Difference between revisions of "Glycosides"

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| 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>
 
| 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>
 
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Revision as of 16:41, 29 August 2016

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 [1]. The release of flavor molecules from glycosides is thought to contribute to the flavor development of aging wines, as well as kriek (cherry) lambic [2]. It is speculated that flavor compounds from hops can also be released from glycosides [3].

Glycosides and Beta-Glucosidase Activity

Monoterpenes and Glycosides

Monoterpenes generally exist as aromatic and flavorful alcohols that tend to smell floral with low odor thresholds (100-400 ppb), and are present in plant material. These alcohols mostly consist of linalool (a major contributor to hop aroma [4]), geraniol, nerol, and linalool oxides, but also includes other monoterpenes such as citronellol, alpha-terpineol, hotrienol, nerol oxide, myrcenol, the ocimenols, and other oxides, aldehydes and hydrocarbons. In wine (and probably sour beer), these alcohols bind with acids to create aromatic monoterpene ethyl esters and acetate esters, and can also be transformed into other types of monoterpenes by yeast metabolism [5][2]. Monoterpenes can also be odorless polyols, some of which can break down easily to produce pleasant aromas. For example, diendiol can break down into hotrienol (tropical, floral, fennel, ginger aroma [6]) and nerol oxide (green, vegetative and floral with a minty undernote [7]). It is thought that some strains of Saccharomyces cerevisiae might be able to produce monoterpenes such as geraniol during fermentation in ways not related to beta-glucosidase activity [8].

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 [9]. These include protecting cells from toxins in the plant and attracting insects via the fragrance of flowers [10].

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 [11][10]. 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 [11]. In fruits, there are mostly just 4 types of flavonol type aglycones: quercetin (found in nearly all fruits), kaempherol (found in 80% of fruit), and less commonly quercetin and isorhamnetin [12] (see 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 the amount that are free in a ratio of 2:1 to 8:1 [11]. 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 bond, discarded parts of plants (peels, stems, skins, etc.) have been used to produce natural flavorings from the remaining and abundant glycosides [10].

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 [11]), or by enzymes called beta-glycosidases. Enzymatic breakdown of glycosides as 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 [11].

Beta-glycosidase enzymes can be added artificially, however there has been much interest in the natural capability of microorganisms to produce beta-glycosidases, particularly 1,4-β-glucosidase [10]. Microorganisms that can break down glycosides by using beta-glucosidases can then access the resulting sugars for fermentation [13]. 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 [10].

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) [10]. However, glycosides in tea leaves that contain disaccharide sugars (cellulose/cellobiose [14]) 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 [11][15]. 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 [16].

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 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 [17].

Activity of Brettanomyces and Saccharomyces

One study screened the beta-glucosidase activity of several strains of Saccharomyces cerevisiae, Saccharomyces pastorianus, and Brettanomyces spp [3]. 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 [3]. However, beta-glucosidase activity for S. cerevisiae is inhibited by pH levels of wine and sour beer (optimal at pH 5) [15]. 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 [18]). 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 [19]), methyl salicylate (minty, wintergreen [20]), 1-octen-3-ol (mushroom, earthy [21]) and cis-3-hexen-1-ol (grassy, melon rind [22]) were released from hop extracts [3]. 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 [23]. 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 [3]. 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) [24][25].

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 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 [2].

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 [26][15]. 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 [17]. 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 [15]. Strains that can metabolize cellobiose tend to also have higher beta-glucosidase activity because the possess an extra gene for beta-glucosidase enzyme production [27].

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 [17].

Cyanogenic Glycosides

(In progress)

All plants contain tiny amounts of hydrogen cyanide (HCN), however some plants also release high amounts of HCN from a class of glycosides called "cyanogenic glycosides". Amygdalin and linamarin are common examples of cyanogenic glycosides. After being released from cyanogenic glycosides, HCN is highly toxic to animals. HCN boils at a relatively low temperature (25.6°C / 78.1°F). The human body is used to breaking down trace amounts of cyanide into the less toxic substance thiocyanate with an enzyme called rhodanese, which then leaves the body via urination [28]. Although there are more than 3,000 plant species that are cyanogenic (a number of them cultivated by farmers perhaps because their cyanogenic properties deter animals from eating them), only a few parts of plants that are considered foods contain enough HCN from cyanogenic glycosides to be considered dangerous (generally, other forms of cyanide are considered more dangerous, such as from exposure to air or water that is polluted with cyanide) [29]. The location of the cyanogenic glycosides and the enzymes that release them are often each located in different (or all) parts of plants, and those locations are diverse across species. In some plants, the cyanogenic glycosides are concentrated in the stems or leaves of the plant and not the seeds (e.g. sorghum, barley, and lima beans). In fruits sometimes the seeds contain concentrated amounts (e.g. black cherry pits), and other times in the fruit itself (e.g. Passiflora edulis). In rosaceous stone fruits, cyanogenic glycosides 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 mature plants, however this is there are a few exceptions where this is the opposite (e.g. some Eucalyptus species, and lima beans). 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. This reaction is stimulated by maceration, and by bacteria in the human gut. The cyanogenic glycosides themselves are not toxic until the HCN is released [30].

Upon learning about cyanogenic glycosides, brewers often question the toxicity of cherry and apricot pits in beer. Assuming full conversion of these glycosides, and that none of the HCN boils off, levels of HCN introduced from cherry and apricot pits are too low to cause harm to adult humans. A lethal dosage of cyanide in humans is estimated to be around 1.52 mg per kilogram of body weight, with 0.56 mg per kilogram of body weight being the lowest recorded (although this lowest figure was obtained from a historical case when the measurements taken may not have been accurate) [31]. High exposure can cause harm to the brain and heart, and can cause comas or death. Exposure to 0.05 mg of cyanide per kilogram of body weight per day for 15-364 days is considered to cause accumulative health risks, such as reproductive, respiratory, neurological, thyroid, and gastrointestinal issues [32]. The EU regulates that alcoholic beverages cannot exceed 1 mg of HCN per ABV percentage (v/v%) per liter [33]. Luk Daenen, a glyoside researcher, calculated that for a 4% ABV alcohol beer, 4 mg of HCN per liter is allowed. With 200 grams of cherries per liter, and the pits being 10-14 grams of that weight, there is 22 - 30.8 amygdalin per liter of beer. This equates to 1.3 - 1.82 mg of HCN per liter of beer, which is less than the 4 mg of HCN per liter EU regulation. Considering that ~42 mg of HCN is required to kill a person that weighs 70 kilograms (154 pounds), that person would need to drink around 23 liters of beer [34]. 350 mL of alcohol would kill a 70 kilogram adult [35]. 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.

Plant mg HCN/kg or mg/liter
Cereal grains and their products 0.001-0.45 [36]
Soy protein products 0.07-0.3 [36]
Soybean hulls 1.24 [36]
Apricot pits, wet weight 89-2170 [36]
Home-made cherry juice from pitted fruits 5.1 [36]
Home-made cherry juice containing 100% crushed pits 23 [36]
Commercial fruit juices
Cherry 4.6 [36]
Apricot 2.2 [36]
Prune 1.9 [36]
Tropical foodstuffs
Cassava(bitter)/dried root cortex 2360-2450 [36][30]
Cassava(bitter)/leaves 300-310 [36][30]
Cassava(bitter)/whole tubers 380-395 [36][30]
Cassava(sweet)/leaves 451-468 [36][30]
Cassava(sweet)/whole tubers 445-462 [36][30]
Gari flour (Nigeria) 10.6-22.1 [36]
Sorghum/whole immature plant 2400-2500 [36][30]
Bamboo/immature shoot tip 7700-8000 [36][30]
Lima beans from Java (coloured) 3000-3120 [36][30]
Lima beans from Puerta Rico (black) 2900-3000 [36][30]
Lima beans from Burma (white) 2000-2100 [36][30]

See Also

Additional Articles on MTF Wiki

External Resources

References

  1. ↑ "Glycoside." Wikipedia. Retrieved 06/27/2016.
  2. ↑ 2.0 2.1 2.2 Evaluation of the glycoside hydrolase activity of aBrettanomyces strain on glycosides from sour cherry (Prunus cerasus L.) used in the production of special fruit beers. Luk Daenen, Femke Sterckx, Freddy R. Delvaux, Hubert Verachtert & Guy Derdelinckx. 2007.
  3. ↑ 3.0 3.1 3.2 3.3 3.4 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.
  4. ↑ Linalool in Hops. MBAA. Stefan Hanke, University Weihenstephan, Germany. 2009.
  5. ↑ Monoterpene alcohols release and bioconversion by Saccharomyces species and hybrids. A. Gamero, P. Manzanares, A. Querol, C. Belloch. 2011.
  6. ↑ "Hotrienol." The Good Scents Company. Retrieved 05/11/2016.
  7. ↑ "Nerol Oxide." The Good Scents Company. Retrieved 05/11/2016.
  8. ↑ De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts. Francisco M. Carrau, Karina Medina, Eduardo Boido, Laura Farina, Carina Gaggero, Eduardo Dellacassa, Giuseppe Versini, Paul A. Henschke. 2005.
  9. ↑ "Glycoside." New World Encyclopedia. Retrieved 05/06/2016.
  10. ↑ 10.0 10.1 10.2 10.3 10.4 10.5 "Glycoconjugated aroma compounds: Occurrence, role and biotechnological transformation." Peter Winterhalter, George K. Skouroumounis. 1997.
  11. ↑ 11.0 11.1 11.2 11.3 11.4 11.5 "Hydrolysis of terpenyl glycosides in grape juice and other fruit juices: a review." Sergi Maicas, José Juan Mateo. May 2005.
  12. ↑ Fruit Phenolics. Jean-Jacques Macheix, Annie Fleuriet. CRC Press, Mar 20, 1990. Pgs 57-61.
  13. ↑ 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.
  14. ↑ "Disaccharides." UC Davis Chemwiki. Retrieved 05/15/2016.
  15. ↑ 15.0 15.1 15.2 15.3 Quantification of Glycosidase Activities in Selected Strains of Brettanomyces bruxellensis and Oenococcus oeni. A. K. Mansfield, B. W. Zoecklein and R. S. Whiton. 2001.
  16. ↑ A survey of glycosidase activities of commercial wine strains of Oenococcus oeni. Antonio Grimaldi, Eveline Bartowsky, Vladimir Jiranek. 2005.
  17. ↑ 17.0 17.1 17.2 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.
  18. ↑ Private correspondence with Daniel Sharp from Oregon State University and Dan Pixley. 05/16/2016.
  19. ↑ "Linalool." The Good Scents Company. Retrieved 05/12/2016.
  20. ↑ "Methyl salicylate." Chemistry World. Retrieved 05/12/2016.
  21. ↑ "1-octen-3-ol." The Good Scents Company. Retrieved 05/12/2016.
  22. ↑ "(Z)-3-hexen-1-ol." The Good Scents Company. Retrieved 05/12/2016.
  23. ↑ World Brewing Congress, 2008. Pg 80. Retrieved 05/13/2016.
  24. ↑ Private correspondence with Richard Preiss by Dan Pixley. 05/16/2016.
  25. ↑ "Hops Oils & Aroma: Uncharted Waters," by Stan Hieronymus. Beer & Brewing. 03/16/2016. Retrieved 05/16/2016.
  26. ↑ Quantification of glycosidase activities in selected yeasts and lactic acid bacteria. H McMahon, B W Zoecklein, K Fugelsang, Y Jasinski. 1999.
  27. ↑ 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
  28. ↑ Cyanogenic Glycosides: Synthesis, Physiology, and Phenotypic Plasticity. Roslyn M. Gleadow and Birger Lindberg Møller. 2014.
  29. ↑ Toxicology Profile for Cyanide. Agency for Toxic Substances & Disease Registry. July 2006. Retrieved 08/25/2016.
  30. ↑ 30.00 30.01 30.02 30.03 30.04 30.05 30.06 30.07 30.08 30.09 30.10 "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.
  31. ↑ Toxicology Profile for Cyanide. Agency for Toxic Substances & Disease Registry. July 2006. Pg 42. Retrieved 08/25/2016.
  32. ↑ Toxicology Profile for Cyanide. Agency for Toxic Substances & Disease Registry. July 2006. Pg 21. Retrieved 08/25/2016.
  33. ↑ [http://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 to flavourings for use in foodstuffs and to source materials for their production (88/388/EEC). The European Food Commission, Food Safety. Retrieved 08/26/2016
  34. ↑ "Use of beta-glucosidase activity for flavour enhancement in specialty beers," slideshow by Luk Daenen. 2012. Retrieved 08/26/2016.
  35. ↑ "Alcohol Poisoning". NZ Health Promotion Agency. Retrieved 08/26/2016.
  36. ↑ 36.00 36.01 36.02 36.03 36.04 36.05 36.06 36.07 36.08 36.09 36.10 36.11 36.12 36.13 36.14 36.15 36.16 36.17 36.18 36.19 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.]
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