, Ritchie Preiss, and Jeff Melo on MTF. 6/4/2015.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1115768398451373/?comment_id=1115817201779826&offset=0&total_comments=34&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Nick Impellitteri on MTF regarding storing Brett on agar plates. 7/24/2015.]</ref>.

Occasional feeding has been shown to keep ''Brettanomyces'' alive in beer for brewers who do not have a lab, ; however, many variables may come into play as far as how effective this will be for individual strains and in different environments. Although no research has been done to indicate what the best practices are for feeding ''Brettanomyces'' to keep it alive in beer, we recommend trying this method: every 3-6 months swirl the vessel so as to suspend all of the yeast and then decant 70-90% of the beer and suspended yeast slurry, and replace it with a 1.040 starter wort with yeast nutrients. This method will discard a lot of the old yeast cells, while retaining enough living cells for replication <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1345924532102424/?comment_id=1345979272096950&reply_comment_id=1346020438759500&comment_tracking=%7B%22tn%22%3A%22R3%22%7D Conversation with Mark Trent and Richard Preiss on MTF regarding occasional feeding of ''Brettanomyces'' to keep it alive. 07/07/2016.]</ref>. Some strains may survive extended periods of aging in beer, ; however, their viability and vitality will be greatly reduced over time. Interestingly, ''Brettanomyces'' remains '''more''' viable over time if it was co-fermented with ''S. cerevisiae'' than if it was fermented without the presence of ''S. cerevisiae''; i.e. [[100%25_Brettanomyces_Fermentation|100% ''Brettanomyces'' beers]] or ''Brettanomyces'' and ''Lactobacillus <ref name="Hubbe" />.