As a home brewer who loves experimentation, I am always looking for new things to try. So, my ears perked up when I saw a video on Brulosophy about sour beer tasting and they mentioned a Brettanomyces beer that was far older than the strong hop aroma let on [1]. As luck would have it, later the same day The Mad Fermentationist posted about how some isolates of Brettanomyces can free additional aromatic compounds from hops that are normally locked away, strengthening and prolonging hops aromas [2]. These additional flavor and aroma compounds are bound up inside other molecules called glycosides.
What are Glycosides?
A glycoside is a molecule consisting of two parts: a carbohydrate and another molecule called an aglycone; a glucoside has glucose as its carbohydrate [3]. Its this aglycone that can be detected by the nose or tongue as an additional aroma or flavor. Unfortunately, aglycones are flavorless when bound up in a glycoside.
An aglycone is not a single thing, but rather a class of things: an aglycone can be any number of aroma or flavor molecules. They can be found in hops, grapes and other fruits, and spices-almost any plant matter. The amounts and types of glycosides in hops are highly dependent on variety [4], much like alpha - acid levels.
Plants form glycosides because aglycones cannot be easily transported through the plant. By binding the aglycone to a carbohydrate, it becomes water soluble and transportable. It's sort of like boxing up a beer bottle for shipment to competition: UPS can't deliver a bottle with stamps on it, but it can deliver a bottle nicely boxed up. It must then be un-boxed later for tasting.
Free the Aglycones!
(This is perhaps the worst rally cry in history.) Aglycones can be broken from the carbohydrate by being put in an acidic environment (acid hydrolysis) or with the help of an enzyme (enzyme catalysis) [3]. Acid hydrolysis is very slow, and frees negligible amounts of aglycones [5]. Additionally, the vast majority of beers are not nearly acidic enough for this process to occur.
So, we are left with enzymes. In general, enzymes are special proteins capable of vastly increasing the rate of specific chemical reactions. Brewers already use many enzymes, primarily in the malting and mashing process to break grain starches into fermentable sugars. To break aglycones from glucosides, the enzyme - beta glucosidase is required. Beta glucosidase is produced by many plants, bacterias, and fungi, including some yeasts [6].
Glucosidase from Yeast
Certain isolates of both Brettanomyces and Saccharomyces have been shown to produce beta glucosidase (and yes, Brettanomyces is a yeast, not a bacteria). But, not all species can. In fact, it is a rare trait, though much more common in Brettanomyces. Even more confusing, not all isolates of the same species are consistent: some isolates might produce beta glucosidase, while others do not. (All standard brewing yeast is of the species S. cerevisiae; WLP001 and Wyeast 1028 are examples of different isolates of this species.).
I have only been able to find evidence of two S. cerevisiae isolates that produce beta glucosidase: UVAFERM 228 (Danstar Lallemand; wine yeast; aka U228) and CMBS LD40 (Center for Malting and Brewing Science, Leuven, Belgium; beer yeast) [7]. While both were shown to release slightly increased aglycone levels during fermentation, neither are available to home brewers so far as I can tell.
Far more Brettanomyces isolates seem to test positive for beta glucosidase. Many studies focus on the species B. custersii (also known as B. bruxellensis [8]). One study found that 7 of 26 tested B. custersii isolates were beta glucosidase active [9]. One isolate in particular, CMBS LD72 (from a lambic fermentation), has been shown not only to increase free aglycone levels, but to increase them above human-detectable levels in beer [5]. This is the only isolate I have found evidence of producing above-threshold levels of free aglycones. But, given the dearth of studies, there are likely many more isolates capable of doing so.
The bad news is none of these isolates are available to the home brewer either. I've talked to researchers from both Wyeast and White Labs and none of the yeasts they offer have been tested for beta glucosidase activity. But, both expressed great interest in testing them, so this information could become available soon. Until then, Michael Tonsmeire suggests pitching many isolates of Brettanomyces into your beer in hopes of getting one or more to produce the enzyme [10].
Glucosidase from Commercial Enzymes
If you'd rather not trust your beer to providence and you can't wait for home brew yeasts to be tested, another option is to use commercially available enzyme formulations already in use by many wine makers. Three popular formulations are Scott Labs Scottzyme BG and Lallzyme BETA [11], and DSM Oenobrands Rapidase AR 2000 [12]. All three are slightly different formulations of pectinase and -glucosidase enzymes.
One study compared AR 2000 with the B. custersii isolate LD72 for its ability to release aromatic volatiles from sour cherries [5]. Of 13 aglycones tested for, the AR 2000 produced greater amounts of 11 of them compared to LD72. In fact, 7 of the 13 compounds were above human flavor threshold in the AR 2000 beer, compared to just two in the Brettanomyces beer. Other isolates of Brettanomyces might perform better, but it certainly shows the potential of the commercial enzyme formulations for freeing aglycones and unlocking additional aromas.
Getting More Flavor
You can get more flavor and aroma out of your hoppy or fruit beers or wines using the power of enzymes. Glucosides are water soluble, meaning hops or fruits added at any point in the process should add bound aglycones to your beer. Dry hopping might prove especially effective to increase glycoside levels without making the beer overwhelmingly bitter. Most glycosides in grapes are in the skins. Fruit beers using extracts only will probably not benefit from treatment by beta glucosidase.
Using a beta glucosidase positive isolate of Brettanomyces will release extra flavor during the course of fermentation and conditioning. If you try a commercial enzyme, be sure to add it according to the directions [11, 12]. This is because enzymes can be inhibited (stopped from working) by sugar, alcohol, pH, or temperature, and different enzymes are inhibited by different factors [13]. Also, there is some evidence that beta glucosidase might result in the breakdown of esters and/or cause slight color loss [14], but commercial formulations are made to limit these effects.
***
Dennis Waldron is the latest writer to join the HomeBrewTalk team! You can find more from him at the Life Fermented Blog, Dennis' personal blog about home brewing, brew science, DIY brewing equipment, and other fermented goodies. Please help me in welcoming Dennis to the team by posting in the comments, and visiting his site.
***
Further Reading - Notes
[1] Brlosophy, "Sours from the other coast: Homebrew tasting," 2014. [Online]. Available: http://brulosophy.com/2014/08/25/sours-from-the-other-coast-homebrew-tasting/
[2] M. Tonsmeire, "New zealand saison and glycosides," 2014. [Online]. Available: http://www.themadfermentationist.com/2014/08/new-zealand-saison-and-glycosides.html
[3] H. Goldstein, P. Ting, A. Navarro, and D. Ryder, "Water-soluble hop flavor precursors and their role in beer flavor," EBC Congress, pp. 5362, 1999.
[4] H. Kollmannsberger, M. Biendl, and S. Nitz, "Occurence of glycosidically bound flavour compounds in hops, hop products and beer," Monatsschrift fr Brauwissenschaft, pp. 839, 2006.
[5] L. Daenen, F. Sterckx, F. Delvaus, H. Verachtert, and G. Derdelinchkx, "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 Research, vol. 8, pp. 110314, 2008.
[6] N. Ranwedzi, "Optimization of -glucosidase activity in recombinant Saccharomyces cerevisiae strains," 2007, Thesis, Master of Science, Stellenbosch University.
[7] S. Crauwels, D. Saison, F. Sterckx, F. Delvaux, H. Verachtert, and G. Derdelinckx, "Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeast," Applied and Environmental Microbiology, vol. 104, pp. 47888, 2007.
[8] J. Barnett, R. Payne, and D. Yarrow, Yeasts: Characteristics and Identification, 3rd ed. Cambridge University Press, 2007.
[9] S. Crauwels, B. Zhu, J. Steensels, P. Busschaert, G. de Samblanx, K. Marchal, K. Willems, K. Verstrepen, and B. Lievens, "Assessing genetic diversity in Brettanomyces yeast using DNA fingerprinting and whole genome sequencing," Applied and Environmental Microbiology, vol. 80, no. 14, pp. 4398413, 2014.
[10] M. Tonsmeire, American Sour Beers: Innovative Techniques for Mixed Fermentations. Brewer's Publications, 2014.
[11] "Scott laboratories 2013 fermentation handbook," 2013. [Online]. Available: http://www.scottlab.com/pdf/ScottLabsHandbook2013.pdf
[12] "Rapidase AR 2000 product data sheet." [Online]. Available: http://www.oenobrands.com/files/PDF/Rapidase/Granulated-Enzymes/Rapidase-AR-2000-Product-Data-Sheet-EN.pdf
[13] N. Jolly, C. Varela, and I. Pretorius, "Not your ordinary yeast: non-Saccharomyces yeasts in wine production uncovered," FEMS Yeast Research, vol. 14, pp. 21537, 2014.
[14] L. Daenen, "Use of beta-glucosidase activity for flavour enhancement in specialty beer," 2012, presentation.
What are Glycosides?
A glycoside is a molecule consisting of two parts: a carbohydrate and another molecule called an aglycone; a glucoside has glucose as its carbohydrate [3]. Its this aglycone that can be detected by the nose or tongue as an additional aroma or flavor. Unfortunately, aglycones are flavorless when bound up in a glycoside.
An aglycone is not a single thing, but rather a class of things: an aglycone can be any number of aroma or flavor molecules. They can be found in hops, grapes and other fruits, and spices-almost any plant matter. The amounts and types of glycosides in hops are highly dependent on variety [4], much like alpha - acid levels.
Plants form glycosides because aglycones cannot be easily transported through the plant. By binding the aglycone to a carbohydrate, it becomes water soluble and transportable. It's sort of like boxing up a beer bottle for shipment to competition: UPS can't deliver a bottle with stamps on it, but it can deliver a bottle nicely boxed up. It must then be un-boxed later for tasting.
Free the Aglycones!
(This is perhaps the worst rally cry in history.) Aglycones can be broken from the carbohydrate by being put in an acidic environment (acid hydrolysis) or with the help of an enzyme (enzyme catalysis) [3]. Acid hydrolysis is very slow, and frees negligible amounts of aglycones [5]. Additionally, the vast majority of beers are not nearly acidic enough for this process to occur.
So, we are left with enzymes. In general, enzymes are special proteins capable of vastly increasing the rate of specific chemical reactions. Brewers already use many enzymes, primarily in the malting and mashing process to break grain starches into fermentable sugars. To break aglycones from glucosides, the enzyme - beta glucosidase is required. Beta glucosidase is produced by many plants, bacterias, and fungi, including some yeasts [6].
Glucosidase from Yeast
Certain isolates of both Brettanomyces and Saccharomyces have been shown to produce beta glucosidase (and yes, Brettanomyces is a yeast, not a bacteria). But, not all species can. In fact, it is a rare trait, though much more common in Brettanomyces. Even more confusing, not all isolates of the same species are consistent: some isolates might produce beta glucosidase, while others do not. (All standard brewing yeast is of the species S. cerevisiae; WLP001 and Wyeast 1028 are examples of different isolates of this species.).
I have only been able to find evidence of two S. cerevisiae isolates that produce beta glucosidase: UVAFERM 228 (Danstar Lallemand; wine yeast; aka U228) and CMBS LD40 (Center for Malting and Brewing Science, Leuven, Belgium; beer yeast) [7]. While both were shown to release slightly increased aglycone levels during fermentation, neither are available to home brewers so far as I can tell.
Far more Brettanomyces isolates seem to test positive for beta glucosidase. Many studies focus on the species B. custersii (also known as B. bruxellensis [8]). One study found that 7 of 26 tested B. custersii isolates were beta glucosidase active [9]. One isolate in particular, CMBS LD72 (from a lambic fermentation), has been shown not only to increase free aglycone levels, but to increase them above human-detectable levels in beer [5]. This is the only isolate I have found evidence of producing above-threshold levels of free aglycones. But, given the dearth of studies, there are likely many more isolates capable of doing so.
The bad news is none of these isolates are available to the home brewer either. I've talked to researchers from both Wyeast and White Labs and none of the yeasts they offer have been tested for beta glucosidase activity. But, both expressed great interest in testing them, so this information could become available soon. Until then, Michael Tonsmeire suggests pitching many isolates of Brettanomyces into your beer in hopes of getting one or more to produce the enzyme [10].
Glucosidase from Commercial Enzymes
If you'd rather not trust your beer to providence and you can't wait for home brew yeasts to be tested, another option is to use commercially available enzyme formulations already in use by many wine makers. Three popular formulations are Scott Labs Scottzyme BG and Lallzyme BETA [11], and DSM Oenobrands Rapidase AR 2000 [12]. All three are slightly different formulations of pectinase and -glucosidase enzymes.
One study compared AR 2000 with the B. custersii isolate LD72 for its ability to release aromatic volatiles from sour cherries [5]. Of 13 aglycones tested for, the AR 2000 produced greater amounts of 11 of them compared to LD72. In fact, 7 of the 13 compounds were above human flavor threshold in the AR 2000 beer, compared to just two in the Brettanomyces beer. Other isolates of Brettanomyces might perform better, but it certainly shows the potential of the commercial enzyme formulations for freeing aglycones and unlocking additional aromas.
Getting More Flavor
You can get more flavor and aroma out of your hoppy or fruit beers or wines using the power of enzymes. Glucosides are water soluble, meaning hops or fruits added at any point in the process should add bound aglycones to your beer. Dry hopping might prove especially effective to increase glycoside levels without making the beer overwhelmingly bitter. Most glycosides in grapes are in the skins. Fruit beers using extracts only will probably not benefit from treatment by beta glucosidase.
Using a beta glucosidase positive isolate of Brettanomyces will release extra flavor during the course of fermentation and conditioning. If you try a commercial enzyme, be sure to add it according to the directions [11, 12]. This is because enzymes can be inhibited (stopped from working) by sugar, alcohol, pH, or temperature, and different enzymes are inhibited by different factors [13]. Also, there is some evidence that beta glucosidase might result in the breakdown of esters and/or cause slight color loss [14], but commercial formulations are made to limit these effects.
***
Dennis Waldron is the latest writer to join the HomeBrewTalk team! You can find more from him at the Life Fermented Blog, Dennis' personal blog about home brewing, brew science, DIY brewing equipment, and other fermented goodies. Please help me in welcoming Dennis to the team by posting in the comments, and visiting his site.
***
Further Reading - Notes
[1] Brlosophy, "Sours from the other coast: Homebrew tasting," 2014. [Online]. Available: http://brulosophy.com/2014/08/25/sours-from-the-other-coast-homebrew-tasting/
[2] M. Tonsmeire, "New zealand saison and glycosides," 2014. [Online]. Available: http://www.themadfermentationist.com/2014/08/new-zealand-saison-and-glycosides.html
[3] H. Goldstein, P. Ting, A. Navarro, and D. Ryder, "Water-soluble hop flavor precursors and their role in beer flavor," EBC Congress, pp. 5362, 1999.
[4] H. Kollmannsberger, M. Biendl, and S. Nitz, "Occurence of glycosidically bound flavour compounds in hops, hop products and beer," Monatsschrift fr Brauwissenschaft, pp. 839, 2006.
[5] L. Daenen, F. Sterckx, F. Delvaus, H. Verachtert, and G. Derdelinchkx, "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 Research, vol. 8, pp. 110314, 2008.
[6] N. Ranwedzi, "Optimization of -glucosidase activity in recombinant Saccharomyces cerevisiae strains," 2007, Thesis, Master of Science, Stellenbosch University.
[7] S. Crauwels, D. Saison, F. Sterckx, F. Delvaux, H. Verachtert, and G. Derdelinckx, "Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeast," Applied and Environmental Microbiology, vol. 104, pp. 47888, 2007.
[8] J. Barnett, R. Payne, and D. Yarrow, Yeasts: Characteristics and Identification, 3rd ed. Cambridge University Press, 2007.
[9] S. Crauwels, B. Zhu, J. Steensels, P. Busschaert, G. de Samblanx, K. Marchal, K. Willems, K. Verstrepen, and B. Lievens, "Assessing genetic diversity in Brettanomyces yeast using DNA fingerprinting and whole genome sequencing," Applied and Environmental Microbiology, vol. 80, no. 14, pp. 4398413, 2014.
[10] M. Tonsmeire, American Sour Beers: Innovative Techniques for Mixed Fermentations. Brewer's Publications, 2014.
[11] "Scott laboratories 2013 fermentation handbook," 2013. [Online]. Available: http://www.scottlab.com/pdf/ScottLabsHandbook2013.pdf
[12] "Rapidase AR 2000 product data sheet." [Online]. Available: http://www.oenobrands.com/files/PDF/Rapidase/Granulated-Enzymes/Rapidase-AR-2000-Product-Data-Sheet-EN.pdf
[13] N. Jolly, C. Varela, and I. Pretorius, "Not your ordinary yeast: non-Saccharomyces yeasts in wine production uncovered," FEMS Yeast Research, vol. 14, pp. 21537, 2014.
[14] L. Daenen, "Use of beta-glucosidase activity for flavour enhancement in specialty beer," 2012, presentation.