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 potentially flavor active compound. These bonds can be broken with exposure to acid, as well as specific enzymes which can be added synthetically or produced naturally by some microorganisms, including some strains of ''Brettanomyces'' <ref>[https://en.wikipedia.org/wiki/Glycoside "Glycoside." Wikipedia. Retrieved 06/27/2016.]</ref>. When the glycosidic bond is broken by an enzyme produced by a microorganism, it can be classified under the generic term as a [https://en.wikipedia.org/wiki/Biotransformation "biotransformation"] <ref>[http://www.sawislibrary.co.za/dbtextimages/17163.pdf Enzymes in Winemaking: Harnessing Natural Catalysts for Efficient Biotransformations - A Review. P. van Rensburg and I.S. Pretorius. 2000.]</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"></ref>. It is speculated that flavor compounds from hops can also be released from glycosides <ref name="Daenen1"></ref>.
==Glycosides and Beta-Glucosidase Activity==
Aglycones have been identified in many fruits and herbs such as grapes, apricots, peaches, yellow plums, quince, sour cherry, passion fruit, kiwi, papaya, pineapple, mango, lulo, raspberry, strawberry, and tea <ref name="Maicas">[http://www.ncbi.nlm.nih.gov/pubmed/15635463 "Hydrolysis of terpenyl glycosides in grape juice and other fruit juices: a review." Sergi Maicas, José Juan Mateo. May 2005.]</ref><ref name="Winterhalter"></ref>. They have been found in different parts of plants, including the green leafy parts, fruit, roots, rhizomes, petals, and seeds. Aglycones in plants are highly complex structures and very diverse, and their percentages can vary from crop to crop. In plants, these include alcohol type aglycones such as terpenols, terpenes, linalool oxides, as well as other flavor precursors including various alcohols, norisoprenoids, phenolic acids and probably volatile phenols such as vanillin <ref name="Maicas"></ref>. In fruits, there are mostly just 4 types of flavonol type aglycones: [https://en.wikipedia.org/wiki/Quercetin quercetin] (found in nearly all fruits), [https://en.wikipedia.org/wiki/Kaempferol kaempherol] (found in 80% of fruit), and less commonly [https://en.wikipedia.org/wiki/Quercetin quercetin] and [https://en.wikipedia.org/wiki/Isorhamnetin isorhamnetin] <ref>[https://books.google.com/books?id=vHqke7F4lWYC&pg=PA59&lpg=PA59&dq=aglycones+in+fruit&source=bl&ots=7Gb10SPZk7&sig=6gaZlwpVaHuteoiVP68zvt6HcpE&hl=en&sa=X&ved=0ahUKEwjk0qW9wp3NAhUCKZQKHfUzDrQQ6AEIKTAC#v=onepage&q=aglycones%20in%20fruit&f=false Fruit Phenolics. Jean-Jacques Macheix, Annie Fleuriet. CRC Press, Mar 20, 1990. Pgs 57-61.]</ref> (see [http://nutrition.ucdavis.edu/content/infosheets/fact-pro-flavonol.pdf this UC Davis PDF] for amounts in different fruit and potential health benefits as antioxidants). In many cases of fruit, the amount of aromatic aglycones that are bound up in glycosides outnumber the amount that are free in a ratio of 2:1 to 8:1 <ref name="Maicas"></ref>. Aglycones that are bound up in glycosides tend to be more water soluble and less reactive once unbound than the naturally free version. By providing enzymes that break the glycosidic bond, discarded parts of plants (peels, stems, skins, etc.) have been used to produce natural flavorings from the remaining and abundant glycosides <ref name="Winterhalter">[http://link.springer.com/chapter/10.1007%2FBFb0102063 "Glycoconjugated aroma compounds: Occurrence, role and biotechnological transformation." Peter Winterhalter, George K. Skouroumounis. 1997.]</ref>.
===Acidic Hydrolysis===
Aglycones can be released from glycosides via a low pH. Generally, this occurs at lower pH's and Daenen et al. (2007) attributed most of the breakdown of glycosides to beta-glucosidase enzymes <ref name="Daenen2" />, but different types of glycosides are more readily broken down at different pH's. For example, ocimenols (lemon, lime <ref>[http://www.thegoodscentscompany.com/data/rw1020581.html "Ocimenol". The Good Scents Company. Retrieved 06/26/2017.]</ref>) are formed from glycoside breakdown at a pH of 1, but terpenes such as linalool, nerol, and geraniol (commonly found in hops and other plant material) are formed at a pH of 3, and alpha-terpineol was formed at both pH values. The acidic breakdown of these glycosides were found to contribute to the flavor of wine during aging <ref name="Maicas"></ref>. Daenen et al. (2007) also found that acidic hydrolysis was responsible for the formation of alpha-ionol, beta-damascenone, linalool and alpha-terpineol and to a lesser extent benzyl alcohol <ref name="Daenen2" />.
In beer, it was found that the lower the pH of the beer the higher the amount of the ketone damascenone (floral, rose <ref>[https://en.wikipedia.org/wiki/Damascenone "Damascenone". Wikipedia. Retrieved 06/26/2017.]</ref>) and dimethyl trisulfide (sulfury cooked onion <ref>[http://www.thegoodscentscompany.com/data/rw1008101.html "Dimethyl trisulfide". The Good Scents Company. Retrieved 06/26/2017.]</ref>). In one study, at a pH of 3, beta-damascenone was reported to be ~27 ppb and dimethyl trisulfide was reported to be 650 ppt. At a pH of 4.2, they were 17 ppb and 300 ppt respectively. At a ph of 5, beta-damascenone was around 12 ppb and dimethyl trisulfide 50 ppt. This was believed to be due to the acidic hydrolysis of glycosides, and it was speculated that other glycosides could have a flavor impact on beers that contain fruit or herbs due to acidic hydrolysis of glycosides <ref>How Low pH Can Intensify ?-Damascenone and Dimethyl Trisulfide Production through Beer Aging. LAURENCE GIJS, FABIENNE CHEVANCE, VESNA JERKOVIC, AND SONIA COLLIN. 2002. DOI: 10.1021/jf020563p.</ref>.
* See also: [[Hops#Glycosides|Glycosides In Hops]].
===Beta-Glucosidase===
Aglycones can also be released chemically from glycosides by either exposure to acid (generally pH of 3 or lower, and different pH's giving different results on which glycosides are broken down; this breakdown of glycosides under low pH has been linked to the slow flavor development of aging wine <ref name="Maicas"></ref>), or by enzymes called beta-glucosidases. Enzymatic breakdown of glycosides has been described as producing a more "natural" flavor in wines versus acidic breakdown. Some fruits have been observed (mostly wine grapes) to have limited beta-glucosidase activity within themselves, however it has been observed as being unstable and having low activity at the low pH of wine and sour beer <ref name="Maicas"></ref>.
Beta-glycosidase enzymes can be added artificially, however there has been much interest in the natural capability of microorganisms to produce beta-glucosidases, particularly 1,4-β-glucosidase <ref name="Winterhalter"></ref>. Microorganisms that can break down glycosides by using beta-glucosidases can then access the resulting sugars for fermentation <ref name="Steensels">[http://www.sciencedirect.com/science/article/pii/S0168160515001865 Brettanomyces yeasts — From spoilage organisms to valuable contributors to industrial fermentations. Jan Steensels, Luk Daenen, Philippe Malcorps, Guy Derdelinckx, Hubert Verachtert, Kevin J. Verstrepen. International Journal of Food Microbiology Volume 206, 3 August 2015, Pages 24–38.]</ref>. There are two major categories of glucosidase activity: endogenous and exogenous. Endogenous enzymatic activity takes place inside of the cell, and exogenous enzymatic activity takes place outside of the cell. Bacteria and fungi that show endogenous glucosidase activity have been shown not to be effective in alcoholic fermentation due to not tolerating low pH (optimum pH of 5), glucose, and/or ethanol. Generally, the flavorless glycosides remain unaffected by yeast fermentation, leaving them unused as a potential source for flavor and aroma <ref name="Winterhalter"></ref>.
Different types of beta-glucosidase enzymes have different optimal pH and temperatures. For example, beta-glucosidase produced from ''A. niger'' is optimal at a pH of 4.5 and a temperature of 58°C (136°F), whereas the enzyme for ''Brettanomyces anomalus'' is optimal at a pH of 5.75 and a temperature of 37°C (98°F) (it was active to some extent between 15°-55°C). The beta-glucosidase enzyme ceases effectiveness below a pH of 4.5 for one strain of ''B anomalus'' studied <ref name="Vervoort"></ref>.
See also:* [http://www.cazy.org/Glycoside-Hydrolases.html Database of glycoside hydrolases.] ===Activity of ''Brettanomyces '' and ''Saccharomyces''=== [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2007.03566.x/full Daenen et al. (2007)] screened the beta-glucosidase activity of several strains of ''Saccharomyces cerevisiae'', ''Saccharomyces pastorianus'', and several ''Brettanomyces'' species. None of the lager brewing strains showed beta-glucosidase activity. Out of 32 strains of ''S. cerevisiae'', only one strain (a wine strain called "U228") showed beta-glucosidase activity, however its activity was repressed in the presence of glucose. This indicates that most ''S. cerevisiae'' strains do not have the capability of producing beta-glucosidase, but it is possible that some very few strains can <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>. Additionally, beta-glucosidase activity for ''S. cerevisiae'' can be inhibited by the pH levels found in wine and sour beer (optimal at pH 5) <ref name="Mansfield"></ref>. All strains of ''S. cerevisiae'' did release another enzyme called beta-glucanase, which led to varying degrees of breaking down some smaller glycosides found in hops (hop extract was tested, not whole hops) containing the aglycones methyl salicylate, 1-octen-3-ol, and cis-3-hexen-1-ol, but not linalool. None of the ''B. bruxellensis'' strains showed this activity, but the researchers only tested strain of ''B. custersianus'' and both of the ''B. anomala'' strains tested did show cell-associated (intracellular) beta-glucosidase activity. In particular, the ''B. custersianus'' strain was tested against glycosides from hops, in which case high amounts of the aglycones linalool (citrus, orange, lemon, floral <ref>[http://www.thegoodscentscompany.com/data/rw1007872.html "Linalool." The Good Scents Company. Retrieved 05/12/2016.]</ref>), methyl salicylate (minty, wintergreen <ref>[http://www.rsc.org/chemistryworld/2015/09/methyl-salicylate-oil-wintergreen-podcast "Methyl salicylate." Chemistry World. Retrieved 05/12/2016.]</ref>), 1-octen-3-ol (mushroom, earthy <ref>[http://www.thegoodscentscompany.com/data/rw1024051.html "1-octen-3-ol." The Good Scents Company. Retrieved 05/12/2016.]</ref>) and cis-3-hexen-1-ol (grassy, melon rind <ref>[http://www.thegoodscentscompany.com/data/rw1005932.html "(Z)-3-hexen-1-ol." The Good Scents Company. Retrieved 05/12/2016.]</ref>) were released from hop extracts <ref name="Daenen1"></ref>. The beta-glucosidase activity was elevated when co-fermenting ''B. custersianus'' with ''S. cerevisiae''. The authors also found dihydroedulan 1 and 2 (elderberry aroma) and theaspirane A and B (woody and campfire aromas), which are classified as norisoprenoids, were released from dry hopping <ref>[http://www.asbcnet.org/events/archives/Documents/2008WBCprogbook.pdf World Brewing Congress, 2008. Pg 80. Retrieved 05/13/2016.]</ref>. ''B. custersianus'' has been isolated from the later stages of lambic fermentation, and it is thought that its ability to produce beta-glucosidase, which gives it the ability to ferment cellobiose and cellotriose, is a possible adaptation from living in oak barrels <ref name="Daenen1"></ref>. Recent studies on hops have linked an increase in fruity thiols from hops (3-mercaptohexan-1-ol and 4-mercapto-4-methylpentan-2-one) being produced during fermentation from the production of beta-lyase enzyme, and this could also explain anecdotal reports of increased fruity aromas from exposing hops to fermentation (see [http://scottjanish.com/genetically-modified-gm-yeast-strains-unlocking-bound-hop-thiols-and-engineering-targeted-fermentation-characteristics/ this article by Scott Janish] on beta-lyase enzymes in GM and non-GM yeast strains). <ref>Private correspondence with Richard Preiss by Dan Pixley. 05/16/2016.</ref><ref>[https://beerandbrewing.com/VuhJRCUAAHMUNfil/article/hops-oils--aroma-uncharted-waters "Hops Oils & Aroma: Uncharted Waters," by Stan Hieronymus. Beer & Brewing. 03/16/2016. Retrieved 05/16/2016.]</ref>. [https://www.academia.edu/23874347/Properties_of_endogenous_%CE%B2_glucosidase_of_a_Saccharomyces_cerevisiae_strain_isolated_from_Sicilian_musts_and_wines Palmeri et al. (2002)] reported one wine strain out of 80 strains of ''S. cerevisiae'' found in Sicilian must that had high beta-glucosidase activity and is active during wine fermentation conditions <ref>[https://www.academia.edu/23874347/Properties_of_endogenous_%CE%B2_glucosidase_of_a_Saccharomyces_cerevisiae_strain_isolated_from_Sicilian_musts_and_wines Spagna, G., Barbagallo, R. N., Palmeri, R., Restuccia, C., & Giudici, P. (2002). Properties of endogenous β-glucosidase of a Saccharomycescerevisiae strain isolated from Sicilian musts and wines. Enzyme and Microbial Technology, 31(7), 1030–1035. https://doi.org/10.1016/S0141-0229(02)00233-8]</ref>. Another study by [http://onlinelibrary.wiley.com/doi/10.1002/jib.418/abstract Sharp et al.] found that both ale and lager yeasts exhibit a wide range of beta-glucosidase activity, but the results should be repeated using a different substrate because the substrate that was used could have been broken down by beta-glucanase enzyme instead of beta-glucosidase <ref name="Sharp_2017">[http://onlinelibrary.wiley.com/doi/10.1002/jib.418/abstract The effect of hopping regime, cultivar and β-glucosidase activity on monoterpene alcohol concentrations in wort and beer. Daniel C. Sharp, Jan Steensels, Thomas H. Shellhammer. 2017.]</ref>Additionally, Lallemand claims that three brewing strains that they offer produce beta-glucosidase enzymes that can promote hop biotransformation, but the details of this activity have not been published to our knowledge (BRY-97, New England, and Belle Saison) <ref>[http://masterbrewerspodcast.com/119-biotransformation Eric Abbot. Master Brewers Association podcast, episode 119. 02/04/2019.]</ref> (~8:50 min in). The same strain of ''B. custersianus'' was screened for beta-glucosidase activity and aglycone byproducts during the refermentation of sour cherries in beer (a very small amount of the byproducts were manufactured by the yeast ''de novo'', particularly linalool, alpha-terpineol, alpha-ionol, and a precursor that leads to beta-damascenone under low pH conditions). Different portions of the cherries were tested: whole cherries with stones (pits), cherry pulp without stones, cherry juice without stones or other solids from the fruit, and the stones alone. Benzaldehyde (almond, cherry stone flavor) was produced during fermentation in all cases, and reduced to benzyl alcohol (almond flavor) and benzyl acetate (fruity, jasmine flavor) by the end of fermentation. There were higher levels of these benzyl based compounds in the whole cherries and cherry stone alone samples, indicating that cherry stones make a big impact on the almond flavors found in cherry sour beers. Methyl salicylate, linalool, alpha-terpineol (pine), geraniol (rose, lime, floral) and alpha-ionol (floral, violet), eugenol (spicy, clove, medicinal) and isoeugenol (fine delicate clove) levels increased in all forms of cherries added except for stones alone, indicating that these aglycones are more present in the flesh and juice of the cherries <ref name="Daenen2"></ref>. Another study that performed whole genome sequencing on a strain of ''B. naardenensis'' found that it had two genes that could potentially allow this species to produce beta-glucosidase, but this was not confirmed in the study <ref>[https://www.mdpi.com/2076-2607/7/11/489 Assembly and Analysis of the Genome Sequence of the Yeast Brettanomyces naardenensis CBS 7540. Ievgeniia A. Tiukova, Huifeng Jiang, Jacques Dainat, Marc P. Hoeppner, Henrik Lantz, Jure Piskur, Mats Sandgren, Jens Nielsen, Zhenglong Gu, and Volkmar Passoth. 2019. DOI: https://doi.org/10.3390/microorganisms7110489.]</ref>. Many strains of ''B. bruxellensis'' have also been found to have varying degrees of intracellular (produced inside the cell only) or parietal (attached to the cell wall) beta-glucosidase activity. At least one mutant strain of ''B. bruxellensis'' found in wine (''D. bruxellensis'' BCRC920084 from the Bioresource Collection and Research Center, Hsin-Chu, Taiwan) has been reported to have high extracellular (excreted out of the cell) production of beta-glucosidase that was very efficient at breaking down a glycoside called "piceid" that released resveratrol, which has been marketed as a dietary supplement <ref>[https://www.ncbi.nlm.nih.gov/pubmed/29389552?dopt=Abstract Characterization of an extracellular β-glucosidase from Dekkera bruxellensis for resveratrol production. Kuo HP, Wang R, Huang CY, Lai JT, Lo YC, Huang ST. 2018.]</ref><ref>[https://en.wikipedia.org/wiki/Resveratrol Wikipedia. "Resveratrol" article. Retrieved 02/09/2018.]</ref>. ''Brettanomyces'' has more strains that can produce beta-glucosidase than other genera of yeast, and the strains generally also have a higher rate of beta-glucosidase activity than other genera of yeast <ref>[http://link.springer.com/article/10.1038/sj.jim.2900720 Quantification of glycosidase activities in selected yeasts and lactic acid bacteria. H McMahon, B W Zoecklein, K Fugelsang, Y Jasinski. 1999.]</ref><ref name="Mansfield"></ref>. Strains with higher beta-glucosidase activity have been isolated from lambic, suggesting that these strains may have an adapted ability to utilize sugar from glycosides <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>. Some ''Brettanomyces'' strains may only be capable of beta-glucosidase activity, and not the other enzymes which are needed to break down disaccharide type glycosides. Additionally, cell death and autolysis can result in an increase in beta-glucosidase activity in solution due to the cell contents being released into solution <ref name="Mansfield"></ref>. Strains that can metabolize cellobiose tend to also have higher beta-glucosidase activity because they possess an extra gene for beta-glucosidase enzyme production <ref name="Crauwels1">[http://link.springer.com/article/10.1007/s00253-015-6769-9 Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains. S. Crauwels, A. Van Assche, R. de Jonge, A. R. Borneman, C. Verreth, P. Troels, G. De Samblanx, K. Marchal, Y. Van de Peer, K. A. Willems, K. J. Verstrepen, C. D. Curtin, B. Lievens. 2015]</ref>. Sensory analysis of beers with cherries or hops have shown that there is a significantly detectable difference between cherry beers that have been exposed to beta-glucosidase from one strain of ''B. anomalus'' versus not exposed to the enzyme, but no significant difference was found in beers hopped with pellets. The cherry beers exposed to the enzyme contained more and above odor threshold eugenol (clove, honey aroma), benzyl alcohol (sweet, flower), benzaldehyde (almond, cherry) than cherry beers that were not exposed to the enzyme. The cherry beers exposed to the enzyme were not only identified in a blind tasting but were also preferred to the cherry beers without exposure to the enzyme, indicating that beta-glucosidase activity in cherry beers provides a significant flavor difference. Other types of beta-glucosidase enzymes released different levels of different flavor compounds, indicating that the source (bacteria or yeast) of the enzyme make a significant difference in the flavors that are produced <ref name="Vervoort"></ref>. The effects of beta-glucosidase on hops may be limited even in ideal conditions using pure beta-glucosidase that is highly efficient in beer where beta-glucosidase activity by yeast is limited. Sharp et al. (2017) determined that hops contain such a small amount of glycosides that their release doesn't contribute much to hop flavor and aroma. While previous studies focused on hop extracts, they studied glycosides in whole leaf hops for the first time and found non-significant levels of hop-derived monoterpenes from glycosides even when using pure beta-glucosidase extracted from almonds. For example, linalool was increased by 16.5 μg/L when using the highest hopping rate, but this amount has little impact on the overall aroma and flavor of the beer. Terpineol, citronellol, nerol, and geraniol were also not increased in significant amounts, however 1-octanol (waxy, green, citrus, orange, aldehydic, fruity <ref>[http://www.thegoodscentscompany.com/data/rw1021071.html Octanol. The Good Scents Company. Retrieved 03/31/2017.]</ref>) was increased significantly <ref name="Sharp_2017" />.
One study screened the beta-glucosidase In 2020, whole genome sequencing and glycosidic activity of several were tested for 84 strains of ''Saccharomyces cerevisiae'', ''Saccharomyces pastorianus'', and ''Brettanomyces'' spp <ref name="Daenen1">by [httphttps://onlinelibrarywww.wileyfrontiersin.comorg/doiarticles/10.11113389/jfmicb.1365-2672.2007.035662020.x00637/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.Colomer et al]</ref>. None of They found that the lager brewing strains showed beta-glucosidase activity. Out of 32 strains capability of ''S. cerevisiaeBrettanomyces'', only one strain (a wine strain called "U228") showed beta-glucosidase activity, however its activity was repressed in the presence of glucose. This indicates that most to break down glycosides is determined by possessing two genes: ''S. cerevisiaeBbBGL1'' strains do not have the capability of producing beta-glucosidase, but it is possible that some very few strains can <ref name="Daenen1"></ref>. However, beta-glucosidase activity for and ''S. cerevisiaeBbBGL2'' is inhibited by pH levels of wine and sour beer (optimal at pH 5) <ref name="Mansfield"></ref>. All strains of The genes called ''S. cerevisiaeBbBGL2'' did release another enzyme called beta-glucanase, which led to varying degrees of breaking have a major role in whether glycosides are broken down some smaller glycosides found in hops (hop extract was tested, not whole hops) containing while the aglycones methyl salicylate, 1-octen-3-ol, and cis-3-hexen-1-ol, but not linalool (it's worth noting that other research using whole hops has shown no significant hop derived aglycones when using beta-glucosidase active role of ''SaccharomycesBbBGL1'' strains; publication yet to be released <ref>Private correspondence with Daniel Sharp from Oregon State University and Dan Pixleyis minimal. 05/16/2016.</ref>). None About half of the ''B. bruxellensis'' strains showed this had some level of glycosidic activity, but although the only tested strain majority was low with a couple of ''B. custersianus'' and both strains that had high levels of the ''B. anomala'' strains tested did show cell-associated (intracellular) beta-glucosidase glycosidic activity. In particular, thus these genes alone do not account for the wide variation in glycosidic activity levels in ''B. custersianusBrettanomyces'' strain was tested against glycosides from hops, in which case high amounts of the aglycones linalool (citrus, orange, lemon, floral <ref>see [httphttps://www.thegoodscentscompanyfrontiersin.comorg/files/Articles/data495404/rw1007872.html "Linalool." The Good Scents Company. Retrieved 05fmicb-11-00637-HTML-r1/12image_m/2016fmicb-11-00637-g007.jpg this figure]</ref>from the study which maps beta-glucosidase activity for the 84 strains that were sequenced), methyl salicylate (minty, wintergreen <ref>[httphttps://www.rscfrontiersin.org/chemistryworldarticles/201510.3389/09/methyl-salicylate-oil-wintergreen-podcast "Methyl salicylatefmicb." Chemistry World2020. Retrieved 0500637/12/2016full Assessing Population Diversity of Brettanomyces Yeast Species and Identification of Strains for Brewing Applications.]</ref>) Marc Serra Colomer, 1-octen-3-ol (mushroomAnna Chailyan, earthy <ref>[http://wwwRoss T.thegoodscentscompany.com/data/rw1024051Fennessy, Kim Friis Olsson, Lea Johnsen, Natalia Solodovnikova and Jochen Forster.html "1-octen-3-ol." The Good Scents Company2020. Retrieved 05DOI: https:/12/2016doi.]<org/ref>) and cis-3-hexen-1-ol (grassy, melon rind <ref>[http://www10.thegoodscentscompany.com3389/data/rw1005932fmicb.html "(Z)-3-hexen-1-ol2020." The Good Scents Company. Retrieved 05/12/201600637.]</ref>) were released from hop extracts <ref name="Daenen1"></ref>. The beta-glucosidase activity was elevated when co-fermenting glucidase levels were also measured for 5 strains (4 strains of ''B. custersianusbruxellensis'' with and 1 strain of ''SB. cerevisiaeanomalus''. The authors also found dihydroedulan 1 and 2 (elderberry aroma) and theaspirane A and B that have the genetic make up to produce this enzyme; most of the enzyme was only produced within the cell (woody and campfire aromasintracellular)for all of the strains tested, which are classified as norisoprenoids, and only small amounts were released from dry hopping <ref>[http://wwwproduced outside of the cells except for one strain of ''B.asbcnet.org/events/archives/Documents/2008WBCprogbook.pdf World Brewing Congress, 2008anomalus''. Pg 80. Retrieved 05/13/2016.]</ref>The enzyme therefore wouldn't act on glycosides outside of the cell unless the cell ruptured. There was also a small amount of beta-glucosidase activity within the cell wall of one of the strains of ''B. custersianusanomalus'' has been isolated from the later stages of lambic fermentation, . They also fermented a dry hopped beer with these strains and it is thought found that its ability to produce the strains with the lowest beta-glucosidase, which gives it activity had the ability lowest conversion of geraniol to ferment cellobiose and cellotrioseβ‐citronellol, is a possible adaptation from living in oak barrels <ref name="Daenen1"></ref>. Recent studies on hops have linked an increase in fruity thiols from hops (3-mercaptohexan-1-ol and 4therefore there was no correlation between beta-mercapto-4-methylpentan-2-one) being produced during fermentation, glucosidase activity and this could also explain anecdotal reports hop biotransformation (although there was evidence of another unidentified type of increased fruity aromas from exposing biotransformation occurring with the hops to fermentation (it is unknown what exactly causes the increase in thiols during fermentationsee [[Brettanomyces#Hop_Biotransformation|''Brettanomyces'' hop biotransformation]]) <ref>Private correspondence with Richard Preiss by Dan Pixley. 05/16/2016.</ref><ref>[https://beerandbrewingonlinelibrary.wiley.com/VuhJRCUAAHMUNfildoi/articlefull/hops-oils--aroma-uncharted-waters "Hops Oils & Aroma: Uncharted Waters10.1002/jib.610 Biotransformation of hop derived compounds by Brettanomyces yeast strains. Marc Serra Colomer, Birgitte Funch, Natalia Solodovnikova, Timothy John Hobley," by Stan HieronymusJochen Förster. Beer & Brewing2020. 03DOI: https:/16/2016doi. Retrieved 05org/1610.1002/2016jib.610.]</ref>.
The same Wang et al. (2018) reported another type of glycosidic activity in one strain of ''B. custersianusbruxellensis'' was screened for beta-glucosidase activity and aglycone byproducts during which is the refermentation of sour cherries in beer (a very small amount conversion of the byproducts glycoside "mogroside V" into an artificial sweetener called siamenoside I. Other yeast and bacteria species were manufactured by the yeast ''de novo'', particularly linalool, alpha-terpineol, alpha-ionol, tested and a precursor that leads to beta-damascenone under low pH conditions)did not find this particular glycosidic activity. Different portions of the cherries were tested[https: whole cherries with stones (pits)//en.wikipedia.org/wiki/Mogroside Mogroside V] is found naturally in some fruit, cherry pulp without stonesspecifically, cherry juice without stones or other solids from the an Asian fruit, and the stones alonecalled [https://en.wikipedia. Benzaldehyde org/wiki/Siraitia_grosvenorii Lo Han Kuo (almond, cherry stone flavor''Siraitia grosvenorii'') was produced during fermentation in all cases, and reduced to benzyl alcohol (almond flavor) and benzyl acetate (fruity, jasmine flavor) by the end of fermentation]. The artificial sweetener siamenoside is 563 times sweeter tasting than 5% sucrose. There were higher levels The specific enzyme responsible for this conversion that this strain of these benzyl based compounds in the whole cherries and cherry stone alone samples''B. bruxellensis'' produced is called ''Db''Exg1 <ref>[https://www.sciencedirect.com/science/article/pii/S0308814618317473 Dekkera bruxellensis, indicating a beer yeast that cherry stones make a big impact on specifically bioconverts mogroside extracts into the almond flavors found in cherry sour beersintense natural sweetener siamenoside I. Methyl salicylateReuben Wang, linaloolYi-Chieh Chen, alphaYun-terpineol (pine)Ju Lai, geraniol (roseTing-Jang Lu, limeShyue-Tsong Huang, floral) and alphaYi-ionol (floral, violet), eugenol (spicy, clove, medicinal) and isoeugenol (fine delicate clove) levels increased in all forms of cherries added except for stones alone, indicating that these aglycones are more present in the flesh and juice of the cherries <ref name="Daenen2">Chen Lo. 2018. DOI: https://doi.org/10.1016/j.foodchem.2018.09.163.]</ref>.
Many strains of ''B. bruxellensis'' have See also been found to have varying degrees of intracellular or parietal (attached to the cell wall) beta-glucosidase activity. ''Brettanomyces'' has more strains that can produce beta-glucosidase than other genera of yeast, and the strains generally also have a higher rate of beta-glucosidase activity than other genera of yeast <ref>:* [[http://link.springer.com/article/10.1038/sj.jim.2900720 Quantification of glycosidase activities Hops#Glycosides|Glycosides in selected yeasts and lactic acid bacteria. H McMahon, B W Zoecklein, K Fugelsang, Y Jasinski. 1999.Hops]]</ref><ref name="Mansfield"></ref>. Strains with higher beta-glucosidase activity have been isolated from lambic, suggesting that these strains may have an adapted ability to utilize sugar from glycosides <ref name="Vervoort">* [http://onlinelibrary.wileyscottjanish.com/wol1/doi/10.1111/jam.13200/abstract Characterization of the recombinant Brettanomyces anomalus βgenetically-modified-gm-glucosidase and its potential for bioflavoring. Yannick Vervoort, Beatriz Herrerayeast-Malaver, Stijn Mertens, Victor Guadalupe Medina, Jorge Duitama, Lotte Michiels, Guy Derdelinckx, Karin Voordeckers, and Kevin J. Verstrepen. 2016.]</ref>. Some ''Brettanomyces'' strains may only be capable of beta-glucosidase activity, and not the other enzymes which are needed to break down disaccharide type glycosides. Additionally, cell death and autolysis can result in an increase in betaunlocking-glucosidase activity in solution due to the cell contents being released into solution <ref name="Mansfield"></ref>. Strains that can metabolize cellobiose tend to also have higher betabound-glucosidase activity because they possess an extra gene for betahop-glucosidase enzyme production <ref name="Crauwels1">[http://link.springer.com/article/10.1007/s00253thiols-015and-6769engineering-9 Comparative phenomics and targeted use of genomics reveals variation in carbon -fermentation-characteristics/ "Genetically Modified (GM) Yeast Strains: Unlocking Bound Hop Thiols and nitrogen assimilation among different Brettanomyces bruxellensis strains. S. CrauwelsEngineering Targeted Fermentation Characteristics, A. Van Assche, R. de Jonge, A. R. Borneman, C. Verreth, P. Troels, G. De Samblanx, K. Marchal, Y. Van de Peer, K. A. Willems, K. J" by Scott Janish. Verstrepen, C. D. Curtin, B. Lievens. 2015]</ref>.
Sensory analysis ===Activity of beers with cherries or hops have shown that there is a significantly detectable difference between cherry beers that have been exposed to beta-glucosidase from one Other Yeasts===A strain of ''B. anomalusCandida glabrata'' versus not exposed was selected in a study for its high beta-glucosidase activity, its tolerance to the enzymeethanol, and its ability to utilize maltose, and was shown to produce novel flavor characteristics in beer fermentation, but no including a significant difference was found increase in beers hopped geraniol <ref>[https://www.sciencedirect.com/science/article/abs/pii/S0308814622026887#f0020 Application of non-Saccharomyces yeasts with pelletshigh β-glucosidase activity to enhance terpene-related floral flavor in craft beer. The cherry beers exposed to the enzyme contained more and above odor threshold eugenol (cloveXiaoyu Han, Qiuxing Qin, Chenyu Li, Xiaoxuan Zhao, Fangxu Song, Mengjiao An, honey aroma)Ying Chen, benzyl alcohol (sweetXiuqin Wang, flower)Weidong Huang, benzaldehyde (almondJicheng Zhan, cherry) than cherry beers Yilin You. 2022.]</ref>. Strains of ''Meyerozyma guilliermondii'' and ''Hanseniaspora uvarum'' that were not exposed isolated from a spontaneous wine fermentation have also been found to the enzymehave positive results when co-fermented with ''S. cerevisiae'' in wine. The cherry beers exposed Specifically, compared to the enzyme were not only identified in a blind tastingcontrol wines, but there were also preferred to higher levels of the cherry beers without exposure to the enzymeterpenes: isopulegol, citronellol, geranylacetone, geraniol, indicating that betatrans-glucosidase activity in cherry beers provides a significant flavor differencenerolidol, and nerol. Other types of This was associated with the high beta-glucosidase enzymes released different levels activity of different flavor compounds, indicating that the source (bacteria or these yeast) of the enzyme make a significant difference in the flavors that are produced strains <ref name="Vervoort">[https://www.frontiersin.org/articles/10.3389/fmicb.2022.845837/full Indigenous Non-Saccharomyces Yeasts With β-Glucosidase Activity in Sequential Fermentation With Saccharomyces cerevisiae: A Strategy to Improve the Volatile Composition and Sensory Characteristics of Wines. Gao Pingping, Peng Shuai, Sam Faisal Eudes, Zhu Yatong, Liang Lihong, Li Min, Wang Jing. 2022.]</ref>. ===Activity of Lactic Acid Bacteria===(To do) http://www.icfsnmalaysia2017.org/ICFSN2017%20proceedings%20-%20pp%2054-60.pdf https://search.proquest.com/docview/752091646?pq-origsite=gscholar https://www.researchgate.net/profile/Prafulla_Mahajan/publication/225362291_Production_of_Cell_Membrane-Bound_a-_and_b-Glucosidase_by_Lactobacillus_acidophilus/links/55fd56cd08aeba1d9f56bdbd.pdf http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2009.04461.x/full https://ifst.onlinelibrary.wiley.com/doi/abs/10.1111/jfpp.16368 https://www.mdpi.com/2076-3921/11/2/305
===Cyanogenic Glycosides===
All plants contain at least tiny amounts of [https://en.wikipedia.org/wiki/Hydrogen_cyanide hydrogen cyanide] ('''HCN'''), however, some plants also release high amounts of HCN from a class of glycosides called "cyanogenic glycosides", also called "cyanoglycosides". [https://en.wikipedia.org/wiki/Amygdalin Amygdalin] and [https://en.wikipedia.org/wiki/Linamarin linamarin] are common examples of cyanogenic glycosides <ref name="Gleadow_2014"></ref>. Amygdalin is sometimes marketed as a cure for cancer by the health food industry, but this claim is not supported by clinical data, and digestion of too much amygdalin can be dangerous <ref>[https://www.quackwatch.org/01QuackeryRelatedTopics/Cancer/laetrile.html Wilson, Benjamin. "The Rise and Fall of Laetrile". Quackwatch.org website. 01/07/2017. retrieved 03/30/2017.]</ref><ref>[http://www.nejm.org/doi/pdf/10.1056/NEJM198201283060403 A Clinical Trial of Amygdalin (Laetrile) in the Treatment of Human Cancer. Charles G. Moertel, M.D., Thomas R. Fleming, Ph.D., Joseph Rubin, M.D., Larry K. Kvols, M.D., Gregory Sarna, M.D., Robert Koch, M.D., Violante E. Currie, M.D., Charles W. Young, M.D., Stephen E. Jones, M.D., and J. Paul Davignon, Ph.D. 1982. DOI: 10.1056/NEJM198201283060403.]</ref><ref>[https://www.infona.pl/resource/bwmeta1.element.springer-2c7e4ea1-3683-3bbd-9394-a98a551e7c63 Laetrile for cancer: a systematic review of the clinical evidence. Stefania Milazzo, Stephane Lejeune, Edzard Ernst. 2007. DOI: 10.1007/s00520-006-0168-9.]</ref><ref>[http://www.sciencedirect.com/science/article/pii/S0944711316000362 Amygdalin, quackery or cure? Roman A. Blahetaa, Karen Nelsonb, Axel Haferkampa, Eva Juengela. 2016.]</ref>. HCN is released from cyanogenic glycosides just like other types of glycosides: beta-glucosidase enzyme or exposure to low pH breaks the bond between a glucose molecule and an unstable compound called "cyanohydrin" (or "alpha-hydroxynnitrile"), which then disassociates into a ketone or benzaldehyde and an HCN molecule. In progresscyanogenic glycosides, this reaction is called "cyanogenesis". Cyanogenesis is stimulated by maceration, and by bacteria in the human gut <ref name="Speijers">[http://www.inchem.org/documents/jecfa/jecmono/v30je18.htm "Cyanogenic Glycosides", First Draft. Dr G. Speijers. National Institute of Public Health and Environmental Protection Laboratory for Toxicology, Bilthoven, The Netherlands. Retrieved 08/25/2016.]</ref>. Although the optimum pH of cyanogenesis (at least for amygdalin)is 5.0 - 5.8, cyongenesis can occur at a wide range of pH values, and can occur in the presence of acid <ref>[http://www.sciencedirect.com/science/article/pii/S0308814601003132 Total cyanide determination of plants and foods using the picrate and acid hydrolysis methods. M Rezaul Haque, J Howard Bradbury. 2002.]</ref>. If seeds containing cyanogenic glycosides are ground up, the coarseness to which they are ground effects how quickly cyanogenesis occurs. Finely ground seeds extract HCN within an hour, where as coarsely ground seeds extract within 24 hours <ref name="tuncel">[http://www.sciencedirect.com/science/article/pii/030881469599841M The effects of grinding, soaking and cooking on the degradation of amygdalin of bitter apricot seeds. G Tunçel, M.J.R Nout, L Brimer. 1995.]</ref>. HCN boils at a relatively low temperature (25.6°C / 78.1°F) <ref name="Gleadow_2014"></ref>. In some cases, soaking, cooking, and/or sometimes fermenting foods with certain bacteria or yeast (this has not been fully documented with ''Saccharomyces'' or ''Brettanomyces'') that contain cyanogenic glycosides allows the HCN to be released, and then subsequent cooking afterwards will boil off the cyanide <ref>[http://www.sciencedirect.com/science/article/pii/016816059400115M International Journal of Food Microbiology. M.J.R. Nout, G. Tunçe, L. Brimer. 1995.]</ref><ref name="Chaouali"></ref>.
All plants contain at least tiny amounts of [https://en.wikipedia.org/wiki/Hydrogen_cyanide hydrogen cyanide] ('''HCN'''), however some plants also release high amounts of HCN After being released from a class of glycosides called "cyanogenic glycosides", also called "cyanoglycosides"HCN is highly toxic to animals. [https://en.wikipedia.org/wiki/Amygdalin Amygdalin] and [https://en.wikipedia.org/wiki/Linamarin linamarin] are common examples of cyanogenic glycosides <ref name="Gleadow_2014"></ref>. HCN The human body is released from cyanogenic glycosides just like other types used to breaking down trace amounts of glycosides: beta-glucosidase enzyme or exposure to low pH breaks cyanide into the bond between a glucose molecule and less toxic substance thiocyanate with an unstable compound enzyme called "cyanohydrin" (or "alpha-hydroxynnitrile")rhodanese, which then disassociates into a ketone or benzaldehyde and an HCN molecule. In cyanogenic glycosides, this reaction is called "cyanogenesis". Cyanogenesis is stimulated by maceration, and by bacteria in leaves the human gut body via urination <ref name="SpeijersGleadow_2014">[http://www.inchemannualreviews.org/documentsdoi/jecfafull/jecmono10.1146/v30je18.htm "annurev-arplant-050213-040027 Cyanogenic Glycosides": Synthesis, Physiology, First Draftand Phenotypic Plasticity. Dr GRoslyn M. Speijers. National Institute of Public Health Gleadow and Environmental Protection Laboratory for Toxicology, Bilthoven, The NetherlandsBirger Lindberg Møller. Retrieved 08/25/20162014.]</ref>. Although the optimum pH there are more than 3,000 plant species that are cyanogenic (a number of cyanogenesis (at least for amygdalinthem cultivated by farmers perhaps because their cyanogenic properties deter animals from eating them) is 5.0 - 5.8, cyongenesis can occur at only a wide range few parts of pH valuesplants that are considered foods contain enough HCN from cyanogenic glycosides to be considered dangerous (generally, and can occur in the presence other forms of acid cyanide are considered more dangerous, such as from exposure to air or water that is polluted with cyanide) <refname="CDC1">[http://www.sciencedirectatsdr.comcdc.gov/sciencetoxprofiles/article/pii/S0308814601003132 Total cyanide determination of plants and foods using the picrate and acid hydrolysis methodstp8.pdf Toxicology Profile for Cyanide. Agency for Toxic Substances & Disease Registry. M Rezaul Haque, J Howard BradburyJuly 2006. 2002Retrieved 08/25/2016.]</ref>. If seeds containing The location of the cyanogenic glycosides and the enzymes that release them are ground upoften each located in different (or all) parts of plants, the coarseness to which they and those locations are ground effects how quickly cyanogenesis occursdiverse across species. Finely ground seeds extract HCN within an hourIn some plants, where as coarsely ground seeds extract within 24 hours <ref>[http://www.sciencedirect.com/science/article/pii/030881469599841M The effects the cyanogenic glycosides are concentrated in the stems or leaves of grinding, soaking the plant and cooking on not the degradation of amygdalin of bitter apricot seeds(e. G Tunçelg. sorghum, M.J.R Noutbarley, L Brimerand lima beans). 1995.]</ref>. HCN boils at a relatively low temperature In fruits sometimes the seeds contain concentrated amounts (25e.6°C / 78g.1°Fblack cherry pits) <ref name="Gleadow_2014"></ref>. In some cases, soaking, cooking, and/or sometimes fermenting foods with certain bacteria or yeast other times in the fruit itself (this has not been documented with ''Saccharomycese.g. '' or ''BrettanomycesPassiflora edulis'') that contain . In rosaceous stone fruits, cyanogenic glycosides allows are located in the seeds, but the beta-glucosidase enzyme that the plant uses to release HCN is located in the roots of the plant. The concentration of cyanogenic glycosides is generally higher in seedling plants compared to be releasedmature plants, and then subsequent cooking afterwards will boil off however there are a few exceptions where this is the cyanide <ref>[http://wwwopposite (e.sciencedirectg.com/science/article/pii/016816059400115M International Journal of Food Microbiologysome ''Eucalyptus'' species, and lima beans). M.J.R. NoutThe HCN potential of plants varies highly depending on the species, G. Tunçestrain, L. Brimer. 1995.]<and climate/ref>environmental conditions of the crop year <ref name="ChaoualiGleadow_2014"></ref>.
After being released Although rare, there have been a few reported deaths due to cyanide poisoning from foods containing cyanogenic glycosides, HCN is highly toxic to animals. The human body is used These reports include deaths from elderberry juice that was thought to breaking down trace amounts of cyanide into contain stems and/or leaves (the less toxic substance thiocyanate with an enzyme called rhodanese, which then stems and leaves contain much higher cyanogenic glycosides than the body via urination berries, and ripe berries by themselves are considered safe) <ref name="Gleadow_2014">[http://www.annualreviewscdc.orggov/mmwr/doipreview/fullmmwrhtml/1000000311.1146/annurev-arplanthtm Poisoning from Elderberry Juice -050213-040027 Cyanogenic Glycosides: Synthesis, Physiology, and Phenotypic PlasticityCalifornia. Roslyn MCDC website. Gleadow and Birger Lindberg Møller 1998. 2014Retrieved 08/30/2016.]</ref>. Although there are more than 3,000 plant species that are cyanogenic apricot kernels (a number of them cultivated by farmers perhaps because their cyanogenic properties deter animals from eating thempits), only choke cherry pits, and improperly processed cassava (a few staple food in parts of plants that are considered foods contain enough HCN from cyanogenic glycosides North Africa) <ref name="who"></ref>. A lethal dosage of cyanide in humans is estimated to be considered dangerous (generallyaround 1.52 mg per kilogram of body weight, other forms with 0.56 mg per kilogram of cyanide are considered more dangerous, such as body weight being the lowest recorded (although this lowest figure was obtained from exposure to air or water that is polluted with cyanidea historical case when the measurements taken may not have been accurate) <ref name="CDC1">[http://www.atsdr.cdc.gov/toxprofiles/tp8.pdf Toxicology Profile for Cyanide. Agency for Toxic Substances & Disease Registry. July 2006. Pg 42. Retrieved 08/25/2016.]</ref>. The location of High exposure can cause light-headedness, nausea, vomiting, stomach cramps, diarrhea, convulsions, harm to the cyanogenic glycosides brain and the enzymes that release them are often each located in different (or all) parts of plantsheart, comas, and those locations are diverse across speciesdeath. In some plants, the cyanogenic glycosides are concentrated in the stems or leaves Exposure to 0.05 mg of cyanide per kilogram of body weight per day for 15-364 days is considered to be the plant and not minimum accumulative cyanide exposure by the seeds (eUS CDC.g. sorghum Accumulative exposure can cause health risks, such as reproductive, respiratory, neurological, barleythyroid, and lima beans)gastrointestinal issues <ref>[http://www.atsdr.cdc.gov/toxprofiles/tp8.pdf Toxicology Profile for Cyanide. In fruits sometimes the seeds contain concentrated amounts (e Agency for Toxic Substances & Disease Registry.g July 2006. black cherry pits), and other times in the fruit itself (e Pg 21.g Retrieved 08/25/2016. ''Passiflora edulis'')]</ref>. In rosaceous stone fruitssome foods, cyanogenic glycosides are located in the such as marzipan and persipan (made from bitter apricot seeds), but the processing of this food destroys the natural beta-glucosidase enzyme that (which denatures at 75°C), leaving the flora in the plant uses human gut to release HCN break down the cyanogenic glycosides. Even if an abnormally large portion of marzipan or persipan is located ingested, the lack of beta-glucosidase along with the high calories in the roots food acts as a slow release of cyanide into the human body which the plantbody can deal with <ref>[http://link.springer.com/article/10. The concentration 1007%2Fs00204-015-1479-8 Bioavailability of cyanide after consumption of a single meal of foods containing high levels of cyanogenic glycosides is generally higher : a crossover study in seedling plants compared to mature plantshumans. Klaus Abraham, Thorsten Buhrke, however this is there are a few exceptions where this is the opposite (eAlfonso Lampen.g 2015. some ''Eucalyptus'' species, and lima beans)]</ref>.
Although rareUpon learning about cyanogenic glycosides, there brewers often question the toxicity of ingredients such as cherry pits or apricot kernels in beer. Cherry pits have traditionally been a few reported deaths due to cyanide poisoning used in [[lambic]] kriek beers in Belgium. However, the dilution of HCN from foods containing cyanogenic glycosidescherry pits in beer results in benign levels. These reports include deaths from elderberry juice Assuming full breakdown of these glycosides, and that was thought to contain stems and/or leaves none of the HCN boils off (the stems and leaves contain much higher cyanogenic glycosides than the berries25.6°C boiling temperature), and ripe berries by themselves levels of HCN introduced from cherry pits are considered safetoo low to cause harm to adult humans. The EU regulates that alcoholic beverages cannot exceed 1 mg of HCN per ABV percentage (v/v%) per liter <ref>[http://wwwec.cdceuropa.goveu/mmwrfood/previewfs/mmwrhtmlsfp/00000311.htm Poisoning from Elderberry Juice -- Californiaaddit_flavor/flav09_en. pdf CDC websiteCOUNCIL DIRECTIVE of 22 June 1988 on the approximation of the laws of the Member States relating to flavourings for use in foodstuffs and to source materials for their production (88/388/EEC). 1998The European Food Commission, Food Safety. Retrieved 08/3026/2016.]</ref>. Luk Daenen, a glycoside researcher, apricot kernels (pits)calculated that for a 4% ABV alcohol beer, 4 mg of HCN per liter is allowed. With 200 grams of cherries per liter, choke cherry and the pitsbeing 10 - 14 grams of that weight, and improperly processed cassava (a staple food in parts there is 22 - 30.8 mg amygdalin per liter of beer. Around 6% of the weight of North Africa) <ref name="who"></ref>amygdalin is converted into HCN. A lethal dosage Assuming maximum extraction of cyanide HCN from the amygdalin glycoside, which is unlikely because the pits are not ground up when used in humans is estimated beer, this equates to be around 1.52 3 - 1.82 mg of HCN per kilogram liter of body weight, with 0beer.56 This amount is less than the 4 mg of HCN per kilogram liter that the EU regulation states. Considering that ~42 mg of body weight being the lowest recorded HCN is required to kill a person that weighs 70 kilograms (although this lowest figure was obtained from a historical case when the measurements taken may not have been accurate154 pounds) , that person would need to drink around 23 liters of beer <refname="daenen">[httphttps://www.atsdruclouvain.cdc.govbe/cps/ucl/doc/inbr/toxprofilesdocuments/tp8presentation-luk-daenen.pdf Toxicology Profile "Use of beta-glucosidase activity for Cyanide. Agency for Toxic Substances & Disease Registryflavour enhancement in specialty beers," slideshow by Luk Daenen. July 2006. Pg 422012. Retrieved 08/2526/2016.]</ref>. High exposure can cause light-headedness, nausea, vomiting, stomach cramps, diarrhea, convulsions, harm to the brain and heart, comas, and death. Exposure to 0.05 mg Considering that 350 mL of cyanide per pure alcohol would kill a 70 kilogram of body weight per day for 15-364 days is considered to be the minimum accumulative cyanide exposure by the US CDC. Accumulative exposure can cause health risks, such as reproductive, respiratory, neurological, thyroid, and gastrointestinal issues adult <ref>[http://www.atsdralcohol.cdcorg.govnz/toxprofilesalcohol-its-effects/tp8.pdf Toxicology Profile for Cyanidehealth-effects/alcohol-poisoning "Alcohol Poisoning". NZ Health Promotion Agency for Toxic Substances & Disease Registry. July 2006. Pg 21. Retrieved 08/2526/2016.]</ref>, the amount of 4% ABV beer required to kill a 70 kg adult from alcohol poisoning is around 8.75 liters. Alcohol would kill such a person far before cyanide poisoning would become a concern. In some foods, such as marzipan and persipan (made from bitter apricot seeds)general, the processing of this food destroys the natural beta-glucosidase enzyme (which denatures at 75°C), leaving the flora potential cyanide in the human gut most plants will become too dilute to have any health problems when added to break down the cyanogenic glycosides. Even if an abnormally large portion of marzipan or persipan is ingestedbeer in normal amounts, the lack of beta-glucosidase along with the however there might still be plants that are extremely high calories in HCN content that should be avoided in beer (see the food acts as a slow release of cyanide into the human body which the body can deal with <ref>[http://linktable below).springer.com/article/10.1007%2Fs00204-015-1479-8 Bioavailability of cyanide after consumption of If there is a single meal of foods concern that an ingredient containing potentially high levels of cyanogenic glycosides: a crossover study HCN could reach unhealthy levels in humans. Klaus Abrahambeer, Thorsten Buhrke, Alfonso Lampenthe beer should be sent for lab analysis so that the HCN levels can be determined before being consumed. 2015.]</ref>.
Upon learning about Some cyanogenic glycosides, brewers often question the toxicity of cherry pits or apricot kernels in beer. Cherry pits foods can have traditionally been used in [[lambic]] kriek beers in Belgium. However, the dilution of HCN from cherry pits in beer results in benign levels. Assuming full breakdown of these their cyanogenic glycosides, and that none of the HCN boils off reduced by cooking them at 230°C for 15 minutes (25.6°C boiling temperature)flaxseed, levels of HCN introduced from cherry pits are too low to cause harm to adult humans. The EU regulates that alcoholic beverages cannot exceed 1 mg of HCN per ABV percentage (v/v%for example) per liter <refname="Chaouali">[http:</ref><ref name="flax"></ec.europa.eu/food/fs/sfp/addit_flavor/flav09_en.pdf COUNCIL DIRECTIVE of 22 June 1988 on the approximation of the laws of the Member States relating ref>, however some amygdalin based cyanogenic plants may have their amygdalin content reduced to flavourings for use in foodstuffs and to source materials for their production about 25% by cooking alone (88/388/EEC). The European Food Commissionapricot seeds, Food Safety. Retrieved 08/26/2016for example; only the cooking temperature of 100°C was tested <ref name="tuncel"></ref>). Luk Daenen, Fermentation by certain species of microbes can have a glycoside researcher, calculated that for a 4% ABV alcohol beer, 4 mg of greater effect on reducing amygdalin to HCN per liter is allowedthan cooking alone. With 200 grams Microbes that have been shown to break down amygdalin include some species of cherries per literlactic acid bacteria including ''Lactobacillus plantarum'', and the pits being 10-14 grams fungi such as ''Endomyces fibuliger'', ''Pichia etchellsii'', and ''Hanseniaspora valbyensis''. Some strains of ''Brettanomyces'' that weight, there is 22 have high beta- 30.8 glucosidase activity might be able to break down amygdalin per liter by around 64%, and some strains of beer''S. Around 6cerevisiae'' might be able to break down up to around 10% of the weight of amygdalin is converted into HCN, but this needs to be verified by science. Assuming maximum extraction of HCN from Once the amygdalin glycoside, which is unlikely because the pits are not ground up when used in beer, this equates to 1.3 - 1.82 mg of broken down into HCN per liter of beer, which is less than the 4 mg of HCN per liter that the EU regulation states. Considering that ~42 mg of HCN is required to kill can then be volatilized off by cooking in a person that weighs 70 kilograms (154 pounds), that person would need to drink around 23 liters of beer ventilated space <ref name="daenen">[httpshttp://wwwlink.uclouvainspringer.becom/cpsarticle/ucl10.1007/doc/inbr/documents/presentation-luk-daenenBF00151870 Simple screening procedure for microorganisms to degrade amygdalin. L. Brimer, G.pdf "Use of beta-glucosidase activity for flavour enhancement in specialty beersTunçel," slideshow by Luk DaenenM. J. R. 2012Nout. Retrieved 08/26/20161993.]</ref>. 350 mL of alcohol would kill a 70 kilogram adult <ref>[http://www.alcoholncbi.orgnlm.nznih.gov/alcohol-its-effectspubmed/health-effects/alcohol-poisoning "Alcohol Poisoning"7710917 Microbial degradation of amygdalin of bitter apricot seeds (Prunus armeniaca). NZ Health Promotion AgencyNout MJ, Tunçel G, Brimer L. Retrieved 08/26/20161995.]</ref><ref name="Daenen1"></ref>. The amount of 4% ABV In normal brewing procedures, however, the beer required to kill a 70 kg adult from alcohol poisoning is around 8.75 liters. Alcohol would kill such a person far before cyanide poisoning would become a concern. In generalnot cooked nor ventilated, the potential cyanide in most foods will become too dilute to have so any health problems, however there might still be plants that are extremely high in HCN content that they is produced by the breakdown of cyanogenic glycosides should be avoided presumed to remain in the beer.
Some cyanogenic foods can have their cyanogenic glycosides reduced by cooking them at 230°C for 15 minutes (flaxseed, for example) <ref name="Chaouali"><===Determining HCN Potential====The [http://nordicfoodlab.org/blog/2013/ref><ref name="flax"><8/ref>, however some amygdalin based cyanogenic plants may have their amygdalin content reduced to about 25% by cooking alone (apricot seeds, hydrogen-cyanide The DIY picric acid test for example). Fermentation by certain species of microbes can have a greater effect on reducing amygdalin to HCN than cooking alone] could help in measuring HCN content in beer. Microbes Make sure that have been shown to break this test is performed against the finished beer after any glycosides would be broken down amygdalin include some species of lactic acid bacteria including ''Lactobacillus plantarum'', and fungi such as ''Endomyces fibuliger'', ''Pichia etchellsii'', and ''Hanseniaspora valbyensis''. Some strains of ''Brettanomyces'' that have high beta-glucosidase activity might be able to break down amygdalin by around 64%Toxicology labs, if available, and some strains of ''S. cerevisiae'' might should be able to break down up to around 10% of amygdalin, but this needs to be verified by scienceoffer services that measure HCN in beer. Once hte amydalin is broken down into The following table also lists potential HCN, content for various food items. For finding the HCN can then be volatilized off by cooking in a ventilated space <ref>[Simple screening procedure for microorganisms to degrade content based on amygdalin. L. Brimercontent, G. Tunçel, M. J. R. Nout. 1993.]</ref><ref>[http://www.ncbi.nlm.nih.gov/pubmed/7710917 Microbial degradation approximately 6% of amygdalin of bitter apricot seeds (Prunus armeniaca). Nout MJ, Tunçel G, Brimer L. 1995.]</ref><ref name="Daenen1"></ref>. In normal brewing procedures, however, the beer by weight is not cooked nor ventilated, so any converted to HCN that is produced by the breakdown of cyanogenic glycosides should be presumed to remain in the beer.
{| class="wikitable sortable"
|-
! Plant !! mg HCN/kg or mg/liter <nowiki>*</nowiki> || Prominent Glycoside
|-
| Cereal grains and their products || 0.001-0.45 <ref name="who">[http://www.who.int/ipcs/publications/cicad/en/cicad61.pdf Concise International Chemical Assessment Document 61. HYDROGEN CYANIDE AND CYANIDES: HUMAN HEALTH ASPECTS. World Health Organization. 2004. Pg 13. Retrieved 08/29/2016.]</ref> ||
|-
| Soybean hulls || 1.24 <ref name="who"></ref> || Linmarin <ref name="soybean"></ref>
|-
| Apricot pits, wet weight || 89-2170 (depends on region/species) <ref name="Chaouali"></ref><ref name="who"></ref> || Amgydalin <ref name="Gleadow_2014"></ref>
|-
| Home-made cherry juice from pitted fruits || 5.1 <ref name="who"></ref> || Amygdalin <ref name="Gleadow_2014"></ref>
|-
| Cherries with pit || 6.5-9.1 <ref name="daenen"></ref> || Amygdalin <ref name="Gleadow_2014"></ref>
|-
| Cherry pits only (black) || 161 <ref name="Bolarinwa_2014" /> || Amygdalin <ref name="Bolarinwa_2014" />
|-
| Cherry pits only (red) || 233 <ref name="Bolarinwa_2014" /> || Amygdalin <ref name="Bolarinwa_2014" />
|-
| Cherry pits only (morello) || 3900 <ref name="VOLDŘICH_1992" /> || Amygdalin <ref name="VOLDŘICH_1992" />
|-
| Apricot pits, wet weight || 89-2170 (depends on region/variety) <ref name="Chaouali"></ref><ref name="who"></ref> || Amgydalin <ref name="Gleadow_2014"></ref>
|-
| Sweet Apricots with kernel (14-24 fruits for 1 kg; single kernel avg weight is 6 grams <ref name="calapricot">[http://www.califapricot.com/marking_regulations.html California Apricots. Marking Regulations. Retrieved 09/01/16.]</ref>) || 4.2 - 7.2 (avg 0.3 mg per kernel <ref name="wiki_apric_kernel">[https://en.wikipedia.org/wiki/Apricot_kernel "Apricot Kernel". Wikipedia. Retrieved 09/01/2016.]</ref>) || Amygdalin <ref name="Gleadow_2014"></ref>
| Bitter Apricots with kernel (14-24 fruits for 1 kg; single kernel avg weight is 6 grams <ref name="calapricot"></ref>) || 25.2 - 43.2 (avg 1.8 mg per kernel <ref name="wiki_apric_kernel"></ref>) || Amygdalin <ref name="Gleadow_2014"></ref>
|-
| Elderflower (leaves/stems) || 1600 <ref name="nordicfoodlab">[https://web.archive.org/web/20190812014649/http://nordicfoodlab.org:80/blog/2013/10/elder-a-love-story "Elder – a love story". Justine de Valicourt. Nordic Food Lab. 10/03/2013. Retrieved 09/07/2016.]</ref> || Sambunigrin (or sometimes prunasin, holocalin, or zierin) <ref name="elderflowers">[https://www.researchgate.net/publication/233262135_Cyanogenic_Glycosides_from_Sambucus_Nigra Cyanogenic Glycosides from Sambucus Nigra. Marina Dellagreca, Antonio Fiorentino, Pietro Monaco, Lucio Previtera & Ana M. Simonet. 2006.]</ref>
|-
| Elderberries (fully ripe; under-ripe will contain more) || 30 <ref name="nordicfoodlab"></ref> || Sambunigrin (or sometimes prunasin, holocalin, or zierin) <ref name="elderflowers"></ref>
|-
| Peach seeds/kernels || 54-2600 (variety dependent; mostly reported between 54-666) <ref name="VOLDŘICH_1992">[https://www.scopus.com/record/display.uri?eid=2-s2.0-84987336720&origin=inward&txGid=713c7a86664b8d2865e54a25eaafc9b5 Cyanogenesis in Canned Stone Fruits. VOLDŘICH, M., KYZLINK, V. 1992. DOI: 10.1111/j.1365-2621.1992.tb05446.x.]</ref><ref name="Bolarinwa_2014">[https://www.sciencedirect.com/science/article/pii/S0308814613016245 Amygdalin content of seeds, kernels and food products commercially-available in the UK. Islamiyat F. Bolarinwa, Caroline Orfila, Michael R.A. Morgan. 2014. DOI: https://doi.org/10.1016/j.foodchem.2013.11.002.]</ref><ref>[https://www.scopus.com/record/display.uri?eid=2-s2.0-0021674858&origin=inward&txGid=ee159dc998e0469bc88f0685e8d41ecb The cyanide content of laetrile preparations, apricot, peach and apple seeds. Holzbecher, M.D., Moss, M.A., Ellenberger, H.A 1984.]</ref> || Amygdalin <ref name="Bolarinwa_2014" />
|-
| Nectarine seeds (summer fire) || 7.2 <ref name="Bolarinwa_2014" /> || Amygdalin <ref name="Bolarinwa_2014" />
|-
| Plum seeds (green) || 1050 <ref name="Bolarinwa_2014" /> || Amygdalin <ref name="Bolarinwa_2014" />
|-
| Plum seeds (black; firar black) || 600 <ref name="Bolarinwa_2014" /> || Amygdalin <ref name="Bolarinwa_2014" />
|-
| Plum seeds (purple; larry anne) || 130 <ref name="Bolarinwa_2014" /> || Amygdalin <ref name="Bolarinwa_2014" />
|-
| Plum seeds (yellow; son gold) || 92 <ref name="Bolarinwa_2014" /> || Amygdalin <ref name="Bolarinwa_2014" />
|-
| Plum seeds (red; laetitia) || 26 <ref name="Bolarinwa_2014" /> || Amygdalin <ref name="Bolarinwa_2014" />
|-
| Apple seeds (royal gala) || 178 <ref name="Bolarinwa_2014" /> || Amygdalin <ref name="Bolarinwa_2014" />
|-
| Pear (conference) || 77 <ref name="Bolarinwa_2014" /> || Amygdalin <ref name="Bolarinwa_2014" />
|-
| Non-Rosaceae fruit seeds (courgette, cucumber, marrow, honey dew melon, squash varieties) || 0.6-12.6 <ref name="Bolarinwa_2014" /> || Amygdalin <ref name="Bolarinwa_2014" />
|-
| '''Commercial fruit juices'''
|-
|}
: <nowiki>*</nowiki> Amounts are averages, or are single examples. Actual levels may vary greatly between strains and growth conditions.
See also:
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1401402279887982/ MTF thread on cyanide potential in beer.]
==See Also==
===Additional Articles on MTF Wiki===
* [[Hops#Hop_Derived_Compounds_In_Beer_and_Biotransformations|Hop Biotransformations]]
* [[Brettanomyces]]
* [[Pediococcus]]
* [[Mixed Fermentation]]
* [[Soured Fruit Beer]]
* [[Soured Herb, Spice, and Vegetable Beer]]
===External Resources===
* [https://www.uclouvain.be/cps/ucl/doc/inbr/documents/presentation-luk-daenen.pdf "Use of beta-glucosidase activity for flavour enhancement in specialty beers," slideshow by Luk Daenen.]
* [http://nordicfoodlab.org/blog/2013/10/elder-a-love-story "Elder: a love story", by Justine de Valicourt (cyanide in elderberries).]
* [http://www.dpi.nsw.gov.au/__data/assets/pdf_file/0013/111190/prussic-acid-poisoning-in-livestock.pdf Overview of HCN in various grass/straw/hay/livestock feed species.]
* [http://scottjanish.com/?s=glycosides Scott Janish blog articles about glycosides.]
==References==