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.

Abstract (Swedish)

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.

• author: Dymek, Katarzyna
• supervisor: Federico Gomez; Petr Dejmek
• opponent: Professor Jäger, Henry, University of Natural Resources and Life Sciences, Vienna, Austria
• organization: Department of Food Technology, Engineering and Nutrition
• publishing date: 2015
• type: Thesis
• publication status: published
• subject: Food Engineering
• pages: 128 pages
• defense location: Lecture hall K:B, Centre for Chemistry and Chemical Engineering, Getingevägen 60, Lund University, Faculty of Engineering, LTH.
• defense date: 2015-02-13 10:15:00
• ISBN: 978-91-7422-384-2
• language: English
• LU publication?: yes
• id: f7d31e00-9370-4e88-96c4-6286fb6e644c (old id 4936425)
• date added to LUP: 2016-04-04 13:59:41
• date last changed: 2024-01-30 10:28:43