Abstract
This thesis aimed to obtain an extensive understanding of the mechanism by which pulsed electric fields (PEF) at low electric field strengths (E ≤ 2 kV/cm) affect the structure and functionality of intact onions. A few studies have investigated the effect of PEF treatment on intact tubers, but little information is known for a complex and multicellular plant organ, made up of layers. Therefore, in this study, onion bulbs and spring onions were chosen as a model system, to understand the PEF induced changes in the tissue integrity and some physiological responses.
The impact of PEF treatment on onion tissue integrity was studied based on ion leakage measurements as well as cell viability staining techniques. PEF treatment (0.3 – 1.8 kV/cm, 0.5 kJ/kg) significantly (P < 0.05) increased the ion leakage (conductivity) in the incubation medium as electric field strength increased. The ion leakage measurements determined in the individual scales of bulb and spring onions indicated that the outer tissue scales were more susceptible to PEF treatment compared to the inner tissue scales due to the positional differences between the scales, with outer scale protecting the inner scales. A similar result was obtained using a cell viability assay. Onion cells present in the outer tissue scales showed more damage as visualised by the amount of cell death, compared to the cells present in the inner tissue region. This indicates that the application of PEF treatment on onion bulbs results in non-homogenous and complex changes in onion structure and cell viability.
This study attempted to identify the potential of volatile compounds as markers of cell membrane damage, using gas chromatography-mass spectrometry (GC-MS) and proton transfer reaction–mass spectrometry (PTR-MS). The changes in the volatile concentrations corresponded to biochemical changes associated with PEF treatment and during storage time. PEF treatment (0.3 – 1.2 kV/cm, 5 kJ/kg) significantly increased the concentrations of propanethial s-oxide (PSO, lachrymatory factor), propenyl propane thiosulfinate (PrPthiosulfinate), 2-methyl-2-pentenal and the disulfides (dipropyl disulfide, propenyl propyl disulfide, methyl propyl disulfide and methyl propenyl disulfide), immediately following PEF treatment (T0), compared to the control (non-PEF treated) samples. In addition, the effect of PEF treatment on the volatile concentrations was much higher after 24 hr of storage at 4 °C (T24). The concentrations of volatile sulfur compounds (such as PrPthiosulfinate, disulfides and trisulfides) increased significantly (P < 0.05), compared to control and sample concentrations detected at T0. The increase in the concentrations of volatile compounds at T0 and T24 was found to be dependent on the applied electric field strength. The maximum concentration (Cmax) of volatile compounds produced in spring onions (Ishikura and Red Bunching) were obtained upon PEF treatment at 1.2 kV/cm whereas, in the Yellow sweet Spanish bulbs, the Cmax was obtained upon PEF treatment at 0.7 kV/cm. The dynamic changes in volatile concentrations were investigated in the spring onions as a function of time following PEF treatment using PTR-MS. The Cmax of the mass ions measured for target volatile compounds and the rate at which volatile concentrations reached steady state were found to be based on the applied electric field strength, significantly different to the control samples. The mass ions were found to reach steady state at different time points, reaching their maximum concentrations either at the start or the end of the analysis (120 min). The results demonstrated that the volatile kinetic trends were due to the relative position of the volatile compound in the onion enzymatic reaction cascade, where the mass ions measured for target compounds of interest were found to either increase or decrease over time to reach completion. Overall, it was found that the concentrations of volatile compounds were predominantly linked to the degree of enzyme – substrate mixing, which corresponded to the applied electric field strength. However, some exceptions were found in the products formed through a mixed chemical reaction cascades, showcasing a different trend. These results suggested that the changes in the onion volatile concentrations were not only correlated to the enzyme initiated reaction cascade but also to physiological responses.
Investigation of cell viability following PEF treatment indicated that there were differences in the degree of cell disruption across the onion tissue regions, based on the applied electric field strength. The cells present in the outer tissue region were dead, and the cells that make the scale of the central core were found to be viable. To evaluate the effect of PEF treatment intensities on the volatile compositions according to the cellular disruption, the volatile compounds were analysed in the inner and outer tissue regions of spring onions. The results showed three different trends based on the concentrations detected in the onion tissue regions, indicating the differences in the physiological response from the onion tissue regions. To further comprehend these volatile trends, several biomarkers related to oxidative damage and antioxidant markers were measured to understand the degree of cell damage.
The effect of PEF treatment at 0.3 kV/cm was found to damage the cells present in the outer tissue region, but the inner tissue region was found to be unaffected. In the outer tissue region, the levels of antioxidant enzymes (SOD, CAT, GPOX, and GR) activities were found to increase significantly. However, no significant changes in the levels of oxidative damage (protein carbonyls and lipid peroxides) markers were observed in the outer tissue region. Volatile compounds such as methyl propenyl disulfide (MPrDS), propyl propenyl trisulfide (PPrTS) and methyl propyl trisulfide (MPTS) were produced in higher concentrations in the inner tissue region, and are suspected to be associated with the physiological response from the viable cells. These results indicate that the metabolically active cells are synthesising new proteins to counteract the oxidative stress. These results suggest that upon PEF induced stress, living cells can change their metabolism to prevent oxidative damage to the cellular components caused by the reactive oxygen species (ROS). In contrast, application of PEF treatment at 0.7 kV/cm resulted in significant accumulation of damage markers and a significant reduction of antioxidant enzyme activities, in both inner and outer tissue regions. This result indicates that the PEF treatment has resulted in extensive cellular disruption in both onion tissue regions, causing oxidation of lipids and denaturation of proteins and enzymes. The volatile compounds such as propanethial s-oxide (PSO), dipropyl disulfide (DPDS), methyl propyl disulfide (MPDS), propyl propenyl disulfide (PPrDS) and dipropyl trisulfide (DPTS), which are associated with cell damage were detected in higher concentrations in the outer tissue region and relatively lower concentrations in the inner tissue region.
This study has demonstrated that the overall PEF induced changes in the structure and physiological function of intact onions could be assessed by evaluating the markers associated with cellular damage and biochemical analysis. Evaluation of the volatile compounds produced in onion tissues makes a unique contribution to the current knowledge in understanding the properties of PEF treated fruit and vegetable tissues. From an industrial point of view, understanding these complex responses will aid in tailoring the activities of antioxidant enzymes, enhanced recovery of phytochemicals, improved texture, flavour and bio-active properties of fruits and vegetables.