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

Abstract: Popular Abstract in English Freezing is a commonly used method of preserving food products including fruits and vegetables. A fresh-like texture and appealing visual appearance of frozen and thawed plant food products are of key importance for customer acceptance. Therefore, the food industry is constantly looking for ways to improve freezing techniques, especially for sensitive plant tissues such as rucola and spinach leaves. These sensitive plant tissues wilt after freezing and thawing due to cell membrane damage and collapse of the cell walls. Introducing a cryoprotectant – a compound such as sugar, which protects the plasma membrane during freezing, thus protecting the cells from freezing induced damage – into plant tissue has been reported to improve the quality of thawed/frozen spinach leaves. The method consists of two steps: vacuum impregnation (VI) followed by the application of a pulsed electric field (PEF). Vacuum impregnation is a technique in which the plant tissue is immersed in a solution of the cryoprotectant and then placed under vacuum. The pressure change causes the removal of the air present in the plant tissue, and its replacement by the solution. Applying an external electric field to the tissue creates pores in the plasma membrane. These pores may be reversible or irreversible, depending on the severity of the applied electric field. Once the pores are open, the cryoprotectant molecules can enter the cell. The combination of VI and PEF may allow more uniform impregnation of the cryoprotectant in the plant tissue. In this thesis the mechanisms governing the introduction of the sugars, used as cryoprotectants, into leaf tissue were studied. Leaves have a heterogeneous structure, made up of cells of different shapes and sizes, arranged in different layers. VI and PEF were optimized to obtain an uniform, and optimal distribution of the cryoprotectant within the complex tissue structure. The optimization of the processes was studied experimentally, using fluorescence microscopy and electrical measurements, as well as theoretically, using a three-dimensional model of the cross section of a leaf. The theoretical results predicted the experimental data well, and the model could thus be used to investigate the mechanisms of VI and PEF in the complex structure of plant tissue. Moreover, the metabolic effects resulting from the introduction of trehalose into the plant tissue were investigated. The results showed an increase in metabolic activity following VI and PEF. The effect of impregnating the leaf tissue with the cryoprotectant on the freezing of leaves was also investigated by monitoring the freezing process with a temperature-sensitive camera, showing that VI with a cryoprotectant followed by PEF influences the ice propagation rate and the freezing temperature of the leaves.