**5.1 Ethyl carbamate**

Ethyl carbamate (EC), also known as urethane, is an ethyl ester of carbamic acid that can be found in several fermented beverages [150] including wines [151]. EC levels in wines can range from n.d.-19 μg/L in white wine, n.d.-54 μg/L in red wine, 14–50 μg/L in fortified wine, and n.d.-58 μg/L in sherry-typewine [152]. EC is classified as a 'probable human carcinogen' by the IARC since 2007 (group 2A) [153]. Although currently there is no harmonised maximum level for EC, some countries have established their criteria for example in Canada the maximum level is 30 μg/L for wine, the Canadian guidelines were adopted by other countries such as Czech Republic, Brazil, France, Germany, and Switzerland. South Korea also set the maximum limit of 30 μg/L only for table wine. For fortified wine, the maximum level of

**189**

**Figure 2.**

*Wine Stabilisation: An Overview of Defects and Treatments*

EC is 100 μg/L in Canada and the Czech Republic, and of 60 μg/L in the US [154]. EC can be produced from at least five precursors, namely urea, citrulline, carbamyl phosphate, cyanic acid and diethyl pyrocarbonate. In turn, urea and citrulline can be respectively generated by yeast and LAB by metabolisation of arginine, a major amino acid found in grape juice and wine [155]. The fermentation conditions, such as pH, temperature, ethanol level, light irradiation, oxygen, storage time, yeast or LAB strains can also affect the formation of EC [156]. For example, lowering the temperature during fermentation and storage, lowering the pH, lowering the ethanol content, and addition of diammonium phosphate as a yeast nutritional supplement reduces the EC concentration. The development of techniques for EC elimination from alcoholic beverages [155] has attracted considerable attention, and enzymatic decomposition methods have been widely employed given their safety and environmentally friendly nature. Two enzymes are used, namely, urease, which is commercially available and can degrade urea, the major precursor of EC [157], and urethanase, which can directly catalyse EC degradation [158]. To reduce EC concentration in wine, the use of acid urease seems to be the most appropriate way to suppress EC formation [19, 159]. Moreover, the efficiency of commercial acid urease treatment varies with several factors, including pH, temperature, the presence, and concentration of inhibitors (malate, ethanol, phenolic compounds), and type of wine [157]. Therefore, the immobilisation of acid urease in chitosan beads enhances the protection against inhibitors, increases the stability of the enzyme, and has the advantage of facilitating enzyme recycling and consequently reducing the cost of its use. EC content can also be effectively reduced by decreasing the

*phenylethylamine; (d) tyramine; (e) putrescine; (f) cadaverine; (g) agmatine; (h) spermine; (i) spermidine;* 

*Main wine potentially toxic compounds. (I) EC; (II) BAs – (a) histamine; (b) tryptamine; (c)* 

*(j) methylamine; (k) dimethylamine; (m) ethylamine; (i) isopentylamine; (III) OTA; (IV) AB1.*

*DOI: http://dx.doi.org/10.5772/intechopen.95245*

#### **Figure 2.**

*Chemistry and Biochemistry of Winemaking, Wine Stabilization and Aging*

proteins used [141–144].

sub-qualities as well as the wine bitterness.

ing compounds during the fermentation process [149].

directly occurs from the precipitation of proanthocyanidins by these protein fining agents and it is influenced by the chemical characteristics of the protein used. The interactions between proanthocyanidins and protein fining agents depend on molecular weight, amino acid composition and surface charge density of the

Different proteins are used for wine fining such as gelatine, egg albumin, isinglass, and casein/potassium caseinate. Different types of gelatine remove different amounts of proanthocyanidins (9–16%) depending on the wine phenolic composition and structural characteristics of the proanthocyanidins and on the gelatine composition and characteristics [142, 143]. It has been generally thought that proteins bind primarily high polymerised tannins as well as high galloylated tannins, and therefore are preferentially removed [141], but some recent work showed that each of the different proteins (gelatine, egg albumin, isinglass, casein) and different size fractions of the same protein class interact differentially with different sizes of tannins [142, 143]. Regardless, allergen labelling may make wine fining with any of the animal-derived products impractical although some effort has been made to evaluate plant-derived proteins [144]. Recent studies of wine astringency demonstrated that tannins must be different two-fold for a trained panel to be able to successfully differentiate the wines [145]. Further, since some of the polymeric pigments can precipitate with protein there is the risk of losing stable colour [146]. As mentioned previously a higher astringency intensity is directly associated with a higher concentration of proanthocyanidins with a higher mean degree of polymerisation [147]. During ageing, astringency perception becomes softer, the reasons for the change in wine astringency could involve a decrease in proanthocyanidin concentration accompanied by a decrease in proanthocyanidins structural changes [148]. Therefore, the phenolic composition could be modulated during the winemaking steps (maceration/fermentation, stabilisation (fining) and ageing) and consequently, it allows the modulation of wine astringency and/or astringency

**5. Origin of potentially toxic compounds and strategies to improve wine** 

In fermented beverages in which a variety of microorganisms exist it may be inevitably the production of toxic products as a result of their metabolism and side reactions, including ethyl carbamate (I, **Figure 2**), biogenic amines (II, **Figure 2**) mycotoxins, namely ochratoxin A (III, **Figure 2**) and aflatoxin B1 (IV, **Figure 2**). They are generally generated due to the incomplete metabolism of nitrogen-contain-

Ethyl carbamate (EC), also known as urethane, is an ethyl ester of carbamic acid that can be found in several fermented beverages [150] including wines [151]. EC levels in wines can range from n.d.-19 μg/L in white wine, n.d.-54 μg/L in red wine, 14–50 μg/L in fortified wine, and n.d.-58 μg/L in sherry-typewine [152]. EC is classified as a 'probable human carcinogen' by the IARC since 2007 (group 2A) [153]. Although currently there is no harmonised maximum level for EC, some countries have established their criteria for example in Canada the maximum level is 30 μg/L for wine, the Canadian guidelines were adopted by other countries such as Czech Republic, Brazil, France, Germany, and Switzerland. South Korea also set the maximum limit of 30 μg/L only for table wine. For fortified wine, the maximum level of

**188**

**safety**

**5.1 Ethyl carbamate**

*Main wine potentially toxic compounds. (I) EC; (II) BAs – (a) histamine; (b) tryptamine; (c) phenylethylamine; (d) tyramine; (e) putrescine; (f) cadaverine; (g) agmatine; (h) spermine; (i) spermidine; (j) methylamine; (k) dimethylamine; (m) ethylamine; (i) isopentylamine; (III) OTA; (IV) AB1.*

EC is 100 μg/L in Canada and the Czech Republic, and of 60 μg/L in the US [154]. EC can be produced from at least five precursors, namely urea, citrulline, carbamyl phosphate, cyanic acid and diethyl pyrocarbonate. In turn, urea and citrulline can be respectively generated by yeast and LAB by metabolisation of arginine, a major amino acid found in grape juice and wine [155]. The fermentation conditions, such as pH, temperature, ethanol level, light irradiation, oxygen, storage time, yeast or LAB strains can also affect the formation of EC [156]. For example, lowering the temperature during fermentation and storage, lowering the pH, lowering the ethanol content, and addition of diammonium phosphate as a yeast nutritional supplement reduces the EC concentration. The development of techniques for EC elimination from alcoholic beverages [155] has attracted considerable attention, and enzymatic decomposition methods have been widely employed given their safety and environmentally friendly nature. Two enzymes are used, namely, urease, which is commercially available and can degrade urea, the major precursor of EC [157], and urethanase, which can directly catalyse EC degradation [158]. To reduce EC concentration in wine, the use of acid urease seems to be the most appropriate way to suppress EC formation [19, 159]. Moreover, the efficiency of commercial acid urease treatment varies with several factors, including pH, temperature, the presence, and concentration of inhibitors (malate, ethanol, phenolic compounds), and type of wine [157]. Therefore, the immobilisation of acid urease in chitosan beads enhances the protection against inhibitors, increases the stability of the enzyme, and has the advantage of facilitating enzyme recycling and consequently reducing the cost of its use. EC content can also be effectively reduced by decreasing the

generation of its precursors. Significant advances have been made via genetic technologies in modifying fermentation strains that produce less EC precursors. Genetic modification approaches have the potential to provide safe, affordable, and effective methods to decrease EC formation. Several studies have shown that the modification of catalytic enzymes, such as urea carboxylase, arginase, and allophanate hydrolase, showed the ability to reduce the concentration of EC [156]. Additionally, the modification of urea permease and amino acid permease, which are regulated by several factors and directly affect the generation of EC precursors have been explored [160]. Since the metabolism pathways related to urea have been fully considered for the high-efficiency minimisation of EC, enhancing the gene expression of DUR1,2 and DUR3, which encode urea degradation enzymes and permease, respectively, is considered to be a viable strategy. In this way, the modification of permease has led to the construction of functionally enhanced urea-importing wine yeast cells, which can continuously express the DUR3 gene and reduce EC level in Chardonnay wine by 81% [161].
