# Antimicrobial Activity of L-Lysine and Poly-L-Lysine with Pulsed Electric Fields
## 4 Discussion  
Pulsed electric fields are commonly applied for microbial inactivation in food processing [31]. The use of PEF with antimicrobial components could lead to new developments for surface infections treatments [34,44]. In this work, we have investigated inactivation of microorganisms by combining Lys and PLL with PEF treatment. Four pathogenic microorganisms with different morphologies and membrane structures were used. Two fungal pathogens, _T. rubrum_ and _C. albicans_, and two bacterial pathogens, _S. aureus_ and _E. coli_ were used. Generally, the susceptibility to electroporation depends on the biological object. Usually, bacteria show higher resistance to PEF when compared to other microorganisms like eukaryotes [45], requiring higher energy/PEF amplitude procedures [46]. Differences in susceptibility were also observed in our studies. The keratinophilic fungus known as dermatophyte _T. rubrum_ was the most susceptible, followed by the Gram-positive bacteria _S. aureus_ and human pathogenic yeast _C. albicans_. Though, the Gram-negative bacteria _E. coli_ was the most resistant demanding the highest energy burst for successful inactivation. Nevertheless, the energy can be controlled by variation of many parameters such as PEF amplitude, number of pulses or the pulsed current. In our study, two amplitudes of 15 and 30 kV/cm were selected and the effect of the number of pulses and pulse duration on the treatment outcome was analyzed. According to our results, higher PEF amplitude resulted in better treatment efficiency using both Lys and PLL. However, to demonstrate the importance of polarization and the effects of electrotransfer on inactivation efficiency various pulse durations were analyzed. It was observed that sub-microsecond pulses show the weakest response of Lys and PLL treatment for studied microorganisms. However, longer pulse duration significantly improved the efficiency of the treatment for all pathogenic microorganisms when the same energy burst was used. The results showed that longer pulses ensure better electrotransfer of Lys and PLL, which can be explained by better pore stability [47] and higher electrophoretic force [48]. In our work, the susceptibility of the microorganisms to Lys and PLL was also studied showing that the Gram-negative bacteria _E. coli_ was the most resistant in both cases. According to several studies [49,50], the mechanism of well-known \(\epsilon\)-PLL and PLL were considered to be similar based on their similar chemical properties. Possibly, positively charged L-Lys amino acid and PLL could electrostatically interact with neutral or negatively charged microorganism membrane systems. [PERSON]'s group [50] observed that the cationic polypeptide could interact with negatively charged cell surface by ionic adsorption inducing the stripping of the lipopolysaccharide-acrylate (LPS) layer, which could increase permeabilization of the outer membrane and lead to rapid cell death. Probably, the cationic amino groups of Lys and PLL interact with the outer membrane of Gram-negative bacteria containing hydrophilic and negatively charged LPS. However, the Gram-negative microorganism membrane is composed of two membranes and a thin layer of peptidoglycan between them, leading to a more resistant structure for the positively charged organic molecules diffusion. For the Gram-positive bacteria, the negative charges are supplied by carboxylate, phosphate or sulphate groups of anionic polymers called teichoic acids. Recently, [PERSON] et al. [51] reported that peptidoglycan structure from the Gram-positive _S. aureus_ can be affected by \(\epsilon\)-PLL. This observation could indicate that Lys and PLL can induce damage through the peptidoglycan layer making the cell wall more fragile. PEF technology can be used synergistically with heat, antimicrobial agents, membrane filtration and ultraviolet radiation to increase the effectiveness of bacterial inactivation prolonging the period of consumption.