Impregnation of Leaf Tissues and its Consequences on Metabolism and Freezing; Study on Vacuum Impregnation and Pulsed Electric Field Treatment

Abstract: Freezing is a widely used method of preserving food products. Efforts are currently being directed towards improving the quality of the sensitive tissues of plant foods such as leaves, after freezing and thawing. One of the methods under investigation is the combination of vacuum impregnation (VI) with cryoprotectants and the application of a pulsed electric field (PEF) to the plant tissue prior to freezing. The main aims of this work were to identify mechanisms for the efficient introduction of a cryoprotectant molecule into the heterogeneous structure of leaf tissue, and to improve our understanding of the consequences of the introduction of this foreign molecule into the tissue regarding cell metabolism, freezing point and ice propagation rate. Leaf tissue is characterized by a high degree of heterogeneity, being composed of cells of different shapes and sizes arranged in easily distinguishable layers interspersed with a branched network of air spaces. It is of key importance to obtain uniform and optimal impregnation of the cryoprotectant molecule in this complex structure. PEF parameters leading to the uniform electroporation of the leaf surface and the bulk tissue were determined experimentally using fluorescence microscopy and electrical resistance measurements, respectively. The results suggest that the level of electroporation is highly influenced by pulse polarity, the number of pulses, and the interval between pulses. To obtain precise information on the electroporation of internally located cells, a three-dimensional numerical model of the cross section of a leaf was developed. Models were constructed using representations of both the untreated and the vacuum impregnated leaf. The models were validated in the frequency domain, where alternating voltage and current at frequencies from 20 Hz to 1 MHz were used to measure the conductivity of the tissue. The models were also validated through measurements of current during electroporation, where a single 250 μs rectangular pulse with amplitudes ranging from 50 to 500 V was applied. Validation of the models showed that both the frequency-dependent conductivity and electroporation are well predicted. The importance of the wax layer and stomata in the model and the pore density in the membranes of specific internal tissues are thoroughly discussed. The metabolic consequences of VI and PEF treatment were explored. The results showed that VI, and the subsequent application of PEF, increased the metabolic activity. It was also shown that VI drastically decreased the porosity of the leaves. However, a small air fraction remained in the tissue, suggesting that the oxygen-consuming pathways are active in the cells after VI. The increase in metabolic activity after VI was accompanied by the accumulation of trehalose-6-phosphate in the cells. The influence of VI with different sugars and PEF on ice propagation rates and freezing temperature was investigated. Leaves impregnated with trehalose, sucrose, glucose and mannitol exhibited significantly lower ice propagation rates and higher freezing temperatures than untreated controls. Leaves subjected to PEF also showed higher freezing temperatures than untreated leaves; however, the ice propagation rate was not influenced by PEF. The combination of VI and PEF resulted in freezing temperatures and ice propagation rates comparable to those for leaves subjected to VI only.