1

Critical synergistic concentration of lecithin phospholipids improve the antimicrobial 2

activity of eugenol against Escherichia coli 3

4

Haoshu Zhanga, Edward G. Dudley

a, b, and Federico Harte

#a 5

a Department of Food Science and

b Center for Molecular Immunology and Infectious Disease, 6

Pennsylvania State University, University Park, PA, USA 7

8

# Address correspondence to Federico Harte, fede@psu.edu 9

AEM Accepted Manuscript Posted Online 25 August 2017Appl. Environ. Microbiol. doi:10.1128/AEM.01583-17Copyright © 2017 American Society for Microbiology. All Rights Reserved.

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Abstract 10

In this study, the effect of individual lecithin phospholipids on the antimicrobial 11

properties of eugenol against E. coli C600 was investigated. We tested five major phospholipids 12

common in soy or egg lecithin (DPPC, DSPC, DPPE, DPPA and DPPS) and one synthetic 13

cationic phospholipid (EPC 18:0). Among six phospholipids, DPPC, DSPC, DPPE, DPPA, and 14

the cationic 18:0 EPC showed critical synergistic concentrations that significantly improve the 15

inactivation effect of eugenol against E. coli after 30 min of exposure. At the critical synergistic 16

concentration, an additional ca. 0.4-1.9 log reduction (ca. 0.66-2.17 log CFU/mL reduction) in 17

microbial population was observed when compared to eugenol-only (control) treatments (ca. 18

0.25 log reduction). In all cases, increasing the phospholipid amount above the critical 19

synergistic concentration (different for each phospholipid) resulted in antimicrobial properties 20

similar to eugenol-only (control) treatments. DPPS did not affect the antimicrobial properties of 21

eugenol at the tested concentrations. The critical synergistic concentration of phospholipids was 22

correlated to their critical micelle concentrations (CMC). 23

Importance 24

Essential oils (EOs) are naturally occurring antimicrobials, with limited use in food due to their 25

hydrophobicity and strong aroma. Lecithin is used as a natural emulsifier to stabilize EOs in 26

aqueous systems. We previously demonstrated that within a narrow critical concentration 27

window, lecithin can synergistically enhance the antimicrobial properties of eugenol. Since 28

lecithin is a mixture of different phospholipids, we aimed to identify which phospholipids are 29

crucial for the observed synergistic effect. This research studies the bioactivity of lecithin 30

phospholipids, contributing to a rational design when using lecithin to effectively control 31

foodborne pathogens in foods. 32

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Introduction 33

In United State alone, foodborne diseases cause ca. 48 million illnesses, 128,000 34

hospitalizations and ca. 3,000 death every year (1). Among all the foodborne disease agents, the 35

World Health Organization (WHO) reports that pathogenic E. coli significantly contributes to 36

this burden (2). The Centers for Disease Control and Prevention (CDC) estimated 265,000 shiga-37

toxin-producing E. coli (STEC) infections, 3,600 U.S. hospitalizations and 30 deaths every year 38

(3). 39

Essential oils (EOs) extracted from plants (e.g., eugenol, carvacrol, thymol) continue to 40

gain attention as natural antimicrobials due to their ‘green’ and ‘clean label’ image (4). EOs are 41

generally recognized as safe (GRAS) but found limited use in food systems due to their 42

hydrophobicity and strong aroma. Many studies showed that after encapsulating EOs in 43

emulsifier micelles or liposomes, both the stability and the antimicrobial properties of the EO 44

improve (4-6). Donsì and co-workers found an increase in D-limonene antimicrobial activity 45

against Lactobacillus delbrueckii and Saccharomyces cerevisiae when encapsulated in the 46

emulsifiers Tween 20 and glycerol monooleate. Lecithin has been widely used in foods (e.g., 47

infant formula and chocolate) as a natural emulsifier (7) to improve physical stability. However, 48

previous reports showed that the antimicrobial properties of EO against E. coli were not 49

enhanced when EOs were combined with lecithin (8, 9) or observed that water-insoluble 50

surfactants (including lecithin) in nanoemulsions diminished the antimicrobial efficacy of EOs 51

(10). On the contrary, we previously reported that the antimicrobial properties of eugenol were 52

significantly improved when lecithin was added at a critical low concentration (11). Nanoscale 53

aggregates (<50 nm) were observed when eugenol was mixed with critical synergistic 54

concentration of lecithin and it was suggested that electrostatic interactions between charged 55

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phospholipids and the negatively charged bacterial cell membrane were responsible of the 56

improved antimicrobial properties. 57

Lecithin is a mixture of various phospholipids including phosphatidylcholine (PC), 58

phosphatidylethanolamine (PE), phosphatidic acid (PA) and phosphatidylserine (PS) (7, 12). Due 59

to the amphiphilic nature of these phospholipids, lamellar aggregates and other structures (e.g., 60

hexagonally arranged rods and tubes) have been reported to form depending on concentration 61

and temperature (13-15). Lecithin phospholipids differ in charged head groups (Table 1), lipid 62

chain length, and degree of saturation (16), resulting in distinct molecular conformations, CMC, 63

and phase transition behaviors (15, 17). Moreover, phospholipids are major components of 64

bacterial cell membrane (18) and previous reports showed that the interaction between bacterial 65

cell and essential oil depended on the cell membrane phospholipid composition (19). Therefore, 66

we hypothesize that not all phospholipids in lecithin are responsible for the enhanced 67

antimicrobial activity of essential oil against bacteria. The aim of this study was to identify the 68

effect of six single phospholipids on the antimicrobial effect of eugenol against E. coli C600, 69

leading to a rational use of phospholipid-based natural antimicrobial systems. 70

Materials and methods 71

Phospholipids 72

From the myriad phospholipids in lecithin, we selected DPPC (1,2-dipalmitoyl-sn-73

glycero-3-phosphocholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DSPC 74

(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPA (1,2-dipalmitoyl-sn-glycero-3-phosphate 75

sodium salt) and DPPS (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine sodium salt) (Avanti, 76

Alabaster, AL; Table 1) to conduct further experiments on the basis of being the major 77

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components in both soy and egg lecithin. The cationic 18:0 EPC (1,2-distearoyl-sn-glycero-3-78

ethylphosphocholine chloride salt) was also obtained from Avanti Polar Lipids, Inc to study the 79

effect of phospholipid charge on the synergistic effect when combined with eugenol. 80

Bacterial strain 81

E. coli C600 (20) was obtained from the Yale E. coli Genetic Stock Center. It is a 82

prophage-cured derivative of E. coli K-12 (21), that showed a positive response to eugenol-83

lecithin buffered systems in a previous study (11). After growing in tryptic soy broth (TSB; 84

Difco, Sparks, MD) under aerobic conditions at 37⁰C for 24 hours, a glycerol stock was made by 85

mixing 1 mL culture aliquots with 0.3 mL 50% v/v glycerol in water solution. The stock was 86

stored in a 2 mL centrifuge tube at -80⁰C until needed for experiments. To make a working 87

culture, the stock culture was grown in TSB under the same condition as previously described 88

until the optical density at 610nm (OD610) reached ca. 0.9, monitored using a DU730 89

spectrophotometer (Beckman Coulter, Pasadena, CA). 90

Eugenol-Phospholipid Preparations 91

The procedure to prepare the eugenol-phospholipid samples followed our previous 92

method (11) with minor modifications. Ten mg/100mL phospholipid stock solutions were 93

prepared by boiling 10 mg each phospholipid in 100mL phosphate buffered saline (PBS; pH ca. 94

7.20) for 7 min and then homogenizing (Ultra-Turrax IKA T-18 basic, Staufen, Germany) at 95

11,000 rpm for 1 min. The solution was then cooled to room temperature and sterile deionized 96

water was added to bring the solution back to 100mL. The stock solution was diluted in PBS 97

subsequently to reach the final phospholipid concentration ranging from 0 to 80 μM. A 0.043% 98

v/v eugenol (previously shown to reduce the population of E. coli C600 at least 0.2 log CFU/mL 99

after a 30 minutes exposure,(11)) dispersed in PBS-phospholipid was prepared by suspending 43 100

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μL eugenol (Acros Oganics, Fairlawn, NJ) in 99 mL dilute phospholipid solution, followed by 101

rotary homogenization (Ultra-Turrax IKA T-18 basic, Staufen, Germany) at 11,000 rpm for 3 102

min. 103

Quantification of bactericidal activity of eugenol-lecithin mixtures 104

After mixing 1 mL working culture (without washing) with 99 mL eugenol-phospholipid 105

mixtures (initial inoculum concentration ca. 6 log CFU/mL; phospholipid concentration refers to 106

the concentration after adding 1 mL working culture), one sample was taken immediately (t=0 107

min) to determine the original inoculum concentration and after 30 minutes (t=30 min) 108

incubation at 37⁰C in a shaking incubator (N1570, Sheldon Manufacturing, Inc., OR) with a 109

speed of 300 rpm to determine the final concentration. No appreciable phase separation was 110

observed in any sample during the time span of the treatments. Each sample (0.1mL) taken at t=0 111

min and t=30 min was serially diluted, plated onto two separate tryptic soy agar plates (TSA; 112

Difco, Sparks, MD), grown overnight at 37⁰C, and counted. Inactivation [Log (CFU/mL)] was 113

calculated followed the equation (1) and plotted versus each phospholipid concentration. Similar 114

experiments without eugenol (phospholipid only) were conducted as controls. Three independent 115

trials at each phospholipid concentration with one replication per trial (measured on duplicate 116

plates) were conducted to quantify the bactericidal activity. 117

Inactivation [Log (CFU/mL)] = Log (CFU/mL)𝑡=0𝑚𝑖𝑛– Log (CFU/mL)𝑡=30𝑚𝑖𝑛 Equation (1) 118

Critical micelle concentration of phospholipids 119

Pyrene was used as a fluorescent probe to measure the CMC of DPPC, DSPC, DPPA and 120

DPPE, following previously reported methods with modifications (22, 23). Serial dilutions were 121

done to the target phospholipid concentrations ranging from 10-11

M to 10-2

M and 2 μM pyrene 122

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(from a stock 0.4 mM pyrene in ethanol solution) was added to the phospholipid-PBS solution. 123

Fluorescence measurements were conducted using a Fluorolog-3 spectrofluorometer (Horiba 124

Scientific, Edison, NJ) with the excitation wavelength set at 331 nm and a fixed scan time at 0.8 125

s. The emission spectra were obtained in the 341-450 nm wavelength range. The ratio of first 126

peak intensity (I1 at around 372nm) and third vibrational peak intensity (I3 at around 383nm) was 127

used as a parameter to indicate the polarity of pyrene’s environment (22). Two independent 128

samples (replicates) were prepared for each phospholipid concentration to measure the 129

fluorescence intensity. 130

Statistical Analysis 131

For all microbial studies, the inactivation (log CFU/mL) after 30 minutes exposure to 132

eugenol-single phospholipid treatment was the response variable. All experiments were analyzed 133

as completely randomized experimental designs with three replicates, except for CMC where 134

two replicates were used. Means were separated using LSD (α=0.05) when ANOVA showed 135

significant treatment effects using Minitab V. 15 (Minitab, State College, PA). 136

Results 137

The inactivation of E. coli C600 after a 30 minutes exposure to a buffer system 138

containing a 0.043% v/v eugenol without (control) or with various concentrations of each 139

phospholipid is shown in Figure 1a-e for DPPC, DSPC, DPPA, DPPE, and DPPS, respectively. 140

Four phospholipids (DPPC, DSPC, DPPA and DPPE) showed similar patterns of bioactivity, i.e., 141

adding the phospholipid to a specific low critical concentration improved the bactericidal effect 142

of eugenol. Beyond this critical synergistic concentration, the synergistic effect was diminished 143

and the microbial inactivation was significantly reduced to levels not significantly different from 144

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eugenol-only control samples. DPPS was the only phospholipid that did not exhibit a critical 145

concentration. When DPPS was added at a 3 log concentration range (from 0.66 μM to 66.97 146

μM), the inactivation effect of eugenol against E. coli was not significantly different from 147

samples containing eugenol-only (p=0.576). 148

The critical synergistic concentration for bioactivity varied with each phospholipid. 149

Adding 1.36 μM to 2.72 μM DPPC to the system significantly improved (p<0.05) the 150

antimicrobial effect of eugenol against E. coli C600 by an additional ca. 0.5 log cycle reduction. 151

Similarly, an additional ca. 1.5 log cycle reduction in E. coli C600 (p<0.05) was obtained with 152

0.63 to 1.90 μM DSPC added. For DPPE, the maximum additional microbial inactivation was 153

reached when 2.17 μM DPPE was introduced to the system, and an additional 1 log cycle 154

reduction was observed when 3.73 μM DPPA was added. Experiments were also conducted 155

using 18:0 EPC, a cationic derivative of DSPC. Figure 1f shows that adding 18:0 EPC below 156

2.34 μM or above 2.93 μM did not change the antimicrobial properties of eugenol. However, at 157

the critical synergistic concentration of 2.34 μM to 2.93 μM, an additional ca. 1.9 log cycle 158

inactivation of E. coli at the same eugenol concentration was found after 30 min of exposure to 159

eugenol. Phospholipid-only experiments (no eugenol) at various concentrations were conducted 160

as negative controls to discard any potential bioactive effect of the phospholipids on E. coli. In 161

all cases, no significant effect of any phospholipid at any concentration was observed on the final 162

microbial population, when compared to the bacterial populations suspended in PBS for 30 min 163

(Figure 1a-e). Based on consistent negative results obtained with all tested phospholipids, we 164

assumed no antimicrobial effect of 18:0 EPC and no control experiment (18:0 EPC only) was 165

conducted (Figure 1f). 166

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Table 1 also shows the critical synergistic concentration and maximum additional 167

antimicrobial effect caused by the various phospholipids tested in this study. The critical 168

synergistic concentration depended on the type of phospholipid and the additional inactivation 169

effect when mixing eugenol together with the phospholipids at the critical synergistic 170

concentration ranged from ca. 0.5 to 2 log CFU/mL. 171

Pyrene was used as a probe to measure the CMC of DPPC, DSPC, DPPE and DPPA, 172

indicating their structure in PBS. The ratio of the first peak (I1) and third peak (I3) in each 173

spectrum was calculated. Figure 2a shows a decrease in the I1/I3 of pyrene when the DPPC was 174

around 1.34x10-6

M which may indicate the CMC of DPPC in PBS. Similarly, results in Figure 175

2b indicated a decrease of I1/I3 started at ca. 1.27x10-9

M for DSPC. There was no clear change in 176

I1/I3 over the tested DPPA concentrations (Figure 2c). In Figure 2d, the I1/I3 of DPPE declined at 177

around 1.45x10-5

M. 178

Discussion 179

Eugenol-only control samples showed lower overall reduction (around 0.2 log CFU/mL), 180

compared to results in our previous study (ca. 2 log CFU/mL (11)), suggesting lower eugenol 181

antimicrobial power. However, the synergistic effect of lecithin was consistent with previous 182

results, i.e., the synergistic effect was observed regardless of the antimicrobial power of the 183

eugenol for results shown in Figures 1a-f. Among the five natural phospholipids tested, DPPC, 184

DSPC, DPPE and DPPA showed synergistic effect with eugenol at critical phospholipid 185

concentrations. The enhanced antimicrobial potency of eugenol was diminished with adding 186

phospholipids to concentration above or below the critical synergistic concentrations. These 187

results were consistent with our previous finding where using lecithin within a narrow 188

concentration window improved the antimicrobial efficacy of eugenol against E. coli. The 189

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mechanism (or mechanisms) behind the observed inactivation patterns are not known. Based on 190

previous Cryo-TEM observations and fluorescence studies, we proposed that the enhanced 191

antimicrobial activity was due to the formation of nano-scale structures (diameter<50 nm) when 192

lecithin was added at critical synergistic concentrations. At the critical phospholipid 193

concentration, the amphiphilic phospholipids self-assembled from monomers to small aggregates 194

where eugenol could favorably partition. These small aggregates with large net surface area and 195

containing positively charged phospholipid head groups would in turn enhance the interaction 196

between eugenol and negatively charged E. coli cell membranes. Adding phospholipids well 197

above the critical synergistic concentration caused the formation of large micron-scale network 198

that could block the net contact between eugenol and target cell membranes and in turn diminish 199

the enhanced antimicrobial properties. 200

Since phospholipids are amphiphilic molecules, they self-assemble to thermodynamically 201

stable structures in aqueous systems. The CMC of phospholipids varies with both the 202

phospholipid’s head group and acyl chain length (24-26). Several studies demonstrated the 203

temperature- and concentration- dependent architectures when phospholipids were suspended in 204

water (27, 28). Even though the conformation of these phospholipids at critical synergistic 205

concentrations was not determined, the CMC of for DPPS, DSPC, DPPE and DPPA was tested 206

as an indicator for the formation of specific structure leading to potential synergistic effect with 207

eugenol. Both surface tension (pendant drop) and pyrene fluorescent probe methods were used to 208

determine the critical micelle concentration of the various phospholipids. However, large 209

variability was found in the surface tension measurement, probably due to the very low 210

phospholipid concentration needed to synergistically improve the antimicrobial properties of 211

eugenol (results not shown). With increasing concentration of each of the tested phospholipids, 212

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the ration I1/I3 of pyrene fluorescence signal was expected to decrease as the CMC was 213

approached, indicating the transferring of the pyrene probe into a more hydrophobic micellar 214

environment vs. water (22, 29). As shown in Figure 2a-d, the CMC for DPPC, DSPC, DPPA and 215

DPPE was ca. 1.36 x10-6

M, ca. 1.27x10-9

M, > 1.49X10-4

M, and ca. 14.5 x10-6

M, respectively. 216

The CMC of DSCP was ca. 3 log cycles below that of DPPC, which may due to the longer 217

hydrocarbon chains in DSPC (30). This was consistent with previous reports by Ross and co-218

workers where the ln(CMC) linearly decreased with increasing in phosphatidylcholine 219

hydrocarbon tail length. In their study, calculated phosphatidylcholine with two fifteen-carbon 220

acyl chains had a larger ln(CMC) value (-24.6) than that of DPPC (sixteen-carbon acyl chain; -221

25.5) (31). The CMC of DPPC in PBS obtained in this study differs from the CMC of DPPC in 222

water (4.6 ± 0.5 x10-10

M) reported previously (31). Since the ionic environment influences 223

micelle formation in an aqueous solution (32, 33), the presence of PBS may explain the 224

discrepancy between the observed vs. reported critical micelle concentration for DPPC. The 225

negatively charged DPPA was expected to show higher CMC consistent with more hydrophilic 226

phosphatides. For instance, the CMC of the zwitterionic spin-labeled lauroylphosphatidylcholine 227

(0.1 mM) is ca. 6 times lower than that of the negatively charged spin-labeled 228

lauroylphosphatidic acid (0.77 mM) in 0.1 M unbuffered NaCl solution (34). 229

With respect to the distinct difference in the CMC of tested phospholipids, it is reasonable 230

to see their different critical synergistic concentrations. Interestingly, the critical synergistic 231

concentration was correlated to critical micelle concentration (Table 1). Both critical synergistic 232

concentration and CMC were in the order: DPPA>DPPE>DPPC>DSPC. The correlation 233

between log (critical synergistic concentration) and log (CMC) was r=0.976 (p<0.01). This 234

supported our hypothesis as discussed above. The synergistic effect between eugenol and 235

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phospholipids may be related to the specific structure formation when adding phospholipids at 236

critical synergistic concentrations. However, for DPPE and DPPC, critical synergistic 237

concentrations were below their CMC. On the contrary, for DSPC and DPPA, critical synergistic 238

concentrations were above their CMC. This indicates that there could be more than one 239

mechanism behind the enhanced antimicrobial property of eugenol when a phospholipid is used. 240

When critical synergistic concentrations were above the CMC, the enhanced antimicrobial 241

property of eugenol may due to the encapsulation of eugenol into phospholipid micelles. 242

However, below the CMC, the phospholipid may form phospholipid-rich ‘patches’ on the 243

eugenol droplets as discussed in our previous study (11). The partition of phospholipids into the 244

E. coli cell membrane and therefore the modification of the membrane fluidity and permeability 245

for hydrophilic compounds is another mechanism that we plan to investigate (35). 246

However, critically thinking about the CMC study, we should notice that the phase 247

behavior of amphiphilic molecules like phospholipids is dynamic and complicated. Recent 248

studies suggest the occurrence of more than one CMC (36) or several “critical aggregation 249

concentrations” (CAC) instead of a single CMC (37). For example, Jana and co-workers stated 250

that after the first change in fluorescence signals indicating a CMC, further phase transition from 251

micelle to liposomes caused additional shifts in the I1/I3 fluorescence signal indicating further 252

aggregation patterns or CAC (37). The CMC value in this study may indicate a specific 253

phospholipid aggregation formation instead of just micelle formation. 254

In contrast with DPPC, DSPC, DPPE, and DPPA, DPPS did not exhibit a synergistic effect 255

on the antimicrobial properties of eugenol against E. coli. The reason is not clear. It is possible 256

that the two negatively charged groups in the polar head of DPPS caused electrostatic repulsion, 257

blocking potential absorption by negatively charged E. coli cell membranes. Even though the 258

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overall net charge of DPPS is the same as DPPA, a previous study showed that zwiterionic 259

phospholipids in water expose charged polar head group outside the self-assembled phospholipid 260

bilayers (38). As a consequence, zwiterionic phospholipid could favorably bind ions with 261

opposite charge. Therefore, two negative charged groups on the polar head of DPPS would 262

potentially cause strong repulsion between eugenol-DPPS aggregates and E. coli cell membranes 263

diminishing the antimicrobial effect. 264

The antimicrobial potency of eugenol with a stable cationic phospholipid 18:0 EPC was 265

tested to further elucidate the effect of charge on the phospholipid synergistic effect. 18:0 EPC 266

has the same acyl chain length and head group than DSPC. The only difference is that 18:0 EPC 267

does not have a negatively charged -O- head group, so it carries positive charges only. We 268

hypothesized that 18:0 EPC at critical synergistic concentration would induce a significantly 269

higher antimicrobial effect of eugenol against E. coli compared to DSPC. However, Table 1 270

showed that the synergistic antimicrobial effects from 18:0 EPC and DSPC were not 271

significantly different so the number of negatively charged group was not important in changing 272

the electrostatic interactions between aggregates and between eugenol-phospholipid and bacterial 273

cells. The electrostatic attraction between eugenol-phospholipid aggregates and E. coli cells may 274

not play a major role in determining the synergistic effect. 275

Furthermore, that maximal inactivation effects achieved by adding DPPC, DSPC, DPPE, 276

DPPA and 18: 0 EPC respectively to the eugenol-in-water system were not the same. 277

Interestingly, what drew our attention was that eugenol with two phospholipids (DSPC and 18:0 278

EPC) containing 18 carbon acyl chains showed significantly higher antimicrobial property 279

against E. coli. This was in consistent with observations by Donsì and co-workers who 280

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concluded that the increase of essential oil antimicrobial activity depended on both concentration 281

and type of the emulsifiers (9). 282

Conclusion 283

According to our findings, not all phospholipids in lecithin synergistically enhance the 284

antimicrobial effect of eugenol. Among all the suggested mechanisms for the enhanced 285

antimicrobial effect of eugenol by adding critical low concentration of phospholipids, the 286

electrostatic interaction between eugenol-phospholipid aggregates and E. coli cells may not be 287

the main mechanism of action, as DSPC and the cationic 18:0 EPC showed similar synergistic 288

action. In terms of designing a more effective antimicrobial delivery system, the most important 289

factor is suggested to be the chain length of phospholipids, as (DSPC) 18:0 showed increased 290

synergistic action vs. DPPC (16:0). However, more sensitive techniques need to be used to 291

characterize the eugenol-phospholipids structure as well as their interactions with E. coli cells to 292

elucidate the mechanisms behind the observed synergistic effect. Until now we are limited by the 293

detection limit of many structure characterization techniques due to the very low concentration 294

of phospholipids leading to synergism.295

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Figure legends: 296

Fig. 1. Inactivation of E. coli C600 after 30 minutes exposure to constant eugenol concentration 297

(0.043 % v/v) and increasing levels of phospholipids ( ) and phospholipid-only controls (298

). All controls showed no inactivation effect. Means followed by a different letter are 299

significantly different using LSD (α<0.05). Error bars are standard deviations for the mean of 300

three independent replicates. 301

Fig. 2. Pyrene 1:3 ratio (I1/I3) versus phospholipids at different concentrations in PBS buffer 302

system. Error bars are standard deviations for the mean of two independent replicates. 303

Table. 1. Chemical structure, highest inactivation, critical synergistic concentrations (CSC) and 304

critical micelle concentrations (CMC) of tested phospholipids. 305

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306

Figure 1 307

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308

Figure 2 309 on March 31, 2020 by guest

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Table 1 310

Name Acronym CAS

number

MW

(Da) Structure

CSC

(μM) CMC

(μM) Highest

Inactivation

[log(CFU/mL

)] 1,2-dihexadecanoyl-sn-

glycero-3-

phosphocholine

DPPC

PC(16:0/16:0) 63-89-8 734.0

2.04 ca. 13.6 0.657±0.08c

1,2-dioctadecanoyl-sn-

glycero-3-

phosphocholine

DSPC

PC(18:0/18:0) 816-94-4 790.1

0.63 ca.

1.27x10-3

1.898±0.247a

1,2-dihexadecanoyl-sn-

glycero-3-phosphate

(sodium salt)

DPPA

PA(16:0/16:0) 169051-60-9 670.9

3.73 > 1.49

x102

1.207±0.152b

1,2-dihexadecanoyl-sn-

glycero-3-

phosphoethanolamine

DPPE

PE(16:0/16:0)

923-61-5 692.0

2.17 ca. 14.5 0.850±0.145b,c

1,2-dihexadecanoyl-sn-

glycero-3-phospho-L-

serine

DPPS

PS(16:0/16:0)

145849-32-7 757.9

None Not

measured

None

1,2-dioctadecanoyl-sn-

glycero-3-

ethylphosphocholine

18:0 EPC 328268-13-9 854.7

2.92 Not

measured

2.168±0.540a

311

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312

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2. Organization WH. 2015. WHO estimates of the global burden of foodborne diseases. WHO: 317 Geneva, Switzerland. 318

3. Control CfD, Prevention. 2014. Reports of selected E. coli outbreak investigations. 319 4. Burt S. 2004. Essential oils: their antibacterial properties and potential applications in foods—a 320

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H7 and Listeria monocytogenes by carvacrol and eugenol encapsulated in surfactant micelles. 323 Journal of Food Protection® 68:2559-2566. 324

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