# Chemical Changes in the Broccoli Volatilome Depending on the Tissue Treatment
## 1 Introduction  
Broccoli is one of the most popular representatives of the _Brassicaceae_ family. Its beneficial properties are well known; they are mainly connected to the presence of bioactive sulphoraphane isothiocyanate, which is a product of glucoraphanin enzymatic hydrolysis. There are numerous examples of recent research proving bioactive properties of isothicyanates which suggest that _Brassica_ vegetables should be included in a daily diet [1, 2, 3].  
Like other plants, broccoli is a source of many other volatile components belonging to different chemical groups: alcohols, aldehydes, sulphides, esters, etc. [4]. Volatiles can be analysed by many available extraction techniques, among which is SPME. The SPME technique has already been used for the extraction of volatiles from plants, and was also proven to be very useful for in vivo studies on volatilome [5]. To the best of our knowledge, none of the studies on extraction and sample preparation techniques are focused on changes in such a complex matrix as plant tissue. SPME, especially in combination with comprehensive (two dimensional) gas chromatography and mass spectrometry (GC\(\times\)GC-ToFMS), gained special attention in profiling volatile compounds in sensomics, volatilomics, and flavoromics studies. This was due to an unrivalled peak capacity of GC\(\times\)GC systems, the spatial separation of peaks, which facilitates a better deconvolution and identification of compounds, especially when fast Time of Flight (ToF) mass spectrometers are used as detectors [6, 7, 8].  
There are some meaningful aspects which need to be considered when one works with fresh plant tissue that are rarely discussed in technique-oriented papers. The mainproblem is in obtaining a \"real\" profile of volatile compounds from fresh plants. Especially important for a flavour study is the instability of the volatile profile which is matrix related. This is mainly associated with the activity of enzymes, which take part in the generation and release of volatiles from plant tissues. Broccoli is an example of a plant known for its bitterness and specific, sharp odour, which are often factors related to consumers' acceptance of this vegetable as well as other _Brassica_[3; 9]. In broccoli, besides the typical odour-active molecules derived from C\({}_{18}\) fatty acids responsible for the typical \"green\" and \"grassy\" aroma [10; 11], there are also sulfur components present as a result of myrosinase and cysteine lyase activity, mainly nitriles and isothi cyanates [12; 13; 14; 15]. The profile of volatile compounds in broccoli (and other _Brassica_) is very dynamic, as it is formed mostly by enzymatic reactions, strongly dependent on environmental conditions (temperature, pH, presence of specific proteins), as well as physical conditions during sampling. These factors influence the overall profile of _Brassica_ volatiles [13].  
The goal of this paper was to evaluate changes in the volatilome of broccoli florets which may be a result of freezing as well as mechanical tissue disruption methods used for sample preparation, along with physica 1 and chemical methods to inhibit enzymatic changes. It was hypothesised that these factors substantially influence the profile of volatile compounds. It was monitored by means of the solid-phase microextraction (SPME) which was optimised for this type of matrix.