## 4 Discussion  
According to many previous studies, the anti-nutrient GSL profile progoitrin was widely detected in the seeds and sprouts of many broccoli cultivars [21; 27; 30]. In order to investigate the variations in GRA and GER as two major health-promoting GSL profiles in broccoli sprouts, the broccoli variety 1001 containing high levels of GRA and GER with progoitrin absent in its seeds [20] was used in this work. The results on the variation in GRA and GER in the broccoli sprouts were basically consistent with earlier studies [31; 32; 33]. The substantial decrease in the content of GRA and GER from 3 d to 6 d was observed in this study and a previous report [34]. [PERSON] et al. (2008) reported a similar trend in both light and dark conditions, with a general reduction in the concentration of aliphatic GSLs during a 7 d period [35]. [PERSON] et al. (2008) found the GRA loss from 2 d to 7 d in broccoli sprouts (dry weight) was 38% for cv. [PERSON], 15% for cv. [PERSON], and 25% for cv. [PERSON] [23]. In _Arabidopsis_, from 2 d to 8 d of seed-to-seedling development, the total GSL content per individual decreased by 30%, indicating GSL turnover [36]. Our previous study on Chinese kale sprouts also demonstrated significant variations in the total GSL content per plant as the sprouts developed [24]. All in all, the fresh broccoli sprouts (3 d-7 d) contained lower concentrations of GRA and GER compared to the germinating seeds (1 d), even though the older seedlings (5 d-7 d) had the potential to produce more GRA and GER than the early-aged seedlings (1 d-3 d). It has been reported recently that sulfur sources in aliphatic GSLs are reallocated by BGLU28 and BGLU30 under sulfur deficiency in the process of seed germination, particularly during early development in Arabidopsis [37]. Therefore, the main loss of GRA and GER may be a consequence of sulfur reallocation during the early stages of seed germination. We found that the concentrations of the breakdown products were much lower compared to the concentrations of GRA and GER at the same time point, and nitriles were the main breakdown products in broccoli seedlings. For consumers, it would be a better choice to intake sprouts treated with boiling water instead of fresh sprouts directly, when GRA and GER can be converted into ITCs by the gut microbes or there is extra intake of TGGs. [PERSON] and [PERSON] (2017) also reported the contents of sulforaphane were no more than 1.2 umol/g in 7-9-day-old seedlings from three varieties [22]. Moreover, they found the levels of sulforaphane nitrile and erucin nitrile were higher than the corresponding ITCs, especially in Marathon and Iron Man. Similarly, [PERSON] et al. (2008) found the primary breakdown product of GRA was sulforaphane nitrile in broccoli seedlings of the Calabrese cultivar from 2 d to 7 d [23]. And the highest level of sulforaphane was lower than 1.5 umol/g in fresh broccoli sprouts. They also observed a general decrease in the sulforaphane and sulforaphane nitrile concentrations during the first 7 days of seedling development based on fresh weight. However, [PERSON] et al. (2012) noted that the content of sulforaphane declined sharply during the first day of seed germination based on dry weight and then increased to the same level as that in the seeds before decreasing again [38]. [PERSON] et al. (2016) suggested that breakdown product analysis was best conducted with fresh plant material, as specifier-protein-mediated product formation was sensitive to freezing [4]. In addition, we found that sulforaphane/sulforaphane nitrile was different from erucin/erucin nitrile during seedling development, especially during seed germination. One possible reason is that the ESPs and NSPs have different binding affinity energies and affinity constants to GRA and GER [39]. In our study, the fresh sprouts were ground in water without adjusting the pH, nor adding other chemicals.