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Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli
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Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli Q1
Hayriye Cetin-Karaca, Melissa C. Newmann
Q3 University of Kentucky Department of Animal and Food Sciences, W. P. Garrigus Building, Lexington,
KY 40546-0215, United States
art i cle i nfo
ab st rac t
Article history:
The present study evaluated the antimicrobial efficacy of natural phenolic compounds (PC)
Received 24 July 2014
extracted from vegetables, fruits, herbs and spices; to inhibit the growth of Gram-negative
Received in revised form
foodborne bacteria which is defined as the minimum inhibitory concentration (MIC).
28 January 2015 Accepted 18 March 2015
Strains of Escherichia coli and Salmonella species were treated with natural PCs including; chlorogenic acid, curcumin, ( ) epicatechin, eugenol, myricetin, quercetin, rutin, thymol, thymoquinone, and xanthohumol. Concentrations of 5, 10, 15, and 20 ppm of each
Keywords:
compound were evaluated by a broth micro-dilution method and the MICs were deter-
Foodborne pathogen
mined by using optical density after 24 and 60 h of incubation. Structural alterations in
Phenolic compounds
treated bacteria were observed via scanning electron microscopy.
Minimum inhibitory concentration
For E. coli, thymoquinone showed the highest inhibition, followed by rutin, ( ) epicatechin and myricetin (MICo20 ppm for all), while Salmonella was most sensitive to
(MIC) Antimicrobial activity
( ) epicatechin (MICo15 ppm), followed by thymoquinone, rutin and myricetin (MICo20 ppm for all) following 60 h of incubation. The results demonstrated that the PCs have varying antimicrobial activities against foodborne pathogens following 24 and 60 h incubation periods. Natural sources of PCs contain major antibacterial components and have great potential to be used as natural antimicrobials and food preservatives, during long term storage. This study highlighted the antimicrobial efficacy of some novel PCs which may replace chemical antimicrobials and preservatives in food or pharmaceutical industry to partially or completely inhibit the growth of bacteria. & 2015 Published by Elsevier Ltd.
1.
Introduction
Spices and aromatic vegetable materials have long been used in food not only for their flavor and aromatic qualities and appetizing effects but also for their preservative and medicinal properties. Since ancient times, they have been used for preventing food spoilage and deterioration and extending the shelf life of foods (Gyawali & Ibrahim, 2014; Shan, Cai, Brooks, & Corke,
2007b). It has been extensively reported that the essential oils and secondary plant metabolites have shown antimicrobial activities against foodborne pathogens (Reichling, Schnitzler, Suschke, & Saller, 2009; Smith-Palmer, Stewart, & Fyfe, 1998). In addition, they have received great attention in recent decades due to their presumed role in the prevention of foodborne diseases, cancer, chronic and cardiovascular diseases, and in the slowdown of the aging process (Lee, Cesario, Wang,
n
Corresponding author. Tel.: þ1 859 257 5881. E-mail address: [email protected] (M.C. Newman).
http://dx.doi.org/10.1016/j.fbio.2015.03.002 2212-4292/& 2015 Published by Elsevier Ltd.
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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Shanbrom, & Thrupp, 2003; Liu, 2003; Nazzaro et al., 2009; Lou, Wang, Zhu, Ma, & Wang, 2011) indicating that, they have diverse beneficial biological functions including antimicrobial and antioxidant activities (Korukluoglu, Sahan, Yigit, Ozer, & Gucer, 2010; Perumalla & Hettiarachchy, 2011). Phenolic compounds (PC) are one of the most diverse groups of secondary metabolites found in a wide variety of fruits, vegetables, nuts, seeds, stems and flowers as well as tea, wine, propolis and honey (Moreno, Scheyer, Romano, & Vojnov, 2006). The exploration of natural antimicrobials for food preservation receives increased attention due to consumer awareness of natural food products and a growing concern of microbial resistance towards conventional food processing and preservation methods. The use of PCs as antimicrobial agents could potentially provide additional benefits, including dual-function effects of both preservation and delivery of health benefits (Cueva et al., 2010). By gaining the fundamental knowledge on the antimicrobial effects of plant derived PCs on pathogenic microorganisms, it is possible to search new strategies to combine the synergic antimicrobial effects of PCs with their natural biological properties. The results may permit the formulation of new products to be used as food preservatives or to be included in the human diet. Thus, the food industry is interested in developing natural components for the total or partial replacement of synthetic antimicrobials (Santas, Almajano, & Carbo, 2010). Mechanisms of action in the bacterial cell of bioactive plant compounds such as degradation of the cell wall (Nychas and Tassou, 1999), damage to cytoplasmic membrane and membrane proteins (Lambert, Skandamis, Coote, & Nychas, 2001), leakage of contents out of the cell, coagulation of cytoplasm, and depletion of the proton motive force (Burt, 2004; Gyawali & Ibrahim, 2014) have been reported. In general, variations in antimicrobial activities against bacteria may reflect differences in cell surface structures between Gram-negative and Grampositive species; Gram-positive being more susceptible to the action of phenolic acids than Gram-negative bacteria (Cueva et al., 2010). Also, the number, type and position of substituents in the benzene ring of the phenolic acids and the saturated side-chain length influence the antimicrobial potential of the phenolic acids against different microorganisms (Gill & Holley, 2006). Inhibitory effects of several natural bioactive compounds from berries and grapes (Puupponen-Pimia et al., 2001; Zanoli & Zavatti, 2008) herbs and spices (Albayrak, Aksoy, Sagdic, & Albayrak, 2012; Shan et al., 2007b; Shan, Cai, Brooks, & Corke, 2007a) and other plant extracts (Pereira et al., 2007b; RaybaudiMassilia, Mosqueda-Melgar, Soliva-Fortuny, & Martin-Belloso, 2009; Santas et al., 2010) were tested against food-relevant bacteria including: E. coli, Salmonella, Listeria, and Staphylococcus. Although the antimicrobial activity of some PCs including: thymol (Lambert et al., 2001; Santas et al., 2010), eugenol (Phanthong, Lomarat, Chomnawangb, & Bunyapraphatsaraa, 2013), carvacrol (Lambert et al., 2001; Santas et al., 2010), quercetin (Yao et al., 2011) and myricetin (Puupponen-Pimia et al., 2001) has been previously reported, the response after the long term exposure was not reported and there are still many unexplored sources. Moreover, thymoquinone and xanthohumol have not been included in antimicrobial studies and antimicrobial activity of chlorogenic acid, curcumin, ( ) epicatechin,
eugenol, myricetin and rutin have not been reported broadly on pathogenic Salmonella and E. coli. Objectives of this study are: (1) to evaluate the antimicrobial activity of selected natural PCs extracted from herbs, spices, vegetables, and fruits against Gram-negative foodborne pathogens: E. coli, E. coli O157:H7, Salmonella paratyphi, Salmonella cholerasuis subsps. cholerasuis and Salmonella Enteritidis, (2) to determine the MIC of the natural PCs and (3) to observe their prolonged antimicrobial activities over extended incubation of 60 h to simulate the long term storage by extending the exposure time of pathogens to PCs.
2.
Materials and methods
2.1.
Preparation of phenolic compounds
The natural PCs used in this study are: chlorogenic acid, curcumin, ( ) epicatechin, eugenol, myricetin, quercetin, rutin, thymol, thymoquinone, and xanthohumol (SigmaAldrich, St Louis, MO, USA). Each compound was prepared 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 0.2 mm filters (Millipore Corporation, Billerica, MA, USA) and stored at 4 1C in sterilized sealed glass containers until needed. All measurements were done at ambient temperature.
2.2. Bacterial strains, culture conditions and preparation of inoculums Foodborne pathogens including: E. coli, F. T. Jones (FTJ), E. coli O157:H7, ATCC 43895 and E. coli O157:H7, ATCC 35150, S. paratyphi, UK Micro 29A (UKM 29A), S. cholerasuis subsp. cholerasuis, ATCC 10708 and S. Enteritidis, UK ( ) H2S were supplied from the American Type Culture Collection and the University of Kentucky. Bacteria were grown and maintained on slants of brain–heart infusion (BHI) agar and stored at 4 1C until needed. Prior to each test, at least three consecutive transfers of the cultures were inoculated in BHI broth and they were incubated overnight at 37 1C. Then, the inoculums were standardized according to a MacFarland 0.5 turbidity standard (108 CFU ml 1) by diluting the sample (Taguri, Tanaka, & Kouno, 2004). Culture growth turbidity, which is indicated by the optical density (OD), was adjusted for each bacterium at a wavelength of 660 nm (OD660) by using the spectrophotometer (BioTek Synergy 4, Winooski, VT, USA) to the final concentrations of approximately 107–108 CFU ml 1. Cell counts were confirmed by using a spiral plating method with Plate Count Agar (PCA) and the Eddy Jet spiral plater (Neutec Group Inc., Farmingdale, NY, USA). The 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).
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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2.3. 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 (Standards, 2004) and modified for the 60 h incubation. Serial dilutions of the compounds (100 ml) were dispensed into 5 ml mueller hinton broth (MHB) to obtain the final concentrations of 5, 10, 15 and 20 ppm (mg l 1). Then, 100 ml of the overnight culture of bacteria was transferred aseptically into MHB. Controls included a compound free MHB broth (growth control), including only culture and a negative control, which contained solvent. Samples were dispensed to the wells of a 96well flat bottom micro-titer plate (Nalge NUNC Int., Corning, NY, USA) and incubated at 37 1C. All experiments were carried out twice in duplicates. After inoculation, the micro-titer plates were read immediately to get the initial OD using a calibrated spectrophotometer at 660 nm wavelength. Prior to each incubation process, the samples in the micro-titer plate were shaken automatically for 10 sec to get a consistent homogeneity. The absorbance was read at every 12-hour intervals for a total 60 hr of incubation period. Minimum inhibitory Concentration (MIC) was defined as the lowest concentration at which no bacterial growth was observed after incubation.
2.4. Investigation of structural changes via scanning electron microscopy Treated and non-treated (control) bacterial cultures were incubated for 24 h at 37 1C. PCs were applied to each strain at their specific MIC level. PCs were filter sterilized through 0.2 μm filters (Thermo Scientific, Nalgene, Rochester, NY, USA) to capture the bacteria for Scanning Electron Microscope (SEM) observations. The procedure was performed according to the description by Kalab, Yang and Denise (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 using Evex Nanoanalysis and Digital Imaging (Evex Analytical Version 2.0.1192, 2006) software.
2.5.
Statistical analysis
The antimicrobial activity of the PCs was subjected to General Linear Model procedure of Statistix 9.0 (2008). Significance level of Po0.05 of the null hypothesis was used to determine the significant variables. Difference between the means was identified by the use of Tukey HSD randomized complete block design between treatments and time.
3.
3
Results
Several PCs were screened for their antimicrobial properties against E. coli (FTJ), two strains of E. coli O157:H7 (ATCC 43895 and ATCC 35150), S. paratyphi (UK Micro 29A), S. cholerasuis subsp. cholerasuis (ATCC 10708) and S. Enteritidis (UK (–) H2S). The MICs at 24 and 60 h of all tested PCs are shown in Table 1. Prolonged antimicrobial effects of PCs and the response of the pathogens to long term exposure were observed with extended incubation time (60 h). Varying degrees of antimicrobial activity was observed with chlorogenic acid (o20 ppm) during 60 h of incubation. It exhibited the lowest MIC (o10 ppm) for all Salmonella strains, compared to other PCs. S. paratyphi and E. coli O157: H7 (ATCC 35150) demonstrated the highest antimicrobial sensitivity to curcumin (MICo5 ppm) during the entire incubation period. However, it showed no antimicrobial effect against E. coli O157:H7; ATCC 43895. All E. coli and Salmonella tested with eugenol exhibited high degrees of inhibition, with MICs: o20 and o15 ppm, respectively. Among the PCs tested, quercetin exhibited the lowest MIC of o5 ppm for all E. coli and o20 ppm for Salmonella species in extended incubation (60 hr). Both E. coli and Salmonella showed varying degrees of antimicrobial sensitivity towards thymol (MICo15 ppm) and xanthohumol (MICo20 ppm). S. paratyphi and S. cholerasuis subsp. cholerasuis were observed to be highly sensitive towards both thymol (MICo15 ppm) and xanthohumol (MICo20 ppm), while S. Enteritidis was consistently resistant during the entire incubation period. Fig. 1a–d shows the antimicrobial activity of thymoquinone against E. coli; FTJ, E. coli O157:H7; ATCC 43895, S. cholerasuis subsp. cholerasuis; ATCC 10708, and S. Enteritidis; ( ) H2S incubated for 60 h. Thymoquinone (MICo20 ppm) exhibited similar and high antimicrobial activity both for E. coli and Salmonella species during the 60 h of incubation, being the most effective one among the PCs tested for E. coli. Rutin inhibited the growth of all E. coli and Salmonella strains showing varying degrees of antimicrobial activity (Fig. 2a–d) with MIC of o20 ppm. ( ) Epicatechin was observed to have high antimicrobial activity against E. coli (Fig. 3a and b) and Salmonella (Fig. 3c and d) when used at o20 and o15 ppm concentrations, respectively. Similarly, myricetin showed a lower MIC (o15 ppm) for Salmonella than E. coli (MICo20 ppm) exhibiting antimicrobial activity through 60 hr of incubation time (Fig. 4a–d). In general, during the 60 h of incubation ( ) epicatechin exhibited the highest growth inhibition in Salmonella, followed by thymoquinone, myricetin and rutin (Figs. 1–4), while thymoquinone showed the highest growth inhibition in E. coli followed by ( ) epicatechin, myricetin and rutin (Figs. 1–4). Despite the fact that all the PCs exhibited antimicrobial activity, the inhibition for each microorganism tested was different, even for the ones from the same family. Thus, it is important to determine the inhibition effects of PCs for selected bacterial strains. In this study, all three strains of E. coli, including non-pathogenic E. coli and E. coli O157:H7 showed consistent antimicrobial sensitivity towards four (eugenol, quercetin, thymol, and thymoquinone, with the MICs of 20, 5, 15, and 20 ppm, respectively) of the ten PCs. Similar antimicrobial consistency
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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Table 1 – MICs (ppm) of PCs against E. coli and Salmonella after 24 and 60 h. Compound
pH
E. coli FTJ
E. coli O157:H7 ATCC 43895
Minimum inhibitory concentrations (ppm) at 24 h Chlorogenic 5.62 o10.0 420.0 acid Curcumin 5.54 o20.0 420.0 Eugenol 5.54 o20.0 o20.0 (–) Epicatechin 5.6 o20.0 420.0 Myricetin 6.46 o15.0 o10.0 Quercetin 7.18 420.0 420.0 Rutin 5.65 o20.0 420.0 Thymol 5.46 o20.0 o15.0 Thymoquinine 5.59 o20.0 420.0 Xanthohumol 8.7 o5.0 o10.0 Minimum inhibitory concentrations (ppm) at 60 h Chlorogenic 5.62 o15.0 o20.0 acid Curcumin 5.54 o20.0 420.0 Eugenol 5.54 o20.0 o20.0 ( ) 5.6 o20.0 o5.0a Epicatechin Myricetin 6.46 o20.0 o10.0 Quercetin 7.18 o5.0 o5.0 Rutin 5.65 o20.0 o5.0 Thymol 5.46 o15.0 o15.0 Thymoquinine 5.59 o20.0 o20.0 Xanthohumol 8.7 o15.0 o15.0
E. coli O157:H7 ATCC 35150
S. paratyphi UK Micro 29A
S. cholerasuis subsp. cholerasuis ATCC 10708
S. Enteritidis UK (–) H2S
o15.0
o5.0
o15.0
420.0
o5.0 o20.0 o15.0 o10.0 o5.0 o15.0 o15.0 o20.0 420.0
o15.0 o20.0 o10.0 o15.0 o20.0 420.0 420.0 420.0 o20.0
o15.0 o20.0 o20.0 o20.0 o15.0 420.0 420.0 o5.0 o20.0
o5.0 o15.0 o20.0 o10.0 420.0 420.0 420.0 o20.0 420.0
o10.0
o10.0
o10.0
o10.0
o5.0 o20.0 o10.0
o5.0 o15.0 o15.0
o15.0 o15.0 o15.0
o20.0 o15.0 o15.0
o15.0 o5.0 o15.0 o15.0 o20.0 420.0
o20.0 o20.0 o20.0 o15.0 o20.0 o20.0
o15.0 o15.0 o15.0 o15.0 o15.0 o15.0
o5.0 o10.0 o15.0 420.0 o20.0 420.0
Active until 12 h of incubation.
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
E. coli Absorbance at 660nm
Absorbance at 660nm
a
0
12
24
36
48
60
E. coli O157:H7
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0
12
24
S. cholerasuis subsp.cholerasuis
0
12
24 36 Time (hours)
48
60
Absorbance at 660 nm
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
36
48
60
Time (hours)
Time (hours)
Absorbance at 660nm
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S. Enteritidis
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0
12
24 36 Time (hours)
48
60
Fig. 1 – Antimicrobial activity of thymoquinone against a) E. coli; FTJ, ( ) 20 ppm; b) E. coli O157:H7; ATCC 43895, ( ) 20 ppm; c) S. cholerasuis subsp. cholerasuis; ATCC 10708, ( ) 15 ppm and d) S. Enteritidis; UK ( ) H2S, ( ) 20 ppm ( ) control. Error bars represent standard deviation from the mean, Po0.05.
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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E. coli Absorbance at 660 nm
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
12
24
36
48
E. coli O157:H7
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
60
0
12
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
S. cholerasuis subsp. cholerasuis
0
12
24 36 Time (hours)
24
36
48
60
Time (hours)
Absorbance at 660 nm
Absorbance at 660 nm
Time (hours)
48
S. Enteritidis
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
60
0
12
24 36 Time (hours)
48
60
E. coli
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Absorbance at 660 nm
Absorbance at 660 nm
Fig. 2 – Antimicrobial activity of rutin against a) E. coli; FTJ, ( ) 20 ppm; b) E. coli O157:H7; ATCC 35150, ( ) 15 ppm; c) S. cholerasuis subsp. cholerasuis; ATCC 10708, ( ) 15 ppm and d) S. Enteritidis; UK ( ) H2S, ( ) 15 ppm ( ) control. Error bars represent standard deviation from the mean, Po0.05.
0
12
24
36
48
E. coli O157:H7
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
60
12
S. paratyphi Absorbance at 660 nm
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
12
24
36
24
36
48
60
48
60
Time (hours)
Time (hours)
Absorbance at 660 nm
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Absorbance at 660 nm
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48
60
Time (hours)
S. Enteritidis
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
12
24 36 Time (hours)
Fig. 3 – Antimicrobial activity of ( ) epicatechin against a) E. coli; FTJ, ( ) 20 ppm; b) E. coli O157:H7; ATCC 35150, ( ) 10 ppm; c) S. paratyphi; UKM 29A, ( ) 15 ppm and d) S. Enteritidis; UK ( ) H2S, ( ) 15 ppm ( ) control. Error bars represent standard deviation from the mean, Po0.05.
was observed for Salmonella strains towards chlorogenic acid, eugenol and ( ) epicatechin, with MICs of 10, 15, and 15 ppm, respectively.
Bacteria treated with PCs were observed by SEM to confirm the antimicrobial efficacy of the PCs along with the morphological changes in the appearance of the cells. Fig. 5 depicts
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
24 36 Time (hours)
48
0
S. cholerasuis subsp. cholerasuis
0
12
24 36 Time (hours)
48
60
E. coli O157:H7
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
60
Absorbance at 660 nm
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
12
Absorbance at 660 nm
Absorbance at 660 nm
E. coli
0
Absorbance at 660 nm
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12
24 36 Time (hours)
48
60
48
60
S. Enteritidis
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
12
24 36 Time (hours)
Fig. 4 – Antimicrobial activity of myricetin against a) E. coli; FTJ, ( ) 20 ppm; b) E. coli O157:H7; ATCC 35150, ( ) 15 ppm; c) S. cholerasuis subsp. cholerasuis; ATCC 10708, ( ) 15 ppm and d) S. Enteritidis; UK ( ) H2S, ( ) 5 ppm ( ) control. Error bars represent standard deviation from the mean, Po0.05.
the SEM images of treated and control samples for two selected bacteria (E. coli O157:H7 and S. cholerasuis subsp. cholerasuis) treated with MICs of chlorogenic acid, myricetin and xanthohumol. It is clearly visible that treatments with PCs (for 24 h) exhibited remarkable antimicrobial activity by damaging the cell wall followed by the leakage of the interior matter in all tested pathogens treated with the PCs. Content of some cells appeared depleted (Fig. 5a2, a3, a4 and b4) and some cells (Fig. 5a2, a3, a4 and b4) even appeared to be empty and the remains were flaccid, which prevents the cells to recover and function as normal. The slimy appearance and rupture of cell wall was observed at chlorogenic acid (15 ppm) in E. coli O157:H7 (Fig. 5a2), myricetin (20 ppm) in S. cholerasuis subsp. cholerasuis (Fig. 5b3), xanthohumol (10 and 20 ppm) in E. coli O157:H7 (Fig. 5a4) and S. cholerasuis subsp. cholerasuis (Fig. 5b4). In Salmonella a decrease in size of cells (Fig. 5b2, b3 and b4) was observed when compared to controls (Fig. 5b1). In general, the bacteria after treatment with PCs showed a lot of adhered material around the cell wall.
4.
Discussion
Although the antimicrobial activity (for 24 hr) of some PCs has been previously reported, this study is the first to evaluate the antimicrobial efficacy of selected PCs during 60 h of incubation. Our findings provide evidence for the prolonged antimicrobial activity of natural plant PCs which might have high potential success in food safety for extended storage time. Recently, Bakathir and Abbas (2011) reported that thymoquinone suspended with H2O have no antimicrobial activity against
E. coli, while in this study, thymoquinone showed remarkable antimicrobial activity (MICo20 ppm) for both E. coli and Salmonella. The inconsistency may be attributed to the different solvents and/or the methods used for preparation of antimicrobial suspension. Rutin's high antimicrobial activity (MICo20 ppm) against both Salmonella and E. coli is in good agreement with the previously published data where Salmonella (Askun, Tumen, Satil, & Ates, 2009a), E. coli O157:H7 and other E. coli (Askun, Tumen, Satil, & Ates, 2009b; Pereira et al., 2007a) strains are shown to be highly susceptible to rutin in vitro. However, it should be noted that Lee and Lee (2010) reported that S. Enteridis and some E. coli strains were not inhibited when treated with rutin (800 mg ml 1) in vitro. Our results for the antimicrobial activity of ( ) epicatechin; including the sensitivity of Salmonella species to () epicatechin (795 mg ml 1) (Taguri et al., 2004) and E. coli being strain dependent are in accordance with the earlier results (Bancirova, 2010; Cueva et al., 2010; Friedman, 2014). Myricetin exhibited a remarkable inhibition for both Salmonella and E. coli being in accordance with previous reports for the inhibition of S. paratyphi, S. Enteridis (Yao et al., 2011; Friedman, 2014) and E. coli with the MIC of o0.5 mg ml 1 (Puupponen-Pimia et al., 2001). Quercetin exhibited the lowest MIC of o5 ppm for E. coli among the tested PCs, which is in good agreement with previous studies (Buddhini, Jones, Ravishankar, & Jaroni, 2014; Santas et al., 2010; Yao et al., 2011). Yao et al. (2011) reported in vitro antimicrobial sensitivity of S. paratyphi and S. Enteridis to quercetin with the MICs 2.07 and 8.28 (mg ml 1), respectively. However, according to Pereira et al. (2007a) E. coli showed antimicrobial resistance against quercetin (100 mg ml 1), extracted from walnuts. The discrepancy in the antimicrobial activity of
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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Treatment
E.coli O157:H7 ATCC 35150
7
Salmonella cholerasuis subsp. cholerasuis ATCC 10708
Control
Chlorogenic acid (15 ppm)
Myricetin (a3; 10ppm b3; 20ppm)
Xanthohumol (15 ppm)
Fig. 5 – Scanning electron microscope observations of E. coli O157:H7 (a1-4) and S. cholerasuis subsp. cholerasuis (b1-4) treated with the natural PCs (1, control; 2, chlorogenic acid; 3, myricetin; 4, xanthohumol).
the current and previous studies may be attributed to the purity of quercetin that can be derived from a diverse variety of plant sources. Also, strains, culture medium and/or the methods used in the experiments could have contributed to antimicrobial resistance of E. coli against quercetin. High antimicrobial activity was observed with chlorogenic acid (MIC was o20 ppm) for all bacteria tested in accordance with the previous studies (Albayrak, Aksoy, Sagdic, & Budak 2010; Babic, Nguyenthe, Amiot, & Aubert, 1994; Xia, Wu, Shi, Yang, & Zhang, 2011); however, it was reported with a MIC of o80 (mg ml 1) against Gram-negatives (Lou et al., 2011). Among the PCs tested, it
exhibited the lowest MIC (o10 ppm), being consistent for all Salmonella. Curcumin's antimicrobial activity was found strain dependent on E. coli, where it did not inhibit E. coli O157:H7; ATCC 43895. However, it showed varying degrees of antimicrobial activity against other tested E. coli and Salmonella confirming those described by Bhawana, Basniwal, Buttar, Jain and Jain (2011). Previously, eugenol was found to be highly antimicrobial against E. coli after being incorporated into alginate-based edible coatings (Raybaudi-Massilia et al., 2009). Moreover, Kim et al. (2011) reported that eugenol (10%) from clove was highly effective against E. coli O157:H7 and Salmonella species on the
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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surface of fresh lettuce, which are in good agreement with our results for eugenol's high antimicrobial activity. Antimicrobial sensitivity of E. coli and Salmonella towards xanthohumol (MICo20 ppm) observed in this study differs from previous studies by Zanoli and Zavatti (2008) and Langezaal, Chandra, and Scheffer (1992) suggesting that xanthohumol (20 mg ml 1) has no antimicrobial activity against E. coli. The inconsistency may be attributed to different sources or purity of xanthohumol and different strains of E. coli. Thymol's consistent antimicrobial activity (MICo15 ppm) among the three treated strains of E. coli is in accordance with those described by Saei-Dehkordi, Tajik, Moradi, and Khalighi-Sigaroodi (2010) for E. coli O157:H7 (MICo2 mg ml 1). However, Bakathir and Abbas (2011) reported no antimicrobial activity for thymol (300 mg ml 1) against E. coli, when prepared with H2O, which could be due to limited solubility of thymol in water. In general, Gram-negative bacteria were reported to be resistant toward many antibacterial substances, due to the hydrophilic surface of their outer membrane and associated enzymes in the periplasmic space, which are capable of breaking down many molecules introduced from outside (Gao, van Belkum, & Stiles, 1999; Shan et al., 2007a). However, the results of this study revealed that the tested Gram-negative pathogens have varying degrees of antimicrobial susceptibility against natural phenolics. Furthermore, SEM observations confirmed the promising antimicrobial efficacy of PCs which caused severe physical damage and significant morphological alteration to all tested foodborne bacteria. Some authors have suggested that the loss of structural integrity and the ability of the membrane to act as a permeability barrier was due to the damage to the cell wall and cytoplasmic membrane (Cueva et al., 2010; Shan et al., 2007a). The distortion of the cell physical structure could expand and destabilize the membrane and increase membrane fluidity, which results in increased permeability (Gyawali & Ibrahim, 2014) and leakage of various vital intracellular constituents, such as ions, ATP, nucleic acids, and amino acids (Lambert et al., 2001; Shan et al., 2007a). It should be noted that the phenolic compounds 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 (Gyawali & Ibrahim, 2014). The inhibition of proton motive force, respiratory chain, electron transfer and substrate oxidation are the possible effects of the aforementioned phenomena. Furthermore, uncoupling of oxidative phosphorylation, inhibition of active transport, loss of pool metabolites, and disruption of synthesis of DNA, RNA, protein, lipid, and polysaccharides could also be observed (Gyawali & Ibrahim, 2014; Shan et al., 2007a).
5.
Conclusion
The present study evaluated the antimicrobial activity of phenolics through 60 h of incubation, while previous studies established MICs for 24 h incubation periods. It has been found that some of the sensitive pathogens (S. paratyphi and E. coli O157:H7) recover and become resistant with extended incubation. Adaptation of these bacteria may lead to resistance to the phenolics that may be caused by gene manipulation which may interfere with long term storage of foods. These findings provide evidence for
the high potential success of natural PCs in food safety for extended storage time (shelf life). It can be concluded that the selected PCs exhibit in vitro antimicrobial activity against E. coli, E. coli O157:H7, S. cholerasuis subsp. cholerasuis, S. paratyphi and S. Enteridis. The sensitivity of the foodborne bacteria to natural PCs depends on bacterial species, the purity and the polyphenol structure of the phenolics as well as the methods used for the experiments. The plant based antimicrobials can be employed as the alternatives for chemical food additives and preservatives since the PCs in the plant extracts have significant inhibitory activity against pathogenic bacteria. However, in order to widely apply PCs as antimicrobials in food systems, their organoleptic impacts, safety and toxicity must be further investigated.
Uncited references
Q2
Jaiswal, Mansa, Prasad, Jena and Negi (2014).
Acknowledgments Research supported by funding from the U. S. Department of Homeland Security, Science & Technology Directorate, Q4 through The National Institute For Hometown Security.
r e f e r e n c e s
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Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli Q1
Hayriye Cetin-Karaca, Melissa C. Newmann
Q3 University of Kentucky Department of Animal and Food Sciences, W. P. Garrigus Building, Lexington,
KY 40546-0215, United States
art i cle i nfo
ab st rac t
Article history:
The present study evaluated the antimicrobial efficacy of natural phenolic compounds (PC)
Received 24 July 2014
extracted from vegetables, fruits, herbs and spices; to inhibit the growth of Gram-negative
Received in revised form
foodborne bacteria which is defined as the minimum inhibitory concentration (MIC).
28 January 2015 Accepted 18 March 2015
Strains of Escherichia coli and Salmonella species were treated with natural PCs including; chlorogenic acid, curcumin, ( ) epicatechin, eugenol, myricetin, quercetin, rutin, thymol, thymoquinone, and xanthohumol. Concentrations of 5, 10, 15, and 20 ppm of each
Keywords:
compound were evaluated by a broth micro-dilution method and the MICs were deter-
Foodborne pathogen
mined by using optical density after 24 and 60 h of incubation. Structural alterations in
Phenolic compounds
treated bacteria were observed via scanning electron microscopy.
Minimum inhibitory concentration
For E. coli, thymoquinone showed the highest inhibition, followed by rutin, ( ) epicatechin and myricetin (MICo20 ppm for all), while Salmonella was most sensitive to
(MIC) Antimicrobial activity
( ) epicatechin (MICo15 ppm), followed by thymoquinone, rutin and myricetin (MICo20 ppm for all) following 60 h of incubation. The results demonstrated that the PCs have varying antimicrobial activities against foodborne pathogens following 24 and 60 h incubation periods. Natural sources of PCs contain major antibacterial components and have great potential to be used as natural antimicrobials and food preservatives, during long term storage. This study highlighted the antimicrobial efficacy of some novel PCs which may replace chemical antimicrobials and preservatives in food or pharmaceutical industry to partially or completely inhibit the growth of bacteria. & 2015 Published by Elsevier Ltd.
1.
Introduction
Spices and aromatic vegetable materials have long been used in food not only for their flavor and aromatic qualities and appetizing effects but also for their preservative and medicinal properties. Since ancient times, they have been used for preventing food spoilage and deterioration and extending the shelf life of foods (Gyawali & Ibrahim, 2014; Shan, Cai, Brooks, & Corke,
2007b). It has been extensively reported that the essential oils and secondary plant metabolites have shown antimicrobial activities against foodborne pathogens (Reichling, Schnitzler, Suschke, & Saller, 2009; Smith-Palmer, Stewart, & Fyfe, 1998). In addition, they have received great attention in recent decades due to their presumed role in the prevention of foodborne diseases, cancer, chronic and cardiovascular diseases, and in the slowdown of the aging process (Lee, Cesario, Wang,
n
Corresponding author. Tel.: þ1 859 257 5881. E-mail address: [email protected] (M.C. Newman).
http://dx.doi.org/10.1016/j.fbio.2015.03.002 2212-4292/& 2015 Published by Elsevier Ltd.
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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Shanbrom, & Thrupp, 2003; Liu, 2003; Nazzaro et al., 2009; Lou, Wang, Zhu, Ma, & Wang, 2011) indicating that, they have diverse beneficial biological functions including antimicrobial and antioxidant activities (Korukluoglu, Sahan, Yigit, Ozer, & Gucer, 2010; Perumalla & Hettiarachchy, 2011). Phenolic compounds (PC) are one of the most diverse groups of secondary metabolites found in a wide variety of fruits, vegetables, nuts, seeds, stems and flowers as well as tea, wine, propolis and honey (Moreno, Scheyer, Romano, & Vojnov, 2006). The exploration of natural antimicrobials for food preservation receives increased attention due to consumer awareness of natural food products and a growing concern of microbial resistance towards conventional food processing and preservation methods. The use of PCs as antimicrobial agents could potentially provide additional benefits, including dual-function effects of both preservation and delivery of health benefits (Cueva et al., 2010). By gaining the fundamental knowledge on the antimicrobial effects of plant derived PCs on pathogenic microorganisms, it is possible to search new strategies to combine the synergic antimicrobial effects of PCs with their natural biological properties. The results may permit the formulation of new products to be used as food preservatives or to be included in the human diet. Thus, the food industry is interested in developing natural components for the total or partial replacement of synthetic antimicrobials (Santas, Almajano, & Carbo, 2010). Mechanisms of action in the bacterial cell of bioactive plant compounds such as degradation of the cell wall (Nychas and Tassou, 1999), damage to cytoplasmic membrane and membrane proteins (Lambert, Skandamis, Coote, & Nychas, 2001), leakage of contents out of the cell, coagulation of cytoplasm, and depletion of the proton motive force (Burt, 2004; Gyawali & Ibrahim, 2014) have been reported. In general, variations in antimicrobial activities against bacteria may reflect differences in cell surface structures between Gram-negative and Grampositive species; Gram-positive being more susceptible to the action of phenolic acids than Gram-negative bacteria (Cueva et al., 2010). Also, the number, type and position of substituents in the benzene ring of the phenolic acids and the saturated side-chain length influence the antimicrobial potential of the phenolic acids against different microorganisms (Gill & Holley, 2006). Inhibitory effects of several natural bioactive compounds from berries and grapes (Puupponen-Pimia et al., 2001; Zanoli & Zavatti, 2008) herbs and spices (Albayrak, Aksoy, Sagdic, & Albayrak, 2012; Shan et al., 2007b; Shan, Cai, Brooks, & Corke, 2007a) and other plant extracts (Pereira et al., 2007b; RaybaudiMassilia, Mosqueda-Melgar, Soliva-Fortuny, & Martin-Belloso, 2009; Santas et al., 2010) were tested against food-relevant bacteria including: E. coli, Salmonella, Listeria, and Staphylococcus. Although the antimicrobial activity of some PCs including: thymol (Lambert et al., 2001; Santas et al., 2010), eugenol (Phanthong, Lomarat, Chomnawangb, & Bunyapraphatsaraa, 2013), carvacrol (Lambert et al., 2001; Santas et al., 2010), quercetin (Yao et al., 2011) and myricetin (Puupponen-Pimia et al., 2001) has been previously reported, the response after the long term exposure was not reported and there are still many unexplored sources. Moreover, thymoquinone and xanthohumol have not been included in antimicrobial studies and antimicrobial activity of chlorogenic acid, curcumin, ( ) epicatechin,
eugenol, myricetin and rutin have not been reported broadly on pathogenic Salmonella and E. coli. Objectives of this study are: (1) to evaluate the antimicrobial activity of selected natural PCs extracted from herbs, spices, vegetables, and fruits against Gram-negative foodborne pathogens: E. coli, E. coli O157:H7, Salmonella paratyphi, Salmonella cholerasuis subsps. cholerasuis and Salmonella Enteritidis, (2) to determine the MIC of the natural PCs and (3) to observe their prolonged antimicrobial activities over extended incubation of 60 h to simulate the long term storage by extending the exposure time of pathogens to PCs.
2.
Materials and methods
2.1.
Preparation of phenolic compounds
The natural PCs used in this study are: chlorogenic acid, curcumin, ( ) epicatechin, eugenol, myricetin, quercetin, rutin, thymol, thymoquinone, and xanthohumol (SigmaAldrich, St Louis, MO, USA). Each compound was prepared 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 0.2 mm filters (Millipore Corporation, Billerica, MA, USA) and stored at 4 1C in sterilized sealed glass containers until needed. All measurements were done at ambient temperature.
2.2. Bacterial strains, culture conditions and preparation of inoculums Foodborne pathogens including: E. coli, F. T. Jones (FTJ), E. coli O157:H7, ATCC 43895 and E. coli O157:H7, ATCC 35150, S. paratyphi, UK Micro 29A (UKM 29A), S. cholerasuis subsp. cholerasuis, ATCC 10708 and S. Enteritidis, UK ( ) H2S were supplied from the American Type Culture Collection and the University of Kentucky. Bacteria were grown and maintained on slants of brain–heart infusion (BHI) agar and stored at 4 1C until needed. Prior to each test, at least three consecutive transfers of the cultures were inoculated in BHI broth and they were incubated overnight at 37 1C. Then, the inoculums were standardized according to a MacFarland 0.5 turbidity standard (108 CFU ml 1) by diluting the sample (Taguri, Tanaka, & Kouno, 2004). Culture growth turbidity, which is indicated by the optical density (OD), was adjusted for each bacterium at a wavelength of 660 nm (OD660) by using the spectrophotometer (BioTek Synergy 4, Winooski, VT, USA) to the final concentrations of approximately 107–108 CFU ml 1. Cell counts were confirmed by using a spiral plating method with Plate Count Agar (PCA) and the Eddy Jet spiral plater (Neutec Group Inc., Farmingdale, NY, USA). The 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).
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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2.3. 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 (Standards, 2004) and modified for the 60 h incubation. Serial dilutions of the compounds (100 ml) were dispensed into 5 ml mueller hinton broth (MHB) to obtain the final concentrations of 5, 10, 15 and 20 ppm (mg l 1). Then, 100 ml of the overnight culture of bacteria was transferred aseptically into MHB. Controls included a compound free MHB broth (growth control), including only culture and a negative control, which contained solvent. Samples were dispensed to the wells of a 96well flat bottom micro-titer plate (Nalge NUNC Int., Corning, NY, USA) and incubated at 37 1C. All experiments were carried out twice in duplicates. After inoculation, the micro-titer plates were read immediately to get the initial OD using a calibrated spectrophotometer at 660 nm wavelength. Prior to each incubation process, the samples in the micro-titer plate were shaken automatically for 10 sec to get a consistent homogeneity. The absorbance was read at every 12-hour intervals for a total 60 hr of incubation period. Minimum inhibitory Concentration (MIC) was defined as the lowest concentration at which no bacterial growth was observed after incubation.
2.4. Investigation of structural changes via scanning electron microscopy Treated and non-treated (control) bacterial cultures were incubated for 24 h at 37 1C. PCs were applied to each strain at their specific MIC level. PCs were filter sterilized through 0.2 μm filters (Thermo Scientific, Nalgene, Rochester, NY, USA) to capture the bacteria for Scanning Electron Microscope (SEM) observations. The procedure was performed according to the description by Kalab, Yang and Denise (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 using Evex Nanoanalysis and Digital Imaging (Evex Analytical Version 2.0.1192, 2006) software.
2.5.
Statistical analysis
The antimicrobial activity of the PCs was subjected to General Linear Model procedure of Statistix 9.0 (2008). Significance level of Po0.05 of the null hypothesis was used to determine the significant variables. Difference between the means was identified by the use of Tukey HSD randomized complete block design between treatments and time.
3.
3
Results
Several PCs were screened for their antimicrobial properties against E. coli (FTJ), two strains of E. coli O157:H7 (ATCC 43895 and ATCC 35150), S. paratyphi (UK Micro 29A), S. cholerasuis subsp. cholerasuis (ATCC 10708) and S. Enteritidis (UK (–) H2S). The MICs at 24 and 60 h of all tested PCs are shown in Table 1. Prolonged antimicrobial effects of PCs and the response of the pathogens to long term exposure were observed with extended incubation time (60 h). Varying degrees of antimicrobial activity was observed with chlorogenic acid (o20 ppm) during 60 h of incubation. It exhibited the lowest MIC (o10 ppm) for all Salmonella strains, compared to other PCs. S. paratyphi and E. coli O157: H7 (ATCC 35150) demonstrated the highest antimicrobial sensitivity to curcumin (MICo5 ppm) during the entire incubation period. However, it showed no antimicrobial effect against E. coli O157:H7; ATCC 43895. All E. coli and Salmonella tested with eugenol exhibited high degrees of inhibition, with MICs: o20 and o15 ppm, respectively. Among the PCs tested, quercetin exhibited the lowest MIC of o5 ppm for all E. coli and o20 ppm for Salmonella species in extended incubation (60 hr). Both E. coli and Salmonella showed varying degrees of antimicrobial sensitivity towards thymol (MICo15 ppm) and xanthohumol (MICo20 ppm). S. paratyphi and S. cholerasuis subsp. cholerasuis were observed to be highly sensitive towards both thymol (MICo15 ppm) and xanthohumol (MICo20 ppm), while S. Enteritidis was consistently resistant during the entire incubation period. Fig. 1a–d shows the antimicrobial activity of thymoquinone against E. coli; FTJ, E. coli O157:H7; ATCC 43895, S. cholerasuis subsp. cholerasuis; ATCC 10708, and S. Enteritidis; ( ) H2S incubated for 60 h. Thymoquinone (MICo20 ppm) exhibited similar and high antimicrobial activity both for E. coli and Salmonella species during the 60 h of incubation, being the most effective one among the PCs tested for E. coli. Rutin inhibited the growth of all E. coli and Salmonella strains showing varying degrees of antimicrobial activity (Fig. 2a–d) with MIC of o20 ppm. ( ) Epicatechin was observed to have high antimicrobial activity against E. coli (Fig. 3a and b) and Salmonella (Fig. 3c and d) when used at o20 and o15 ppm concentrations, respectively. Similarly, myricetin showed a lower MIC (o15 ppm) for Salmonella than E. coli (MICo20 ppm) exhibiting antimicrobial activity through 60 hr of incubation time (Fig. 4a–d). In general, during the 60 h of incubation ( ) epicatechin exhibited the highest growth inhibition in Salmonella, followed by thymoquinone, myricetin and rutin (Figs. 1–4), while thymoquinone showed the highest growth inhibition in E. coli followed by ( ) epicatechin, myricetin and rutin (Figs. 1–4). Despite the fact that all the PCs exhibited antimicrobial activity, the inhibition for each microorganism tested was different, even for the ones from the same family. Thus, it is important to determine the inhibition effects of PCs for selected bacterial strains. In this study, all three strains of E. coli, including non-pathogenic E. coli and E. coli O157:H7 showed consistent antimicrobial sensitivity towards four (eugenol, quercetin, thymol, and thymoquinone, with the MICs of 20, 5, 15, and 20 ppm, respectively) of the ten PCs. Similar antimicrobial consistency
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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Table 1 – MICs (ppm) of PCs against E. coli and Salmonella after 24 and 60 h. Compound
pH
E. coli FTJ
E. coli O157:H7 ATCC 43895
Minimum inhibitory concentrations (ppm) at 24 h Chlorogenic 5.62 o10.0 420.0 acid Curcumin 5.54 o20.0 420.0 Eugenol 5.54 o20.0 o20.0 (–) Epicatechin 5.6 o20.0 420.0 Myricetin 6.46 o15.0 o10.0 Quercetin 7.18 420.0 420.0 Rutin 5.65 o20.0 420.0 Thymol 5.46 o20.0 o15.0 Thymoquinine 5.59 o20.0 420.0 Xanthohumol 8.7 o5.0 o10.0 Minimum inhibitory concentrations (ppm) at 60 h Chlorogenic 5.62 o15.0 o20.0 acid Curcumin 5.54 o20.0 420.0 Eugenol 5.54 o20.0 o20.0 ( ) 5.6 o20.0 o5.0a Epicatechin Myricetin 6.46 o20.0 o10.0 Quercetin 7.18 o5.0 o5.0 Rutin 5.65 o20.0 o5.0 Thymol 5.46 o15.0 o15.0 Thymoquinine 5.59 o20.0 o20.0 Xanthohumol 8.7 o15.0 o15.0
E. coli O157:H7 ATCC 35150
S. paratyphi UK Micro 29A
S. cholerasuis subsp. cholerasuis ATCC 10708
S. Enteritidis UK (–) H2S
o15.0
o5.0
o15.0
420.0
o5.0 o20.0 o15.0 o10.0 o5.0 o15.0 o15.0 o20.0 420.0
o15.0 o20.0 o10.0 o15.0 o20.0 420.0 420.0 420.0 o20.0
o15.0 o20.0 o20.0 o20.0 o15.0 420.0 420.0 o5.0 o20.0
o5.0 o15.0 o20.0 o10.0 420.0 420.0 420.0 o20.0 420.0
o10.0
o10.0
o10.0
o10.0
o5.0 o20.0 o10.0
o5.0 o15.0 o15.0
o15.0 o15.0 o15.0
o20.0 o15.0 o15.0
o15.0 o5.0 o15.0 o15.0 o20.0 420.0
o20.0 o20.0 o20.0 o15.0 o20.0 o20.0
o15.0 o15.0 o15.0 o15.0 o15.0 o15.0
o5.0 o10.0 o15.0 420.0 o20.0 420.0
Active until 12 h of incubation.
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
E. coli Absorbance at 660nm
Absorbance at 660nm
a
0
12
24
36
48
60
E. coli O157:H7
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0
12
24
S. cholerasuis subsp.cholerasuis
0
12
24 36 Time (hours)
48
60
Absorbance at 660 nm
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
36
48
60
Time (hours)
Time (hours)
Absorbance at 660nm
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S. Enteritidis
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0
12
24 36 Time (hours)
48
60
Fig. 1 – Antimicrobial activity of thymoquinone against a) E. coli; FTJ, ( ) 20 ppm; b) E. coli O157:H7; ATCC 43895, ( ) 20 ppm; c) S. cholerasuis subsp. cholerasuis; ATCC 10708, ( ) 15 ppm and d) S. Enteritidis; UK ( ) H2S, ( ) 20 ppm ( ) control. Error bars represent standard deviation from the mean, Po0.05.
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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E. coli Absorbance at 660 nm
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
12
24
36
48
E. coli O157:H7
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
60
0
12
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
S. cholerasuis subsp. cholerasuis
0
12
24 36 Time (hours)
24
36
48
60
Time (hours)
Absorbance at 660 nm
Absorbance at 660 nm
Time (hours)
48
S. Enteritidis
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
60
0
12
24 36 Time (hours)
48
60
E. coli
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Absorbance at 660 nm
Absorbance at 660 nm
Fig. 2 – Antimicrobial activity of rutin against a) E. coli; FTJ, ( ) 20 ppm; b) E. coli O157:H7; ATCC 35150, ( ) 15 ppm; c) S. cholerasuis subsp. cholerasuis; ATCC 10708, ( ) 15 ppm and d) S. Enteritidis; UK ( ) H2S, ( ) 15 ppm ( ) control. Error bars represent standard deviation from the mean, Po0.05.
0
12
24
36
48
E. coli O157:H7
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
60
12
S. paratyphi Absorbance at 660 nm
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
12
24
36
24
36
48
60
48
60
Time (hours)
Time (hours)
Absorbance at 660 nm
435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494
Absorbance at 660 nm
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48
60
Time (hours)
S. Enteritidis
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
12
24 36 Time (hours)
Fig. 3 – Antimicrobial activity of ( ) epicatechin against a) E. coli; FTJ, ( ) 20 ppm; b) E. coli O157:H7; ATCC 35150, ( ) 10 ppm; c) S. paratyphi; UKM 29A, ( ) 15 ppm and d) S. Enteritidis; UK ( ) H2S, ( ) 15 ppm ( ) control. Error bars represent standard deviation from the mean, Po0.05.
was observed for Salmonella strains towards chlorogenic acid, eugenol and ( ) epicatechin, with MICs of 10, 15, and 15 ppm, respectively.
Bacteria treated with PCs were observed by SEM to confirm the antimicrobial efficacy of the PCs along with the morphological changes in the appearance of the cells. Fig. 5 depicts
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
24 36 Time (hours)
48
0
S. cholerasuis subsp. cholerasuis
0
12
24 36 Time (hours)
48
60
E. coli O157:H7
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
60
Absorbance at 660 nm
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
12
Absorbance at 660 nm
Absorbance at 660 nm
E. coli
0
Absorbance at 660 nm
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12
24 36 Time (hours)
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60
48
60
S. Enteritidis
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
12
24 36 Time (hours)
Fig. 4 – Antimicrobial activity of myricetin against a) E. coli; FTJ, ( ) 20 ppm; b) E. coli O157:H7; ATCC 35150, ( ) 15 ppm; c) S. cholerasuis subsp. cholerasuis; ATCC 10708, ( ) 15 ppm and d) S. Enteritidis; UK ( ) H2S, ( ) 5 ppm ( ) control. Error bars represent standard deviation from the mean, Po0.05.
the SEM images of treated and control samples for two selected bacteria (E. coli O157:H7 and S. cholerasuis subsp. cholerasuis) treated with MICs of chlorogenic acid, myricetin and xanthohumol. It is clearly visible that treatments with PCs (for 24 h) exhibited remarkable antimicrobial activity by damaging the cell wall followed by the leakage of the interior matter in all tested pathogens treated with the PCs. Content of some cells appeared depleted (Fig. 5a2, a3, a4 and b4) and some cells (Fig. 5a2, a3, a4 and b4) even appeared to be empty and the remains were flaccid, which prevents the cells to recover and function as normal. The slimy appearance and rupture of cell wall was observed at chlorogenic acid (15 ppm) in E. coli O157:H7 (Fig. 5a2), myricetin (20 ppm) in S. cholerasuis subsp. cholerasuis (Fig. 5b3), xanthohumol (10 and 20 ppm) in E. coli O157:H7 (Fig. 5a4) and S. cholerasuis subsp. cholerasuis (Fig. 5b4). In Salmonella a decrease in size of cells (Fig. 5b2, b3 and b4) was observed when compared to controls (Fig. 5b1). In general, the bacteria after treatment with PCs showed a lot of adhered material around the cell wall.
4.
Discussion
Although the antimicrobial activity (for 24 hr) of some PCs has been previously reported, this study is the first to evaluate the antimicrobial efficacy of selected PCs during 60 h of incubation. Our findings provide evidence for the prolonged antimicrobial activity of natural plant PCs which might have high potential success in food safety for extended storage time. Recently, Bakathir and Abbas (2011) reported that thymoquinone suspended with H2O have no antimicrobial activity against
E. coli, while in this study, thymoquinone showed remarkable antimicrobial activity (MICo20 ppm) for both E. coli and Salmonella. The inconsistency may be attributed to the different solvents and/or the methods used for preparation of antimicrobial suspension. Rutin's high antimicrobial activity (MICo20 ppm) against both Salmonella and E. coli is in good agreement with the previously published data where Salmonella (Askun, Tumen, Satil, & Ates, 2009a), E. coli O157:H7 and other E. coli (Askun, Tumen, Satil, & Ates, 2009b; Pereira et al., 2007a) strains are shown to be highly susceptible to rutin in vitro. However, it should be noted that Lee and Lee (2010) reported that S. Enteridis and some E. coli strains were not inhibited when treated with rutin (800 mg ml 1) in vitro. Our results for the antimicrobial activity of ( ) epicatechin; including the sensitivity of Salmonella species to () epicatechin (795 mg ml 1) (Taguri et al., 2004) and E. coli being strain dependent are in accordance with the earlier results (Bancirova, 2010; Cueva et al., 2010; Friedman, 2014). Myricetin exhibited a remarkable inhibition for both Salmonella and E. coli being in accordance with previous reports for the inhibition of S. paratyphi, S. Enteridis (Yao et al., 2011; Friedman, 2014) and E. coli with the MIC of o0.5 mg ml 1 (Puupponen-Pimia et al., 2001). Quercetin exhibited the lowest MIC of o5 ppm for E. coli among the tested PCs, which is in good agreement with previous studies (Buddhini, Jones, Ravishankar, & Jaroni, 2014; Santas et al., 2010; Yao et al., 2011). Yao et al. (2011) reported in vitro antimicrobial sensitivity of S. paratyphi and S. Enteridis to quercetin with the MICs 2.07 and 8.28 (mg ml 1), respectively. However, according to Pereira et al. (2007a) E. coli showed antimicrobial resistance against quercetin (100 mg ml 1), extracted from walnuts. The discrepancy in the antimicrobial activity of
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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Treatment
E.coli O157:H7 ATCC 35150
7
Salmonella cholerasuis subsp. cholerasuis ATCC 10708
Control
Chlorogenic acid (15 ppm)
Myricetin (a3; 10ppm b3; 20ppm)
Xanthohumol (15 ppm)
Fig. 5 – Scanning electron microscope observations of E. coli O157:H7 (a1-4) and S. cholerasuis subsp. cholerasuis (b1-4) treated with the natural PCs (1, control; 2, chlorogenic acid; 3, myricetin; 4, xanthohumol).
the current and previous studies may be attributed to the purity of quercetin that can be derived from a diverse variety of plant sources. Also, strains, culture medium and/or the methods used in the experiments could have contributed to antimicrobial resistance of E. coli against quercetin. High antimicrobial activity was observed with chlorogenic acid (MIC was o20 ppm) for all bacteria tested in accordance with the previous studies (Albayrak, Aksoy, Sagdic, & Budak 2010; Babic, Nguyenthe, Amiot, & Aubert, 1994; Xia, Wu, Shi, Yang, & Zhang, 2011); however, it was reported with a MIC of o80 (mg ml 1) against Gram-negatives (Lou et al., 2011). Among the PCs tested, it
exhibited the lowest MIC (o10 ppm), being consistent for all Salmonella. Curcumin's antimicrobial activity was found strain dependent on E. coli, where it did not inhibit E. coli O157:H7; ATCC 43895. However, it showed varying degrees of antimicrobial activity against other tested E. coli and Salmonella confirming those described by Bhawana, Basniwal, Buttar, Jain and Jain (2011). Previously, eugenol was found to be highly antimicrobial against E. coli after being incorporated into alginate-based edible coatings (Raybaudi-Massilia et al., 2009). Moreover, Kim et al. (2011) reported that eugenol (10%) from clove was highly effective against E. coli O157:H7 and Salmonella species on the
Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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surface of fresh lettuce, which are in good agreement with our results for eugenol's high antimicrobial activity. Antimicrobial sensitivity of E. coli and Salmonella towards xanthohumol (MICo20 ppm) observed in this study differs from previous studies by Zanoli and Zavatti (2008) and Langezaal, Chandra, and Scheffer (1992) suggesting that xanthohumol (20 mg ml 1) has no antimicrobial activity against E. coli. The inconsistency may be attributed to different sources or purity of xanthohumol and different strains of E. coli. Thymol's consistent antimicrobial activity (MICo15 ppm) among the three treated strains of E. coli is in accordance with those described by Saei-Dehkordi, Tajik, Moradi, and Khalighi-Sigaroodi (2010) for E. coli O157:H7 (MICo2 mg ml 1). However, Bakathir and Abbas (2011) reported no antimicrobial activity for thymol (300 mg ml 1) against E. coli, when prepared with H2O, which could be due to limited solubility of thymol in water. In general, Gram-negative bacteria were reported to be resistant toward many antibacterial substances, due to the hydrophilic surface of their outer membrane and associated enzymes in the periplasmic space, which are capable of breaking down many molecules introduced from outside (Gao, van Belkum, & Stiles, 1999; Shan et al., 2007a). However, the results of this study revealed that the tested Gram-negative pathogens have varying degrees of antimicrobial susceptibility against natural phenolics. Furthermore, SEM observations confirmed the promising antimicrobial efficacy of PCs which caused severe physical damage and significant morphological alteration to all tested foodborne bacteria. Some authors have suggested that the loss of structural integrity and the ability of the membrane to act as a permeability barrier was due to the damage to the cell wall and cytoplasmic membrane (Cueva et al., 2010; Shan et al., 2007a). The distortion of the cell physical structure could expand and destabilize the membrane and increase membrane fluidity, which results in increased permeability (Gyawali & Ibrahim, 2014) and leakage of various vital intracellular constituents, such as ions, ATP, nucleic acids, and amino acids (Lambert et al., 2001; Shan et al., 2007a). It should be noted that the phenolic compounds 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 (Gyawali & Ibrahim, 2014). The inhibition of proton motive force, respiratory chain, electron transfer and substrate oxidation are the possible effects of the aforementioned phenomena. Furthermore, uncoupling of oxidative phosphorylation, inhibition of active transport, loss of pool metabolites, and disruption of synthesis of DNA, RNA, protein, lipid, and polysaccharides could also be observed (Gyawali & Ibrahim, 2014; Shan et al., 2007a).
5.
Conclusion
The present study evaluated the antimicrobial activity of phenolics through 60 h of incubation, while previous studies established MICs for 24 h incubation periods. It has been found that some of the sensitive pathogens (S. paratyphi and E. coli O157:H7) recover and become resistant with extended incubation. Adaptation of these bacteria may lead to resistance to the phenolics that may be caused by gene manipulation which may interfere with long term storage of foods. These findings provide evidence for
the high potential success of natural PCs in food safety for extended storage time (shelf life). It can be concluded that the selected PCs exhibit in vitro antimicrobial activity against E. coli, E. coli O157:H7, S. cholerasuis subsp. cholerasuis, S. paratyphi and S. Enteridis. The sensitivity of the foodborne bacteria to natural PCs depends on bacterial species, the purity and the polyphenol structure of the phenolics as well as the methods used for the experiments. The plant based antimicrobials can be employed as the alternatives for chemical food additives and preservatives since the PCs in the plant extracts have significant inhibitory activity against pathogenic bacteria. However, in order to widely apply PCs as antimicrobials in food systems, their organoleptic impacts, safety and toxicity must be further investigated.
Uncited references
Q2
Jaiswal, Mansa, Prasad, Jena and Negi (2014).
Acknowledgments Research supported by funding from the U. S. Department of Homeland Security, Science & Technology Directorate, Q4 through The National Institute For Hometown Security.
r e f e r e n c e s
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Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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Please cite this article as: Cetin-Karaca, H., & Newman, M. C. Antimicrobial efficacy of plant phenolic compounds against Salmonella and Escherichia Coli. Food Bioscience (2015), http://dx.doi.org/10.1016/j.fbio.2015.03.002
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