Antimicrobial Efficacy of Natural Phenolic Compounds against Gram Positive Foodborne Pathogens

Protection of food from pathogens and spoilage organisms has been achieved by a variety of methods. Due to consumer preference, health and economic concerns in recent years, there is considerable interest to employ natural antimicrobials as an alternative to control the growth of microorganisms. This study evaluates the antimicrobial efficacy of natural plant derived phenolic compounds (PDPC) including chlorogenic acid, coumarin, curcumin, ellagic acid, (-) epicatechin, eugenol, rosmarinic acid, rutin, tannic acid, thymol, thymoquinone, and xanthohumol) as preservatives in food products. Several strains of Bacillus, Listeria and Clostridium species were treated with 12 natural PDPCs. Concentrations of 5, 10, 15, and 20 ppm of each compound were evaluated by broth micro-dilution method and the MICs were determined by using optical density after 24 and 60 hours of incubation. Thymoquinone, xanthohumol and ellagic acid demonstrated the highest antimicrobial efficacy (MIC <20 ppm). Structural alterations in treated bacteria were observed via scanning electron microscopy. The results demonstrated that the PDPCs have varying antimicrobial activities against both aerobic and anaerobic Gram-positive foodborne pathogens following 24 hour and 60 hour incubation periods, respectively. Natural sources of phenolic compounds contain major antimicrobial components and have great potential to control the growth of pathogens and be used as natural antimicrobials and food preservatives for extended storage. This study highlighted the antimicrobial efficacy of some PDPCs which may replace the artificial antimicrobials and preservatives in food industry to partially or completely control or inhibit the growth of harmful bacteria.

limited due to the unacceptable organoleptic changes (Cowan, 1999) they may induce when used in high doses (Friedman, 2014, Weerakkody et al., 2011. Thus, there is an increasing demand for accurate knowledge of the minimum inhibitory (effective) concentrations (MIC) of plant derived phenolic compounds (PDPCs) to enable a balance between the sensory acceptability and antimicrobial efficacy that can be achieved with both in vitro and applied studies (Lambert et al., 2001, Phantong et al., 2013. Although the antimicrobial activity of some PDPCs has been previously studied (Cueva et al., 2010;Kim et al., 2011) the response after long term exposure has not been reported. This study is the first to reveal the antimicrobial efficacy of thymoquinine, xanthohumol and coumarin. Also, antimicrobial activity of chlorogenic acid, curcumin, (-) epicatechin, eugenol, thymol, tannic acid and rutin on pathogenic Listeria monocytogenes, Bacillus spp. and Clostridium spp. needs to be studied more.
The main objectives of this study were (1) to evaluate the antimicrobial efficacy of selected natural PDPCs against Gram-positive foodborne pathogens: Bacillus subtilis, Bacillus cereus, Bacillus (Paenibacillus) polymyxa, L. monocytogenes, Clostridium perfringens, Clostridium butyricum and Clostridium sporogenes (2) to determine the MIC of the natural PDPCs and (3) to observe their prolonged antimicrobial activities over the prolonged incubation of 60 h to simulate the long term storage by extending the exposure time of pathogens to PDPCs.

Plant Derived Phenolic Compounds
Twelve different natural PDPCs were used in the study including: chlorogenic acid, coumarin, curcumin, ellagic acid, eugenol, (-) epicatechin, rosmarinic acid, rutin, tannic acid, thymol, thymoquinone, and xanthohumol (Sigma-Aldrich, St Louis, MO, USA). All of the compounds are commercially available (powder form) and they were ≥95% pure. Each compound was dissolved in ethanol, (95%) (Decon Laboratories, King of Prussia, PA, USA) except for thymoquinone, which was prepared with dimethyl sulfoxide 99.9% (DMSO) (Fisher Scientific, Fair Lawn, NJ, USA). The final phenolic solution was adjusted to approximately pH 5.00 using HCl (15%) to ensure that the pH would not affect the bacterial growth. All solutions were filter sterilized using 0.22 μm filters (Millipore Corporation, Billerica, MA, USA) and stored at 4°C in sterilized sealed glass containers until needed. All experiments were done at ambient temperature.

Bacterial Strains, Culture Conditions and Preparation of Inoculum
A total of 9 foodborne pathogen strains including B. subtilis (ATCC 6051), B. cereus (ATCC 11778), B. polymyxa (ATCC 842), C. perfringens (R. Newsome Research), C. butyricum (ATCC 8260), C. sporogenes (ATCC 7955), and 3 strains of L. monocytogenes (ATCC 7644,UK Animal Diagnostic Lab,and ATCC 49594) were supplied from the American Type Culture Collection and the University of Kentucky. Listeria and Bacillus were grown and maintained on slants of Brain-Heart Infusion (BHI) agar and Clostridium spp. were maintained on thioglycollate medium (TM) anaerobically at 4°C until needed. Prior to each test, the isolates were sub-cultured at least three times before inoculating in BHI for aerobic bacteria and Reinforced Clostridial Broth (RCB) for Clostridium spp. Culture growth turbidity, which is indicated by the optical density (OD), was standardized for each bacterium at a wavelength of 660 nm (OD 660 ) by using the spectrophotometer (BioTek Synergy 4, Winooski, VT, USA). For the initial bacterial count approximately 10 7 -10 8 CFU/ml was targeted and cell counts were confirmed by using the Eddy Jet spiral plater (Neutec Group Inc., Farmingdale, NY, USA) with Plate Count Agar (PCA). The bacterial counts were determined by the Flash and Go plate reader (Neutec Group Inc., Farmingdale). All microbiological media and supplements used in the study were supplied from Difco Laboratories (Sparks, MD, USA) unless otherwise specified.

Determination of Minimum Inhibitory Concentrations of Phenolic Compounds
Micro broth dilution technique of Antimicrobial Susceptibility testing was performed as outlined in the National Committee for Clinical Laboratory Standards (NCCLS, 2004) and modified for 60 h incubation (Cetin-Karaca & Newman, 2015). Serial dilutions of the compounds (100 µl) were dispensed into 5 ml Mueller Hinton Broth (MHB) and RCB for Clostridium to obtain the final concentrations of 5, 10, 15 and 20 ppm (mg l -1 ). Then, 100 µl of the overnight culture of bacteria was transferred aseptically into MHB/RCB. Compound free inoculated MHB/RCB with and without the solvent were served as growth control and negative control, respectively. One hundred and fifty µl of each sample, including the controls, was dispensed to the wells of a 96-well flat bottom micro-titer plate (Nalge NUNC Int., Corning, NY, USA) and incubated at 37°C. Clostridium spp. were incubated anaerobically at 37°C in BBL anaerobe jars with GasPak EZ anaerobe system (Becton and Dickinson, Sparks, MD, USA). All experiments were carried out three times in duplicates.
After inoculation, the micro-titer plates were read immediately to get the initial OD using a calibrated spectrophotometer (BioTek Synergy 4) at 660 nm wavelength. Prior to each incubation process, the samples in the micro-titer plate were shaken automatically for 10 seconds to get a consistent homogeneity. The absorbance was read at 12-hour intervals for a 60 h incubation period. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of the compound that visibly inhibited the bacterial growth in comparison with the control (Waśko et al., 2014). Antimicrobial activities of PDPCs towards selected pathogens were demonstrated as absorbance difference relative to the control (%, treatment/control).

Investigation of Structural Changes of Cells via Scanning Electron Microscopy
MICs of PDPCs were applied to bacterial cultures and they were incubated for 24 hr at 37°C. Suspensions were filter sterilized through 0.22 μm filters (Thermo Scientific, Nalgene, Rochester, NY, USA) to capture the bacteria and the same filters with bacteria were used for scanning electron microscope (SEM) observations as described by Kalab et al. (2008).
The filters containing thin layers of bacterial specimens were fixed with glutaraldehyde fixative (6%) (E.M. Grade, SPI Supplies Inc., West Chester, PA, USA) and then dehydrated using serial dilutions of ethanol; 20%, 40%, 60%, 80%, and 100%, followed by hexamethyldisilazane (Sigma-Aldrich). The specimens were prepared and sputter-coated with carbon using a plasma coating system for SEM (Hummer VI Sputtering System; Technics, Union City, CA, USA). The morphology of bacterial cells was examined in Hitachi S-800 SEM (Tokyo, Japan) and captured images were analyzed by Evex Nano-analysis and Digital Imaging (Evex Analytical Version 2.0.1192, 2006) software.

Statistical Analysis
All the experiments were repeated three times and the data were calculated as means ± SD. The antimicrobial efficacy of the PDPCs was subjected to General Linear Model procedure of Statistix 9.0 (2008). Differences in the means of Bacillus spp., L. monocytogenes and Clostridium spp. absorbance (OD) influenced by the presence of phenolic compounds were determined by the use of Tukey HSD (P <0.05).

Antimicrobial Activity against Bacillus spp.
The MICs of selected PDPCs at 60 h are shown in Table 1. Although the antimicrobial activity (for 24 h) of some PCs has been previously reported (Cueva et al., 2010;Jianu et al., 2012;Kim et al., 2011), this study is the first to evaluate the antimicrobial efficacy of selected PDPCs during 60 h of extended incubation. The prolonged antimicrobial activity of PDPCs and the response of the pathogens to long term exposure was observed with the extended 60 h incubation time, which might provide evidence for high potential success in food safety for products stored for extended periods.

Antimicrobial Activity against Clostridium spp.
MICs of tested PDPCs against Clostridium spp. which demonstrated variations in antimicrobial efficacy; curcumin, thymol and xanthohumol being the most and eugenol being the least efficient PDPC (Table 1).
Antimicrobial efficacy of PDPCs against anaerobic bacteria including Clostridium spp. haven't been reported broadly, therefore antimicrobial effectiveness of curcumin, coumarin, ellagic acid, (-) epicatechin, tannic acid, thymoquinone and xanthohumol are first being reported in this study.

Antimicrobial Activity against L. monocytogenes
The MICs of selected PDPCs against L. monocytogenes (LM1-3) are presented in Table 1. Tannic acid with MIC <20 ppm showed strong antimicrobial activity following 60 hours of incubation. According to Pyla et al. (2010) starch-based films impregnated with tannic acid caused a 2.72-log decrease in L. monocytogenes in 48 hours incubation period. MIC of eugenol was determined as <10 ppm in accordance with eugenol's efficacy in www.ccsenet.org/jfr Journal of Food Research Vol. 4, No. 6; reducing the proliferation of L. monocytogenes on the surface of fresh lettuce (Kim et al., 2011) and when incorporated into alginate-based edible coatings (Raybaudi-Massilia et al., 2009).
Moreover, thymol was previously found to inhibit Listeria (>7 mm inhibition zones) (Tanis et al., 2009), whereas, in this study, neither thymol nor rosmarinic acid showed any antimicrobial activity (P <0.05) against L. monocytogenes (20 ppm) at 60 h. This should be due to the different methods (agar diffusion) and higher concentrations used in those studies. Also, the chemical composition of PDPCs may vary due to the variety of plant used, harvesting period and agricultural methods used and the type of extraction method and solvent used.

Structural Observations via Scanning Electron Microscope
Treated samples of bacteria were observed by SEM to confirm the antimicrobial effects of the PDPCs along with the morphological changes in the appearance of the cells. Figure 8 illustrates the SEM images of two selected bacteria (B. subtilis and L. monocytogenes) treated with MICs of chlorogenic acid, thymoquinone and xanthohumol. It is clear that PDPCs caused severe damage to the bacteria via degradation of cell wall followed by the disruption of cytoplasmic membrane and membrane proteins which cause leakage of cell contents. Similar observations along with coagulation of cytoplasm and depletion of proton motive force were also reported by Cetin-Karaca & Newman (2015) and Burt (2004). B. subtilis cells were observed to decrease in size (Fig. 8A2, A3) when compared to controls (Fig. 8A1). Rupture of the cell wall and slimy appearance was clearly observed with L. monocytogenes (Fig. 8B2, B3). A lot of adhered material around the cells (Fig. 8A3, A4 and B2) and even some empty cells (Fig. 8B2) were observed. Gram-positive bacteria were reported to be more sensitive toward antibacterial substances than gram-negative bacteria (Burt, 2004;Cueva et al., 2010;Shan et al., 2007). This is likely due to hydrophilic surface of gram-negative bacteria on their outer membrane (Burt 2004;Shan et al., 2007) and a unique periplasmic space not found in gram-positive bacteria (Cueva et al., 2010). SEM observations confirmed the severe physical damage and considerable morphological alteration to all tested foodborne pathogens treated with the PDPCs. It was reported that as a part of the mechanism of action, PCs might bind to the cell surface and then penetrate to the target sites, possibly the phospholipid bilayer of the cytoplasmic membrane and membrane-bound enzymes (Cetin-Karaca & Newman, 2015;Gyawali & Ibrahim, 2014). Furthermore, the inhibition of proton motive force, respiratory chain, electron transfer and substrate oxidation could also be observed (Gyawali & Ibrahim, 2014;Shan et al 2007a). Also, the number and position of www.ccsenet.org/jfr Journal of Food Research Vol. 4, No. 6;2015 24 substitutions in the benzene ring of the PCs and the saturated side-chain length influence the antimicrobial potential of the PCs against different microorganisms (Gill & Holley, 2006).

Conclusion
This study evaluated the antimicrobial activity of PDPCs through 60 hours of incubation, longer than the previous studies established MICs for 24 hours incubation periods. Our findings provide evidence for the high potential success of natural PDPCs in food safety for extended storage time. However it was also found that some of the sensitive pathogens including Bacillus and Clostridium spp. recover and become resistant with extended incubation periods, while LM1 was resistant from the beginning of the incubation. Thus, long term effects of these bacteria should be investigated carefully, since adaptation of pathogens may lead to resistance to PCs which may be initiated by gene manipulation and interfere with extended storage of foods. It can be concluded that the selected PDPCs exhibit in vitro antimicrobial activity against L. monocytogenes, B. subtilis, B. cereus, B. polymyxa, C. perfringens, C. sporogenes and C. butyricum even when utilized at low concentrations (5-20 ppm). Moreover, it should be noted that despite being from the same family each individual strain has its own growth and sensitivity characteristics against the PDPCs. In fact, PCs derived from plant extracts have the potential inhibitory activity against pathogenic bacteria and they can be superior alternatives for replacing chemicals used in food preservation. However, to be widely applied in food systems as antimicrobials, more systematic investigations should be done on their organoleptic impact, issues of safety and toxicity.