Synergistic Effect of Ultrasonication on Antimicrobial Activity of Cecropin P1 Against Escherichia Coli

Maya Fitriyanti, Purdue University

Abstract

Ultrasound has recently been used in the food industry to develop various effective and reliable processing applications such as extraction of intracellular material and desinfection. Antimicrobial peptides are one of the most promising alternatives to antibiotics for targeting pathogens without developing resistance. Recent studies have shown that both low frequency (20-100 kHz) ultrasonication and antimicrobial peptides (AMPs) treatment processes have a significant advantage in inactivating bacterial cells than the conventional heat treatment due to higher food texture quality of the final product. However, the effect of the combined process has not been fully investigated in complex matrices such as food. In this study, deactivation of Escherichia coli in different concentrations of milk and orange juice were performed using three different treatments: low frequency ultrasonication (20 kHz) at different power levels using a commercial probe type ultrasonicator, antimicrobial peptide Cecropin P1, and combination of both. The results of all samples showed that the combined treatment is more efficient, reducing the cell density of E. coliup to four orders of magnitude, compared to individual treatments. However, the milk concentration results in lower synergistic effect. This is believed to be due to complexation of milk proteins with Cecropin P1 thus resulting in less availability of the latter for antimicrobial action. This dependence was not observed in orange juice samples. Ultrasonication resulted in insignificant decrease in viscosity, total color difference (TCD), and vitamin C for both milk and orange juice except at higher power level of 160 W at longer exposure time (60 min). In the second part of the study, pore formation in 1,2-Dimyristoyl-snglycero-3-phosphocholine (DMPC)/cholesterol liposome induced by Cecropin P1 was investigated by monitoring the dynamics of fluorescence dye leakage. A critical peptide concentration was required for dye leakage with the rate of leakage being dependent on peptide concentration above a critical value. A lag time was required for dye leakage for low peptide concentrations, which decreased at sufficiently higher peptide concentrations eventually approaching zero. Size distribution of liposomes exposed to peptides of different concentrations indicated that toroidal pore formation with accompanied stretching of liposomes may have occurred at low peptide concentrations. At much higher peptide concentrations, however, pore formation may be due to combined action of toroidal pores and solubilization of lipids by peptides into micelles that is consistent with carpet mechanism. In the third study, we investigated the synergistic effect of ultrasonication and antimicrobial action of Cecropin P1 using a batch and continuous cylindrical ultrasonic processing system. The deactivation of E. coliin PBS (pH 7.4) were performed using three different treatments: ultrasound (22 kHz) at different power levels (1 - 8 watts) and different exposure times (5, 10, and 15 minutes), Cecropin P1 (20 µg/ml), and combination of both. The results showed that the combined treatment at higher power level (8 watts) for 15 minutes is more efficient, reducing the cell density to six orders of magnitude, compared to individual treatments. Our results on the effect of different frequencies (14, 22, and 47 kHz) also shown that combination of higher frequency (47 kHz) and Cecropin P1 for one minute of exposure time were able to deactivate more cells (up to six orders of magnitude) compare to combined treatment with 14 and 22 KHz ultrasound for one minute. Continuous flow ultrasonic processing system using this cylindrical transducer of 22 kHz with power level of 7 W and 7.5 W also resulted in cell reduction up to four orders of magnitude for residence time of 15 min and up to five orders of magnitude for residence time of 34 min respectively. A mathematical model for the description of interaction of antimicrobial peptide with a lipid bilayer in the presence of ultrasonication is presented. The model considers the growth and collapse of bubbles created by cavitation. The interaction of pressure waves created by bubble collapse with lipid bilayer leading to the formation and growth of pores in the absence as well as in the presence of antimicrobial peptides are described to demonstrate synergistic action. The model is able to predict the effects of pressure amplitude, sonication frequency, surface tension, physical properties of the bilayer such as line tension, bending modulus and physical properties of antimicrobial peptide such as net charge and hydrophobicity. The time of disintegration of phospholipids leading to pore formation is found to be smaller at higher pressure amplitudes, lower line tensions, higher surface tensions and higher frequencies. The destabilization of pores due to pressure fluctuation by antimicrobial peptides is mainly due to electrostatic interactions in the pore lined with proteins. The model is also able to predict deactivation of bacterial cells as a result of pore formation due to pressure waves created by ultrasonication.

Degree

Ph.D.

Advisors

Narsimhan, Purdue University.

Subject Area

Energy|Acoustics|Food Science|Microbiology|Pharmaceutical sciences

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