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→Determining HCN Potential
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''===
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 (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>). 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 fermentationfrom the production of beta-lyase enzyme, 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 see [http://scottjanish.com/genetically-modified-gm-yeast-strains-unlocking-bound-hop-thiols during -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>. 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>.
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" />.
In 2020, whole genome sequencing and glycosidic activity were tested for 84 strains of ''Brettanomyces'' by [https://www.frontiersin.org/articles/10.3389/fmicb.2020.00637/full Colomer et al]. They found that the capability of ''Brettanomyces'' to break down glycosides is determined by possessing two genes: ''BbBGL1'' and ''BbBGL2''. The genes called ''BbBGL2'' have a major role in whether glycosides are broken down, while the role of ''BbBGL1'' is minimal. About half of the strains had some level of glycosidic activity, although the majority was low with a couple of strains that had high levels of glycosidic activity, thus these genes alone do not account for the wide variation in glycosidic activity levels in ''Brettanomyces'' (see [https://www.frontiersin.org/files/Articles/495404/fmicb-11-00637-HTML-r1/image_m/fmicb-11-00637-g007.jpg this figure] from the study which maps beta-glucosidase activity for the 84 strains that were sequenced) <ref>[https://www.frontiersin.org/articles/10.3389/fmicb.2020.00637/full Assessing Population Diversity of Brettanomyces Yeast Species and Identification of Strains for Brewing Applications. Marc Serra Colomer, Anna Chailyan, Ross T. Fennessy, Kim Friis Olsson, Lea Johnsen, Natalia Solodovnikova and Jochen Forster. 2020. DOI: https://doi.org/10.3389/fmicb.2020.00637.]</ref>. The beta-glucidase levels were also measured for 5 strains (4 strains of ''B. bruxellensis'' and 1 strain of ''B. anomalus'') that have the genetic make up to produce this enzyme; most of the enzyme was only produced within the cell (intracellular) for all of the strains tested, and only small amounts were produced outside of the cells except for one strain of ''B. anomalus''. 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. anomalus''. They also fermented a dry hopped beer with these strains and found that the strains with the lowest beta-glucosidase activity had the lowest conversion of geraniol to β‐citronellol, and therefore there was no correlation between beta-glucosidase activity and hop biotransformation (although there was evidence of another unidentified type of biotransformation occurring with the hops[ (see [[Brettanomyces#Hop_Biotransformation|''Brettanomyces'' hop biotransformation]]) <ref>[https://onlinelibrary.wiley.com/doi/full/10.1002/jib.610 Biotransformation of hop derived compounds by Brettanomyces yeast strains. Marc Serra Colomer, Birgitte Funch, Natalia Solodovnikova, Timothy John Hobley, Jochen Förster. 2020. DOI: https://doi.org/10.1002/jib.610.]</ref>.
Wang et al. (2018) reported another type of glycosidic activity in one strain of ''B bruxellensis'' which is the conversion of the glycoside "mogroside V" into an artificial sweetener called siamenoside I. Other yeast and bacteria species were tested and did not find this particular glycosidic activity. [https://en.wikipedia.org/wiki/Mogroside Mogroside V] is found naturally in some fruit, specifically, an Asian fruit called [https://en.wikipedia.org/wiki/Siraitia_grosvenorii Lo Han Kuo (''Siraitia grosvenorii'')]. The artificial sweetener siamenoside is 563 times sweeter tasting than 5% sucrose. The specific enzyme responsible for this conversion that this strain of ''B. bruxellensis'' produced is called ''Db''Exg1 <ref>[https://www.sciencedirect.com/science/article/pii/S0308814618317473 Dekkera bruxellensis, a beer yeast that specifically bioconverts mogroside extracts into the intense natural sweetener siamenoside I. Reuben Wang, Yi-Chieh Chen, Yun-Ju Lai, Ting-Jang Lu, Shyue-Tsong Huang, Yi-Chen Lo. 2018. DOI: https://doi.org/10.1016/j.foodchem.2018.09.163.]</ref>.
See also :* [[Hops#Glycosides|Glycosides in Hops]].* [http://scottjanish.com/genetically-modified-gm-yeast-strains-unlocking-bound-hop-thiols-and-engineering-targeted-fermentation-characteristics/ "Genetically Modified (GM) Yeast Strains: Unlocking Bound Hop Thiols and Engineering Targeted Fermentation Characteristics," by Scott Janish.]
===Activity of Lactic Acid Bacteria===
| 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>
* [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==