# Chemical Changes in the Broccoli Volatilome Depending on the Tissue Treatment
## 3 Discussion  
Freezing is a common practice both for the storage of vegetables for culinary purposes and for experiments in metabolite profiling or fingerprinting. Storing frozen vegetables in temperatures below 0 \({}^{\circ}\)C (\(-\)15 to \(-\)25 \({}^{\circ}\)C) is a way to preserve their nutritional value. Often, samples of vegetables for analytical purposes are also stored at this temperature range. Grinding plant tissues in liquid nitrogen is a popular and often used method prior to extracting metabolites. As shown in plant tissue, \"freezing\" and \"defrosting/thawing\" may have a significant impact on the volatilome. This indicates that in the case of living tissue, there is no possibility to compare results concerning fresh vegetables with results concerning frozen (\(-\)20 \({}^{\circ}\)C) and thawed vegetables due to the significant differences  
Figure 6: Influence of enzymatic reactions quenching in broccoli florets on extracted volatiles. Each bar graph is expressed as mean value \(\pm\) standard deviation. \({}^{\star}\)\(p\)\(\leq\) 0.05, \({}^{\star\star}\)\(p\)\(\leq\) 0.01, \({}^{\star\star}\)\(p\)\(\leq\) 0.001, \({}^{\star\star}\)\(p\)\(\leq\) 0.0001 (according to the ANOVA test—Turkey’s test). between the samples. The most relevant distinctions were observed in the LOX pathway metabolites and the glucosinolates and myrosinase system products. This problem is worth attention, considering the health benefits of isothiances consumption. The problem of methanethiol and dimethyl disulphide formation in crushed, homogenized, and frozen-thawed tissues of broccoli florets has been already investigated [14]. These volatiles were formed in crushed florets; however, their formation was inhibited in frozen-thawed tissues. The inhibition of DMDS production during the freeze-thawing cycle was explained as a result of a pH drop in the tissues, adherence of DMDS into the tissue surface, and a weakening of cysteine sulphoxide lyase activity under acidic conditions. With a tissue pH of 8.0 in crushed, homogenized, but most significantly in frozen-thawed tissues, DMDS dominated compared to tissues without a buffer [17]. It is known that the optimum pH for myrosinase is strongly dependent on its source. Myrosinase from Brussel sprouts was characterized by an optimum pH of 6.0-6.5, while myrosinase from white cabbage had an optimym pH of 8.0. For the enzyme from mustard or rapeseed, on the other hand, the maximum activity was noted in the pH range of 4.5-4.9 [18; 19]. In the case of broccoli, which was the subject of this study, the optimal pH was between 6.5 and 7 [20]. The experiment with protein separation confirms the hypothesis that nitriles do not have an autonomic hydrolytic enzyme activity. However, this step does not explain the source of the nitriles in the broccoli sample after freezing. Moreover, it does not support the hypothesis that glucosinolates are a source of a high nitrile concentration after freezing. In the sample preparation aspect of the study, it was shown that blending had a substantial influence on the increase of released volatile metabolites, especially isothi cyanates, aldehydes, and alcohols. As expected, the extract from more fragmented tissue gave a more abundant chromatogram. The damage of plant tissue leads to the release of a complex blend of volatiles responsible for the specific odour of disrupted plant tissue. \"Green leaf volatiles,\" which are mostly C6 aldehydes, alcohols, and esters, are products of the distribution of fatty acids by lipoxygenase [11]. Besides \"green\" aroma compounds, typical for Brassica vegetables, there are isothi cyanates and nitriles which are degradation products of glucosinolates [13]. The abundance of several groups of volatiles was monitored in fresh vegetables, vegetables with EDTA addition, vegetables frozen and ground in liquid nitrogen, and in cooked broccoli. In Figure 6, the presented chemical groups were selected based on the observed changes of peak areas after enzyme inhibition.