Glycosides

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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]. When the glycosidic bond is broken by an enzyme produced by a microorganism, it can be classified under the generic term as a "biotransformation" [2]. The release of flavor molecules from glycosides is thought to contribute to the flavor development of aging wines, as well as kriek (cherry) lambic [3]. It is speculated that flavor compounds from hops can also be released from glycosides [4].

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 [5]), 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 [6][3]. 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 [7]) and nerol oxide (green, vegetative and floral with a minty undernote [8]). 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 [9].

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 [10]. These include protecting cells from toxins in the plant and attracting insects via the fragrance of flowers [11].

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 [12][11]. 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 [12]. 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 [13] (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 outnumber the amount that are free in a ratio of 2:1 to 8:1 [12]. 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 [11].

Acidic Hydrolysis

Aglycones can be released from glycosides via a low pH. Generally, this occurs at lower pH's, but different types of glycosides are more readily broken down at different pH's. For example, ocimenols (lemon, lime [14]) 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 [12].

In beer, it was found that the lower the pH of the beer the higher the amount of the ketone damascenone (floral, rose [15]) and dimethyl trisulfide (sulfury cooked onion [16]). In one study, at a pH of 3, 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, 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 glycosdes [17].

Beta-Glucosidase

Aglycones can also be released chemically from glycosides 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 [12].

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 [11]. Microorganisms that can break down glycosides by using beta-glucosidases can then access the resulting sugars for fermentation [18]. 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 [11].

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) [11]. However, glycosides in tea leaves that contain disaccharide sugars (cellulose/cellobiose [19]) 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 [12][20]. 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 [21].

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 [22].

Activity of Brettanomyces and Saccharomyces

One study screened the beta-glucosidase activity of several strains of Saccharomyces cerevisiae, Saccharomyces pastorianus, and Brettanomyces spp [4]. 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 [4]. However, beta-glucosidase activity for S. cerevisiae is inhibited by pH levels of wine and sour beer (optimal at pH 5) [20]. 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 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 [23]). 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 [24]), methyl salicylate (minty, wintergreen [25]), 1-octen-3-ol (mushroom, earthy [26]) and cis-3-hexen-1-ol (grassy, melon rind [27]) were released from hop extracts [4]. 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 [28]. 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 [4]. 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) [29][30].

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 [3].

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 [31][20]. 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 [22]. 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 [20]. Strains that can metabolize cellobiose tend to also have higher beta-glucosidase activity because they possess an extra gene for beta-glucosidase enzyme production [32].

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 [22].

The effects of beta-glycosidase 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 suppressed. 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. Terineol, citronellol, nerol, and geraniol were also not increased in significant amounts, however 1-octanol (waxy, green, citrus, orange, aldehydic, fruity [33]) was increased significantly [23].

See also Glycosides in Hops.

Cyanogenic Glycosides

All plants contain at least tiny amounts of hydrogen cyanide (HCN), however, some plants also release high amounts of HCN from a class of glycosides called "cyanogenic glycosides", also called "cyanoglycosides". Amygdalin and linamarin are common examples of cyanogenic glycosides [34]. Amygdalin is often 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 [35][36][37][38]. 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 cyanogenic glycosides, this reaction is called "cyanogenesis". Cyanogenesis is stimulated by maceration, and by bacteria in the human gut [39]. 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 [40]. 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 [41]. HCN boils at a relatively low temperature (25.6°C / 78.1°F) [34]. 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 [42][43].

After being released from cyanogenic glycosides, HCN is highly toxic to animals. 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 [34]. 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) [44]. 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 there are a few exceptions where this is the opposite (e.g. some Eucalyptus species, and lima beans). The HCN potential of plants varies highly depending on the species, strain, and climate/environmental conditions of the crop year [34].

Although rare, there have been a few reported deaths due to cyanide poisoning from foods containing cyanogenic glycosides. These reports include deaths from elderberry juice that was thought to contain stems and/or leaves (the stems and leaves contain much higher cyanogenic glycosides than the berries, and ripe berries by themselves are considered safe) [45], apricot kernels (pits), choke cherry pits, and improperly processed cassava (a staple food in parts of North Africa) [46]. 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) [47]. 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 of cyanide per 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 [48]. In some foods, such as marzipan and persipan (made from bitter apricot seeds), the processing of this food destroys the natural beta-glucosidase enzyme (which denatures at 75°C), leaving the flora in the human gut to break down the cyanogenic glycosides. Even if an abnormally large portion of marzipan or persipan is ingested, the lack of beta-glucosidase along with the high calories in the food acts as a slow release of cyanide into the human body which the body can deal with [49].

Upon learning about cyanogenic glycosides, brewers often question the toxicity of cherry pits or apricot kernels in beer. Cherry pits 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 glycosides, and that none of the HCN boils off (25.6°C boiling temperature), 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%) per liter [50]. Luk Daenen, a glycoside 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 mg amygdalin per liter of beer. Around 6% of the weight of amygdalin is converted into HCN. Assuming maximum extraction of HCN from 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 HCN per liter of beer. This amount 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 a person that weighs 70 kilograms (154 pounds), that person would need to drink around 23 liters of beer [51]. Considering that 350 mL of pure alcohol would kill a 70 kilogram adult [52], 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 general, the potential cyanide in most plants will become too dilute to have any health problems when added to beer in normal amounts, however there might still be plants that are extremely high in HCN content that should be avoided in beer (see the table below).

Some cyanogenic foods can have their cyanogenic glycosides reduced by cooking them at 230°C for 15 minutes (flaxseed, for example) [43][53], however some amygdalin based cyanogenic plants may have their amygdalin content reduced to about 25% by cooking alone (apricot seeds, for example; only the cooking temperature of 100°C was tested [41]). Fermentation by certain species of microbes can have a greater effect on reducing amygdalin to HCN than cooking alone. Microbes that have been shown to break 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%, and some strains of S. cerevisiae might be able to break down up to around 10% of amygdalin, but this needs to be verified by science. Once the amygdalin is broken down into HCN, the HCN can then be volatilized off by cooking in a ventilated space [54][55][4]. In normal brewing procedures, however, the beer is not cooked nor ventilated, so any HCN that is produced by the breakdown of cyanogenic glycosides should be presumed to remain in the beer.

Plant mg HCN/kg or mg/liter * Prominent Glycoside
Cereal grains and their products 0.001-0.45 [46]
Soy protein products 0.07-0.3 [46] Linmarin [56]
Soybean hulls 1.24 [46] Linmarin [56]
Home-made cherry juice from pitted fruits 5.1 [46] Amygdalin [34]
Home-made cherry juice containing 100% crushed pits 23 [46] Amydalin [34]
Almonds (wild bitter) 1062-4690 [43][57] Amygdalin [58]
Almonds (sweet domesticated) 25 [43] Amygdalin [58]
Cherries with pit 6.5-9.1 [51] Amygdalin [34]
Apricot pits, wet weight 89-2170 (depends on region/species) [43][46] Amgydalin [34]
Sweet Apricots with kernel (14-24 fruits for 1 kg; single kernel avg weight is 6 grams [59]) 4.2 - 7.2 (avg 0.3 mg per kernel [60]) Amygdalin [34]
Bitter Apricots with kernel (14-24 fruits for 1 kg; single kernel avg weight is 6 grams [59]) 25.2 - 43.2 (avg 1.8 mg per kernel [60]) Amygdalin [34]
Elderflower (leaves/stems) 1600 [61] Sambunigrin (or sometimes prunasin, holocalin, or zierin) [62]
Elderberries (fully ripe; under-ripe will contain more) 30 [61] Sambunigrin (or sometimes prunasin, holocalin, or zierin) [62]
Commercial fruit juices
Cherry 4.6 [46] Amygdalin [34]
Apricot 2.2 [46] Amygdalin [34]
Prune 1.9 [46] Amygdalin [34]
Tropical foodstuffs
Cassava(bitter)/dried root cortex 2360-2450 [46][39] Linamarin [34]
Cassava(bitter)/leaves 300-310 [46][39] Linamarin [34]
Cassava(bitter)/whole tubers 380-395 [46][39] Linamarin [34]
Cassava(sweet)/leaves 451-468 [46][39] Linamarin [34]
Cassava(sweet)/whole tubers 445-462 [46][39] Linamarin [34]
Gari flour (Nigeria) 10.6-22.1 [46] Linamarin [34]
Sorghum/whole immature plant 2400-2500 [46][39] Dhurrin [34]
Bamboo/immature shoot tip 7700-8000 [46][39] Taxiphyllin [63]
Lima beans from Java (coloured) 3000-3120 [46][39] Linmarin [56]
Lima beans from Puerto Rico (black) 2900-3000 [46][39] Linmarin [56]
Lima beans from Burma (white) 2000-2100 [46][39] Linmarin [56]
Flaxseed 910 [53] Linamarin, linustatin and neolinustatin [53]
* Amounts are averages, or are single examples. Actual levels may vary greatly between strains and growth conditions.

See also:

See Also

Additional Articles on MTF Wiki

External Resources

References

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