predominant glucosinolates in mustard seeds; <sup>a</sup> aliphatic olefin, <sup>b</sup> aromatic aryl, <sup>c</sup> S-containing, <sup>d</sup> aliphatic branched chain, <sup>e</sup> aromatic indol.


#### **Table 2.** Glucosinolate content in mustard species.

DW dry weight, FW fresh weight.

Sinalbin and sinigrin whose chemical structure was elucidated by Ettlinger and Lundeen [48,49] belong to the main glucosinolates in mustard seeds. In brown mustard (*B. juncea*) and black mustard (*B. nigra*) seeds sinigrin is the predominant compound degrading to allyl-isothiocyanate (AITC) after hydrolysis, whereas, the main glucosinolate of white mustard (*S. alba*) is sinalbin yielding 4-hydroxybenzyl isothiocyanate (29) (Figure 3).

The concentration of glucosinolates and their breakdown products in mustard plants, especially in the seeds, is largely dependent on the activity of myrosinase or related bacterial enzymes or further chemical transformation prior to consumption [26].

In case of mustard as food one has to distinguish between the intake of whole notprocessed seeds as seasoning in several dishes, and processed seeds, for example as mustard flour made from ground, peeled and non-degreased seeds or as oil made by pressing the seeds. Prepared mustard is mostly composed of a mixture of ground mustard seed and/or mustard flour and/or mustard cake [9,10].

In general, glucosinolates are relatively stable in the seeds until cell damage results in chemical degradation involving myrosinase-catalysed hydrolysis. Thermal treatment leads to inactivation of myrosinase via denaturation [50] and preserve most of the glucosinolates. Van Eylen et al. showed that the optimum temperature of myrosinase activity of mustard seeds from *S. alba* was at 60 ◦C, however the activity decreased when the temperature of the experimental system was higher and was influenced by several other factors such as pH or presence of ascorbic acid [51].

**Figure 3.** Main glucosinolates and corresponding hydrolysis products in brown (*B. juncea*), black (*B. nigra*) and white (*S. alba*) mustard seeds.

This occurs for example when whole mustard seeds are added as spice to dishes which are cooked, like soups or stews. In this case, intact glucosinolates might reach the colon, however a certain amount of the glucosinolates seem to be hydrolysed in the stomach or can be absorbed via passive transport or via diffusion in the small intestine. In the colon, several bacteria strains are able to hydrolyse glucosinolates to form isothiocyanates, amines or nitriles depending on the type of bacterial myrosinase-like activity. Usually, cooking reduces the concentration of glucosinolates, partly through thermal breakdown and partly to leaching of the intact glucosinolates and their derivatives into the cooking liquid. Besides, heat treatment inhibits the activity of myrosinase through denaturation of the enzyme [52,53]. Therefore, the processing method can make a very large difference, both to the intake of glucosinolates, and to the bioavailability of their breakdown products.

The pattern of intact glucosinolates and breakdown products upon heat treatment is influenced by the thermal stability of the corresponding molecules. For instance, indol glucosinolates—one of the best-known representatives is progoitrin—seem to be less stable than aliphatic ones such as sinigrin, which is the predominant glucosinolate in black and brown mustard seeds. However, sinigrin totally disappears upon boiling, while 5-vinyloxazolidine-2 thione (derived from progoitrin) and 3-methyl-sulphinylpropylisothiocyanate (derived from glucoiberin) may partly escape decomposition [54].

Comparison between different mustard seed preparations showed that the seed alone had higher sinigrin and lower content of the breakdown product isothiocyanate compared to mustard preparations, whereas the wholegrain-style mustard contained lower sinigrin and higher isothiocyanate levels. This suggests that during mustard preparation, the enzyme myrosinase can break down sinigrin in the presence of high salinity and acidity from added ingredients, such as salt or vinegar [55]. Isothiocyanates are volatile and water-soluble. It can be expected that they largely disappear after processing steps using high temperatures and soaking in water, but those processes are commonly not included during mustard preparation.

Interestingly, in a thesis, it was shown that beside allyl-isothiocyanate derived from sinigrin, sulforaphane derived from glucoraphanin also corresponds to the most abundant breakdown product detected in different samples of table mustard. In this case,

concentration of allyl-isothiocyanates ranged from 4 to above 200 mg/100 g fresh weight whereas sulforaphane ranges from 24 to above 188 mg/kg. These unexpected results of sulforaphane quantification may be explained by the fact that table mustard is a mixture and probably also contain glucoraphanin-containing ingredients [56].

Consumption of mustard seeds, either whole or after preparation, may lead to the intake of intact glucosinolates as well as their different breakdown products. One has to note, that the pungent taste of different prepared mustard samples strongly depends on levels of allyl-isothiocyanates [57]. The dietary intake of glucosinolates in general have been reported ranging from 2 to 29 mg/day depending on population groups and countries [58]. However, dietary intake of glucosinolates through the consumption of mustard as well as exposure data on different types of glucosinolates, especially of sinigrin and sinalbin, and their corresponding breakdown products such as allyl-isothiocyanates, are not available.

#### 2.1.1. Toxicological and Antinutritional Effects

Many plants of the Brassicaceae family containing glucosinolates are used for human and animal nutrition. Glucosinolates themselves are biologically inactive, however various breakdown products exert a variety of toxicological and antinutritional effects in animals whereas the information on adverse effects in humans is limited.

Numerous studies confirm the negative effects that occur in animals after feeding of glucosinolate-containing rapeseed cake (de-oiled seeds). After oil extraction from oilseed crops, such as rapeseed (*B. napus)* or white mustard (*S. alba)*, the hydrophilic glucosinolates remain in the seed meal fraction (by-product) used in animal diets [25]. The presence of the glucosinolates sinigrin and progoitrin in the diet is strongly associated with bitter taste and is responsible for reduced palatability corresponding to a lower intake of glucosinolate containing animal diets [26,59].

Although progoitrin is a non-bitter glucosinolate, it produces more profound bitter taste compared to sinigrin due its degradation to the extremely bitter compound goitrin by myrosinase or by heat treatment [60]. In Annex I of Directive 2002/32/EC, vinyl thiooxazolidone (5-vinyloxazolidine-2-thione (5-VOT), known as goitrin) and volatile mustard oil expressed as allyl-isothiocyanate are listed as undesirable substances with maximal amounts for animal feed. Humans are also sensitive to the strong flavours of glucosinolate breakdown products, and these compounds are therefore important determinants of intentionally or unintentionally flavour [61].

The main toxic effects in animals can be described as decreased feed intake, growth depression, enlargement of liver and kidneys, structural and functional alteration of the thyroid gland, and reprotoxic effects such as embryo mortality in mammals and reduced egg production in birds, amongst which the adverse effects on thyroid metabolism are the most thoroughly studied. Even though the extent of the detrimental effects both on productivity and on health vary within the animal species and the amount of glucosinolates, it is suggested that the formation of isothiocyanates, thiocyanates, oxazolidinethiones and nitriles from glucosinolates strongly contribute to the observed toxicological effects [62,63].
