# Chemical Changes in the Broccoli Volatilome Depending on the Tissue Treatment
## 2 Results
### Enzyme Activity Assays  
The changes in volatile composition after freezing in liquid nitrogen or at \(-20\)\({}^{\circ}\)C could be caused by the activity of selected enzymes. In the case of broccoli, those changes were mainly related to nitrile and aldehyde concentrations. Therefore, special attention was placed on myrosinase and lipoxygenase. To check the myrosinase activity in broccoli after freezing, two trials were compared: fresh broccoli and broccoli after freezing in liquid nitrogen. The sinigrin solution was added to the enzyme extract from the vegetable because broccoli does not contain this glucosinolate; the detailed scheme is presented in Supplementary Materials, namely Figure S4. That is why broccoli could be a model matrix in which the hydrolysis of sinigrin (added to it) might be observed. The amount of glucosinolate hydrolysis products formed were measured using SPME. The zero-tests were extracts without the addition of sinigrin, in which no allyl isothiarate or alternative breakdown products of 3-butenonitrile sinigrin or the corresponding epitonitrile were noted.  
Figure 2: Microscopic images of broccoli tissue made in a plane perpendicular to the axis of the stem for three types of samples: (**a**,**d**) control sample, (**b**,**e**) sample subjected to rapid freezing in liquid nitrogen, (**c**,**f**) sample subjected to slow freezing (\(-20\)\({}^{\circ}\)C). Characteristic tissue damage that occurs during slow freezing of samples has been highlighted in a separate colour in part (**c**).  
A significant amount of allyl isothiocyanate was found in the test with the addition of sinigrim to the extract from the fresh broccoli (Figure 4A). In the sample after freezing in liquid nitrogen, the amount of this isothiamate was as high as in the non-frozen sample. In the next stage, in order to separate myrosinase from epitho-specific and nitrile-specific proteins whose mass does not exceed 50 kDa [16], AMICON filters were used to separate 100 kDa proteins, enabling separation of myrosinase (whose mass is about 200 kDa) from epithio-specific and nitrile-specific proteins. A significant amount of allyl isothiamate was formed after the addition of sinigrim to the upper layer containing  
Figure 3: Analysis mosaic of microscopic images of tissue made of broccoli in a plane perpendicular to the axis of the stem for three types of treatment samples. For analysis, the objects were divided into groups depending on the distance from the edge of the sample (the division into groups is shown in the colour maps in the upper part of the drawing). The graphs show the average values of the geometrical parameters of the cells within the designated groups, the horizontal axis indicates the distance in micrometres from the right edge of the image (from the stem epidermis).  
myrosinase (Figure 4B). The larger peak on the chromatogram was probably caused by a higher myrosinase concentration after filtration. On the other hand, after the addition of sinigrin to the lower layer, in which the nitrile-specifier proteins were present, no degradation products of glucosinolate were observed (Figure 4C).  
Figure 4: Chromatograms from enzyme activity assay experiment, presenting the amount of sinigrin degradation products-ally isothioxane. (**A**) the broccoli extract before proteins separation with sinigrin addition, (**B**) extract after separation (the upper layer, with myrosinase) with sinigrin addition, (**C**) extract after separation (containing nitrile-specifier proteins) with sinigrin addition.