The effectivity of vacuum impregnation with glycerol and secondary metabolites on electroporated arugula leaves to improve freezing stability.

Abstract: Freeze damage is a common issue in many food items. The cellular dehydration caused by freezing can be prevented through addition of a cryoprotectant. Research has shown that it is possible to improve the freezing tolerance of plant materials by addition of cryoprotectants, which forms hydrogen bonds with water molecules. However, the cryoprotect needs to be in the cell to function. Which is where vacuum impregnation can be applied. Vacuum impregnation is a method of transfecting substances through pressure fluctuations. As the pressure drops, gas will be forced out of the intracellular space and solutes can be transfected into the intracellular space until an equilibrium is formed. However, the cryoprotectant needs to be within the cell membrane, where the vacuole is. Small pores can be formed on the cell membrane by using pulsed electric field technology (PEF). Hydrophilic pores are formed in the lipid bilayer of a cell membrane when exposed to electric pulses. As a result, hydrophobic pores are formed in the cell membrane by spontaneous thermal fluctuations of membrane lipids. If the voltage is too high or the number of pulses is too high, the cell dies. In some cases, with the right parameters, reversible electroporation can be achieved. This means that the cell is able to recover to its original state, with a closed cell membrane. In this study, the effect of vacuum impregnation of arugula with glycerol solutions with secondary metabolites, in combination with PEF on the recovery rate, PEF survival rate, and freezing survival rate of arugula cells was studied. The electroporation causes solutes to leech out of the cell, which can be measured by measuring the resistance. 100 Hz was used to measure the resistance of the arugula. At higher frequencies, the electrical current goes through the cell, whereas with lower frequencies, the electrical current goes around the cell, where the leeched out solutes will reduce the resistance. This reduction in resistance can be picked up by measuring the resistance over time, and as the cell recovers, the solutes are taken up by the cell and the resistance increases again. Microscopy showed that bipolar electroporation with a pulse width of 1000µs, a pulse space of 20µs at 1000 V/cm with 1000 loops gave homogenous electroporation in arugula cells. Using these pulsed electric field and vacuum impregnation with different substances it has been shown that after only electroporation 40% of the leaves survived and recovery of the cells took 8 hours, no leaves survived the freezing experiment. Vacuum impregnation of different substances reduced the recovery time up to 3 hours and increased the freezing resistance from 0% to 10%.