# Chemical Changes in the Broccoli Volatilome Depending on the Tissue Treatment
## 2 Results
### The Influence of Freezing and Thawing on the Profile of Volatile Compounds  
The results of the GC\(\times\)GC-ToFMS analysis of fresh broccoli and the same vegetable after freezing and thawing are shown in Figure 1. The comparison of profiles of volatile compounds obtained from fresh broccoli with that of frozen and later thawed broccoli showed significant differences. In freshly cut florets, 3-hexen-1-ol, 1-hexanol dominated, and in the sample analysed after freezing and thawing, the greatest peak areas were observed for: hexanal, 2-hexanal, 2,4-heptdiagnenal, 1-penten-3-one, 1-pentanol, as well as 3-hexen-1-ol (however, in smaller amounts than in the fresh sample) and furan 2-ethyl (Table S1). The meaningful difference related to the LOX pathway metabolites (Figure 1a). There was a change in the proportion between aldehydes and alcohols in the volatile fraction. (Figure 1a) The experiment showed that in fresh vegetables, the main products were alcohols, which were metabolites in the LOX pathway, resulting from aldehydes reduction catalysed by alcohol dehydrogenase. While in frozen/thawed samples, the main products from the LOX pathway were aldehydes: hexanal, 3-hexenal, and 2-hexenal, among others (Table S1), which suggests that alcohol dehydrogenase is effectively stopped by low temperature and there is no catalysing reduction of aldehydes after thawing. According to applied statistics, a t-test was performed and \(p\) = 0.00005 was observed for alcohol concentration. No meaningful probability (\(p\) = 0.18) was noted for aldehydes which could be caused by a high standard deviation value (Figure 1, Table S4). The profile of compounds was the same regardless of the use of liquid nitrogen or freezing at \(-\)20 \({}^{\circ}\)C.  
Figure 1: Influence of freezing-thawing of broccoli florets on the amount of extracted (**a**) LOX derived and (**b**) myrosinase derived volatiles by SPME, compared to those extracted from fresh florets. Each bar graph is expressed as mean value \(\pm\) standard deviation. \({}^{*}\)\(p\)\(\leq\) 0.05, \({}^{**}\)\(p\)\(\leq\) 0.01, \({}^{***}\)\(p\)\(\leq\) 0.0001 (\(t\)-test). The difference between samples was significant regarding the products of glucosinolates degradation, as presented in Figure 1b. In fresh vegetables, the isothiyanates were the main products resulting from glucosinolates hydrolysis, while in frozen vegetables, the number of nitriles was growing rapidly compared to fresh tissue. The differences between isothiyanates and nitriles in fresh and freeze-thawed vegetables were significant in both cases \(p<0.05\) (isothiocyanates: \(p=0.02\); nitriles: \(p=0.003\), according to \(t\)-test results) (Figure 1, Table S4)  
Our results indicate that there are broader consequences of freezing-thawing on broccoli volatilome. Figure S1 (supplementary material), together with supplementary Table S1, show a more detailed insight into the profile of volatile compounds extracted from fresh and frozen-thawed broccoli florets. Some characteristic compounds for fresh and frozen-thawed broccoli tissues can be elucidated from the graph regarding peak areas (intensities) of these metabolites. For fresh broccoli, there are (Z)-3-hexene-1-ol (168), 1-hexenol (42), 3-ethyl-1, 5-octalienone (164), thiocyanic acid methyl ester (478), 1-butene, 4-isotiocyanate (36), and 3-hexenol-1-acetate (170). For frozen and thawed florets, the most abundant were 2-ethylfuran (354), 1-pentene-3-one (65), and 1-pentene-3-ol (64). The detailed list of the compounds differentiating both samples based on GC\(\times\)GC-ToFMS results was listed in supplemental materials as Table S1. Compounds (489 in total) are listed alphabetically and colour-coded, where the heat scale ranges from deep blue (0 intensity) to red (highest intensities). From the comprehensive GC\(\times\)GC-ToFMS data, initially, 768 features were compared for frozen-thawed and fresh broccoli. First, compounds including siloxanes, TMS derivatives, and perfluorinated compounds were discarded on the assumption that they were mainly column artifacts.