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Brettanomyces

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''Brettanomyces'' strains may possess both alpha and beta-glucosidases. Beta-glucosidase is intracellular (works on sugars that are passed into the cell through the cell wall), while alpha-glucosidase is both intracellular and extracellular (released into the environment by the cell). <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><ref name="Kumara_1993">[http://aem.asm.org/content/59/8/2352.short Localization and Characterization of α-Glucosidase Activity in Brettanomyces lambicus. H. M. C. Shantha Kumara, S. De Cort and H. Verachtert. 1993.]</ref> These enzymes allow ''Brettanomyces'' strains to break down a broad range of sugars, including long-chain carbohydrate molecules (polysaccharides, dextrins, and cellulose/cellobiose), and to liberate glycosidically bound sugars which are unfermentable to ''Saccharomyces'' yeasts. <ref name="Steensels"></ref><ref>[http://www.scribd.com/doc/277758178/Insight-into-the-Dekkera-anomala-YV396-genome Insight into the Dekkera anomala YV396 genome. Samuel Aeschlimann. Self-published on Eureka Brewing Blog. Spet 2015.]</ref>.
Extracellular and intracellular alpha-glucosidase activity has been shown to break down sugars up to 9-12 chain carbons in one strain of ''B. lambicus'' (now classified as ''B. bruxellensis''), which is partly responsible for the slow, over-attenuation of wort that some strains of ''Brettanomyces'' an achieve in beers such as lambic and American sour beers <ref name="yakobson_introduction"></ref><ref name="smith_divol_2016"></ref>. Alpha-glucosidases are the enzymes that allow them to break down maltose, turanose, melezitose, and trehalose, as well as dextrins such as maltotetraose and maltopentaose. These enzymes work by cleaving off glucose that can be directly consumed by the cell, leaving a shorter chain sugar behind which is then further broken down. In the case of extracellular alpha-glucosidase activity, this breakdown of complex sugars occurs outside of the cell and may benefit other microorganisms if present such as lactic acid bacteria. These dextrins are left over after a normal ''Saccharomyces'' fermentation <ref name="Steensels"></ref>. Some other polysaccharides can be fermented by ''Brettanomyces'', including starch, laminarin, and pectin <ref name="Crauwels1"></ref>. The more complex the starch or sugar, the slower it is hydrolyzed by the alpha-glucosidase enzymes. The optimal pH for the alpha-glucosidase enzyme produced by one strain of ''B. bruxellensis'' was 6 and at a temperature of 39-40°C (102-104°F), and its activity was greatly reduced below a pH of 4.5 and above 8 (although citric acid was used as a buffer, and its effects on the enzyme was not compared to other acids), which might contribute to slower ''Brettanomyces'' fermentation in acidic beers <ref name="Kumara_1993" />. ''B. bruxellensis'' and ''B. nanus'' can also produce oligo-1,6-glucosidase, which hydrolyze the alpha-1,6 linkages in starch and glycogen to produce oligosaccharides, and then further break down these oligosaccharides to produce sugars with alpha-1,4 linkages (for example, maltose, in the case of starches from malted barley <ref>[https://www.ncbi.nlm.nih.gov/books/NBK22396/ Complex Carbohydrates Are Formed by Linkage of Monosaccharides. Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. 2002.]</ref>). These alpha-1,4 linkages (maltose) are then further broken down by the alpha-glucosidases. Unlike for some domesticated diastatic ''S. cerevisiae'' strains, this ability for ''Brettanomyces'' to break down starches has occurred in the wild without domestication <ref name="roach_2019" /><ref>"Amylases". H. Taniguchi, Y. Honnda, in Encyclopedia of Microbiology (Third Edition), 2009.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3049768678384659/ Ben Sykes. Milk The Funk Facebook post on ''Brettanomyces'' evolution to ferment dextrins. 11/13/2019.]</ref>.
Beta-glucosidases can break down the beta-glycosidic bond in disaccharides (cellulose, cellobiose, and gentiobiose) <ref name="ucdavis_chemwiki">[http://chemwiki.ucdavis.edu/Core/Organic_Chemistry/Carbohydrates/Disaccharides "Disaccharides." UC Davis Chemwiki. Retrieved 05/15/2016.]</ref><ref name="smith_divol_2016"></ref>, as well as glycosides. Glycosides are sugar molecules connected to other organic compounds such as acids, alcohols, and aldehydes which are flavor and aroma inactive due to the sugar molecule attached. By cleaving off the sugar molecule through beta-glucosidase activity, ''Brettanomyces'' species can liberate these compounds (called aglycones) into their aroma-active and flavor-active states, or states that may become flavor and aroma active through further modification <ref>Daenen et al., 2008. 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. FEMS Yeast Res. 8, 1103-1114.</ref>. Therefore some ''Brettanomyces'' strains are believed to be able to produce novel flavors and aromas from hops, fruits, and fruit pits that ''Saccharomyces'' yeasts cannot produce. In addition, the liberated aroma and flavor active compounds may be further processed by ''Brettanomyces'' through ester production or destruction pathways. See [[Brettanomyces#Glycosides_and_Beta-Glucosidase_Activity|Beta-Glucosidase Activity]] for more information.

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