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Glycosides are a very diverse group of non-volatile and flavorless molecules that generally encompass any molecule that has a sugar bound to a non-sugar molecule (thus separating them from polysaccharides). The sugar (monosaccharide or oligosaccharide) component of the molecule is known as the "glycone", and the non-sugar component is known as the "aglycone". By breaking the glycosidic bond of a glycoside, the aglycone component is released. The aglycone component of glycosides are often polyphenols or the floral monoterpene alcohols described above. Glycosides can be categorized based on their glycone (glucose vs fructose), type of glycosidic bond (α-glycosides or β-glycosides), or by their aglycone (alcoholic, anthraquinone, coumarin, cyanogenic, flavonoid, phenolic, aponins, steroidal/cardiac, steviol, or thioglycosides). Glycosides play important roles in living organisms, especially many types of plants which store glycosides in their tissue and then break the bond between the sugar and non-sugar aglycone when the aglycone is needed for certain biological functions <ref>[http://www.newworldencyclopedia.org/entry/Glycoside "Glycoside." New World Encyclopedia. Retrieved 05/06/2016.]</ref>. These include protecting cells from toxins in the plant and attracting insects via the fragrance of flowers <ref name="Winterhalter"></ref>.
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 out number 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 glicosidic 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>.
===Beta-Glucosidase===
Aglycones can be released 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-glycosidasesglucosidases. Enzymatic breakdown of glycosides as 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-glycosidasesglucosidases, 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>.
Exogenous beta-glycosidase activity has been shown to be much more effective at releasing aglycones from glycosides in bacteria and fungi. For glycosides which contain a glucose, which is the majority, beta-glucosidase cleaves the sugar, thus releasing the aglycone. For glycosides that contain disaccharides, usually another enzyme must be present to first break down the disaccharide before the beta-glucosidase can release the aglycone (beta-xylosidase, alpha-arabinosidase, alpha-rhamnosidase, or beta-apiosidase) <ref name="Winterhalter"></ref>. However, glycosides in tea leaves that contain disaccharide sugars (cellulose/cellobiose <ref name="ucdavis_chemwiki">[http://chemwiki.ucdavis.edu/Core/Organic_Chemistry/Carbohydrates/Disaccharides "Disaccharides." UC Davis Chemwiki. Retrieved 05/15/2016.]</ref>) have been observed to be broken down without the use of these other enzymes; the beta-glucosidase cleaves the aglycone from the disaccharide on its own. Some species of yeast (''Debaryomyces castelli'', ''D. hansenii'', ''D. polymorphus'', ''Kloeckera apiculata'', ''Hansenula anomala'', and ''Brettanomyces'' spp), bacteria (''Oenococcus oeni''), and fungi (''Aspergillus niger'') have been found to have strain dependent beta-glucosidase activity, however several inhibitors for glucosidase activity vary for different strains of microbes. These inhibitors include the presence of glucose, pH, temperature, ethanol, and phenols <ref name="Maicas"></ref><ref name="Mansfield">[https://theses.lib.vt.edu/theses/available/etd-07262001-172630/unrestricted/Mansfieldthesis.pdf Quantification of Glycosidase Activities in Selected Strains of Brettanomyces bruxellensis and Oenococcus oeni. A. K. Mansfield, B. W. Zoecklein and R. S. Whiton. 2001.]</ref>. For example, for some strains of ''O. oeni'', as little as 10mg/L of glucose is enough to inhibit beta-glucosidase activity, or the presence of alcohol or typical wine pH (3.0 - 4.0) was enough to inhibit. Other strains of ''O. oeni'' are not inhibited by some or all of these inhibitors <ref>[http://www.sciencedirect.com/science/article/pii/S0168160505003296 A survey of glycosidase activities of commercial wine strains of Oenococcus oeni. Antonio Grimaldi, Eveline Bartowsky, Vladimir Jiranek. 2005.]</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), where as 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>.
===Activity of Brettanomyces and Saccharomyces===
One study screened the beta-glucosidase activity of several strains of ''Saccharomyces cerevisiae'', ''Saccharomyces pastorianus'', and ''Brettanomyces'' spp <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>. 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"></ref>. However, beta-glucosidase activity for ''S. cerevisiae'' is inhibited by pH levels of 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 (it's worth noting that other research using whole hops has shown no significant hop derived aglycones when using beta-glucosidase active ''Saccharomyces'' strains; publication yet to be released <ref>Private correspondence with Daniel Sharp from Oregon State University and Dan Pixley. 05/16/2016.</ref>). None of the ''B. bruxellensis'' strains showed this activity, but the 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, and this could also explain anecdotal reports of increased fruity aromas from exposing hops to fermentation (it is unknown what exactly causes the increase in thiols during fermentation) <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>.
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, jasmin 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>.
Many strains of ''B. bruxellensis'' have 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 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 the 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>.
| Lima beans from Java (coloured) || 3000-3120 <ref name="who"></ref><ref name="Speijers"></ref>
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| Lima beans from Puerta Puerto Rico (black) || 2900-3000 <ref name="who"></ref><ref name="Speijers"></ref>
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| Lima beans from Burma (white) || 2000-2100 <ref name="who"></ref><ref name="Speijers"></ref>