inhibition of foodborne pathogens and spoiling bacteria by essential oil and extracts of erigeron...

INHIBITION OF FOODBORNE PATHOGENS AND SPOILINGBACTERIA BY ESSENTIAL OIL AND EXTRACTS OF ERIGERON

RAMOSUS (WALT.) B.S.P.

ATIQUR RAHMAN1,2 and SUN CHUL KANG1,3

1Department of BiotechnologyDaegu University

Kyoungsan, Kyoungbook 712-714, Korea

2Department of Applied Chemistry and Chemical TechnologyIslamic University

Kushtia 7003, Bangladesh

Accepted for Publication January 5, 2008

ABSTRACT

The antibacterial potential of essential oil and methanolic extracts ofErigeron ramosus (Walt.) B.S.P. was evaluated. Thirty-one components repre-senting 95.3% of the total oil were identified, of which b-caryophyllene(24.0%), a-humulene (14.5%), 1,8-cineole (9.0%), eugenol (7.2%), globulol(7.1%), caryophyllene oxide (5.2%), d-cadinene (5.0%), a-copaene (4.9%)and widdrol (2.0%) were the major components. The antibacterial activity ofessential oil and methanolic extracts of E. ramosus was determined in vitrousing the agar diffusion method and minimum inhibitory concentration deter-mination test against 14 (seven gram-positive and seven gram-negative) food-borne bacteria. The essential oil (5 mL/mL, corresponding to 1,000 ppm/disc),methanol extract and its different organic subfractions (7.5 mL/mL, corre-sponding to 1500 ppm/disc) of E. ramosus displayed a great potential ofantibacterial activity against all gram-positive bacteria: Staphylococcusaureus (ATCC 6538 and KCTC 1916), Listeria monocytogenes (ATCC 19116,ATCC 19118, ATCC 19166 and ATCC 15313) and Bacillus subtilis ATCC6633 and four gram-negative bacteria: Pseudomonas aeruginosa KCTC 2004,Enterobacter aerogenes KCTC 2190 and Escherichia coli (0157:H7 ATCC43888 and ATCC 8739). The zones of inhibition of different concentrations ofessential oil and methanolic extracts against the tested bacteria were found inthe range of 10.1~22.3 mm, and MIC values were recorded between 62.5 and500 mg/mL.

3 Corresponding author. TEL: +82-53-850-6553; FAX: +82-53-850-6559; EMAIL: [email protected]

Journal of Food Safety 29 (2009) 176–189.© 2009, The Author(s)Journal compilation © 2009, Wiley Periodicals, Inc.

176

PRACTICAL APPLICATIONS

The use of essential oil and organic extracts of Erigeron ramosus (Walt.)B.S.P. as antibacterial agents will be suitable for applications on the foodindustry as natural preservatives or flavoring to control foodborne pathogens.They can be used as growth inhibitors of Listeria monocytogenes, Staphylo-coccus aureus, Bacillus subtilis, Escherichia coli, Enterobacter aerogenes andPseudomonas aeruginosa, some important foodborne pathogens and spoilingbacteria. The main reason for their suitability is their natural origin, whichconsumers find comforting and which is beneficial for the environment, andthe very low risk that pathogens will develop resistance to the mixture ofcomponents that make up the oil and extracts with their apparent diversity ofantibacterial mechanisms. These beneficial characteristics could increase foodsafety and shelf life.

INTRODUCTION

Illness caused by the consumption of contaminated foods has a wideeconomic and public health impact worldwide (Mead et al. 1999). Manypathogenic microorganisms such as Listeria monocytogenes, Staphylococcusaureus, Bacillus subtilis, Escherichia coli, Salmonella sp. and Pseudomonasaeruginosa have been reported as the causal agents of foodborne diseases(McCabe-Sellers and Samuel 2004). A variety of different chemical and syn-thetic compounds have been used as antimicrobial agents to inhibit bacteria infoods. Due to the identified and potential toxicity of chemical food preserva-tives, there have been increased demands for food preservatives from naturalsources. The demands for more natural antimicrobials have driven foodscientists to investigate the effectiveness of inhibitory compounds, such asessential oils (Nguefack et al. 2004), and extracts from plants (Nasar-Abbasand Halkman 2004; Shin et al. 2004). Essential oils are a complex mixture ofcompounds, mainly monoterpenes, sesquiterpenes and their correspondingoxygenated derivatives (alcohols, aldehydes, esters, ethers, ketones, phenolsand oxides) from plants, which are widely known for their scents and flavors.Plant-derived essential oils have been long used as flavoring agents or preser-vatives in foods, beverages and confectionary products, and also have a broadspectrum of in vitro antimicrobial activities (Conner 1993). In general, plant-derived essential oils are considered as nonphytotoxic compounds and poten-tially effective against microorganisms (Pandey et al. 1982). Thus, essentialoils and plant extracts are promising natural antimicrobial agents with poten-tial applications in food industries for controlling of foodborne pathogens andspoiling bacteria.

177ANTIBACTERIAL ACTIVITY OF ERIGERON RAMOSUS

The genus Erigeron is a member of the Compositae (Asteraceae) familyand contains more than 400 species. Erigeron ramosus (Walt.) B.S.P. is anindigenous weed from northern America and Canada, widely found in fields(Nesom 1989), and it has been introduced to many parts of the world (Holmet al. 1979). This species is also commonly found all over in Korea. Thechemical constituents of the genus Erigeron plants such as Erigeron annuus,Erigeron philadelphicus and Erigeron sumatrensis have been previouslyinvestigated and shown to contain monoterpenoids, diterpenoid, sesquiterpe-noids, triterpenoids, sterols and phenolic compounds (Miyazawa et al. 1981;Waddell et al. 1983; Oh et al. 2002; Iijima et al. 2003). Secondary metabolitesisolated from E. annuus showed inhibitory effects on seed germination, withpotential value as agriculturally useful products (Oh et al. 2002). However,there are no reports available in the literature on the analyses of essential oiland antibacterial activity of the oil and extracts of E. ramosus (Walt.) B.S.P.occurring in Korea.

Therefore, the aims of the present study were (1) to examine the chemicalcomposition of the essential oil of E. ramosus (Walt.) B.S.P.; and (2) toevaluate the antibacterial activity of essential oil and methanolic extractsagainst a range of organisms comprising food spoilage and foodborne bacteria,with emphasis on the possible future uses of the essential oil and plant extractsas alternative antibacterial compounds.

MATERIALS AND METHODS

Plant material

The leaves, stems and flowers of E. ramosus (Walt.) B.S.P. were collectedfrom Kyungsan city area of the Republic of Korea in June 2006. The plant wasidentified on the basis of morphological features and by the database presentin the library at the Department of Biotechnology, Daegu University, Republicof Korea, and a voucher specimen has been deposited in the herbarium of theDepartment of Biotechnology, Daegu University, Republic of Korea.

Isolation of the essential oil

The air-dried flower parts (250 g) of E. ramosus (Walt.) B.S.P. wassubjected to hydrodistillation for 3 h using a Clevenger-type apparatus. The oilwas dried over anhydrous sodium sulphate and preserved in a sealed vial at 4Cuntil further analysis.

Preparation of crude methanolic extracts

The air-dried leaves and stems of E. ramosus (Walt.) B.S.P. were pulver-ized into powdered form. The dried powder (50 g) was extracted three times

178 A. RAHMAN and S.C. KANG

with 80% methanol (200 mL ¥ 3) at room temperature, and the solvents fromthe combined extracts were evaporated by a vacuum rotary evaporator (EYELAN-1000, Tokyo Rikakikai Co. Ltd., Tokyo, Japan). The methanol extract (5.7 g)was suspended in water and extracted successively with hexane, chloroform andethyl acetate to give hexane (1.9 g), chloroform (1.2 g) and ethyl acetate (0.8 g),and residual methanol fractions (0.7 g), respectively. Solvents (analyticalgrade) for extraction were obtained from commercial sources.

Gas chromatography-mass spectrometry (GC-MS)/MS analysis

The GC-MS analysis of the essential oil was performed using a ShimadzuGC-MS (GC-17A, Shimadzu, Kyoto, Japan), equipped with a ZB-1 MS fusedsilica capillary column (30 m ¥ 0.25 mm i.d., film thickness 0.25 mm). ForGC-MS detection, an electron ionization system with an ionization energy of70 eV was used. Helium gas was used as the carrier gas at a constant flow rateof 1 mL/min. Injector and MS transfer line temperature were set at 220 and290C, respectively. The oven temperature was programmed from 50 to 150Cat 3C/min, then held isothermal for 10 min and finally raised to 250C at10C/min. Diluted samples (1/100, v/v, in methanol) of 1.0 mL was injectedmanually in the splitless mode. The relative percentage of the oil constituentswas expressed as percentages by peak area normalization.

Identification of compounds of the essential oil was based on GC reten-tion time on a ZB-1 capillary column, computer matching of mass spectra withthose of standards (Wiley 6.0 data of GC-MS system), and, whenever possible,by co-injection with authentic compounds (Adam 2001).

Microorganisms

The following food-spoiling and foodborne bacterial strains were used inthe antimicrobial tests: S. aureus ATCC 6538, S. aureus KCTC 1916, L.monocytogenes (ATCC 19116, ATCC 19118, ATCC 19166 and ATCC 15313),B. subtilis ATCC 6633, P. aeruginosa KCTC 2004, Enterobacter aerogenesKCTC 2190, E. coli ATCC 8739, E. coli 0157:H7 ATCC 43888, E. coli 0157(human), Salmonella enteritidis KCTC 12021 and S. typhimurium KCTC2515. The strains were obtained from the Korea Food and Drug Administra-tion (KFDA), Daegu, South Korea. Listeria monocytogenes strains were main-tained on BHI agar (brain heart infusion, Becton Dickinson, Sigma, St. Louis,MO) medium at 4C. The other strains were maintained on Luria Broth (LB)agar medium (Acumedia Manufacturers, Inc., Lansing, MI) at 4C.

Antibacterial activity assay

The agar diffusion method (Murray et al. 1995) was used for antibacterialassay. Petri plates were prepared by pouring 20 mL of BHI agar and LB agar


(1.0% trypton, 0.5% yeast extract, 1.0% NaCl, 1.5% agar) medium and allowedto solidify. Plates were dried, and 1 mL of standardized inoculum suspensionwas poured and uniformly spread. The excess inoculum was drained out andthe inoculum was allowed to dry for 5 min. A Whatman No. 1 sterile filterpaper disc (6 mm diameter) was impregnated with 5 mL/mL of essential oil(1,000 ppm/disc) and 7.5 mL/mL of MeOH extract and its derived subfractions(1500 ppm/disc). Negative controls were prepared using the same solventemployed to dissolve the samples. Standard reference antibiotics, tetracyclineand streptomycin (10 mg/disc, each from Sigma-Aldrich Co., St. Louis, MO),were used as positive controls for the tested bacteria. Antibacterial activity wasevaluated by measuring the diameter of the zones of inhibition against thetested bacteria. Each assay in this experiment was replicated three times.

Minimum inhibitory concentration (MIC)

MIC of essential oil, methanol, and methanol-derived subfractions ofhexane, chloroform and ethyl acetate, was tested by the twofold serial dilutionmethod (Chandrasekaran and Venkatesalu 2004). The test samples of oil,methanol extract and its derived subfractions were incorporated into 1 mL BHIbroth and LB medium to get a concentration of 1,000 mg/mL, and seriallydiluted to achieve 500, 250 125, 62.5 and 31.25 mg/mL, respectively. A 10-mLstandardized suspension of each tested organism (108 cfu/mL) was transferredto each tube. The control tubes contained only bacterial suspension and wereincubated at 37C for 24 h. The lowest concentration of the test samples, whichdid not show any growth of tested organism after macroscopic evaluation, wasdetermined as MIC.

RESULTS

Chemical composition of the essential oil

The hydrodistillation of the air-dried flower parts of E. ramosus (Walt.)B.S.P. gave the dark yellowish oil with a yield of 0.4% (w/w). GC-MS analysesof the oil led to the identification of 31 different components, representing95.3% of the total oil. The identified compounds are listed in Table 1 accordingto their elution order on a ZB-1 capillary column. The oil contains a complexmixture consisting of mainly oxygenated mono- and sesquiterpene hydro-carbons. The major compounds detected were b-caryophyllene (24.0%),a-humulene (14.5%), 1,8-cineole (9.0%), eugenol (7.2%), globulol (7.1%),caryophyllene oxide (5.2%), d-cadinene (5.0%), a-copaene (4.9%) andwiddrol (2.0%). Geraniol (1.9%), spathulenol (1.4%), viridiflorol (1.3%),nerolidol (0.8%), trans-b-farnesene (0.6%), ledol (0.6%), b-selinene (0.5%)


and n-nonanal (0.5%) were also found to be the minor components of E.ramosus (Walt.) B.S.P. oil in the present study.

In vitro antibacterial activity

The in vitro antibacterial activity of essential oil, methanol extract andmethanol-derived subfractions of E. ramosus (Walt.) B.S.P. against the

TABLE 1.COMPONENTS OF ESSENTIAL OIL OF ERIGERONRAMOSUS (WALT.) B.S.P. IDENTIFIED BY GC-MS

Peak no. Components Percentagein total oil

1 2-phenethyl alcohol 0.62 Eugenol 7.23 b-chamigrene 0.64 a-humulene 14.55 Nerolidol 0.86 g-cadinene 0.67 a-copaene 4.98 Aromadendrene 1.39 b-caryophyllene 24.0

10 d-cadinene 5.011 Trans-b-farnesene 0.612 Aromadendrene epoxide 0.513 Caryophyllene oxide 5.214 3-p-menthen-9-ol 0.415 (Z)-5-pentadecen-7-yne 0.516 b-selinene 0.517 Spathulenol 1.418 (Z)-6-hexadecen-4-yne 0.419 Widdrol 2.020 Globulol 7.121 Viridiflorol 1.322 Ledol 0.623 n-nonanal 0.524 Ledane 0.525 Geraniol 1.926 Myristic acid 1.027 Patchulane 0.228 1,8-cineole (eucalyptol) 9.029 Dibutyl phthalate 0.330 Stearic acid 0.731 Palmitic acid 1.2

Total 95.3

GC-MS, gas chromatography-mass spectrometry.


employed bacteria was qualitatively and quantitatively assessed by the pres-ence or absence of inhibition zones. According to the results given in Table 2,a total of 14 food spoilage and foodborne bacterial strains, including sevengram-positive and seven gram-negative bacteria, were tested. The oil exhib-ited antibacterial activity against all seven gram-positive and four gram-negative bacteria at the concentration of 5 mL/mL (1,000 ppm/disc). The oilexhibited a potent inhibitory effect against S. aureus ATCC 6538, S. aureusKCTC 1916, L. monocytogenes (ATCC 19116, ATCC 19118, ATCC 19166and ATCC 15313), B. subtilis ATCC 6633, P. aeruginosa KCTC 2004, E.aerogenes KCTC 2190, E. coli ATCC 8739 and E. coli 0157:H7 ATCC 43888,with diameter zones of inhibition of 22.3~12.2 mm, as shown in Table 2.Methanol extract of E. ramosus (Walt.) B.S.P. and its derived subfractions alsorevealed a great potential of antibacterial activity against all seven gram-positive and four gram-negative bacteria, at the concentration of 7.5 mL/mL,corresponding to 1500 ppm/disc (Table 2). Methanol extract showed thestrongest antibacterial effect against S. aureus (ATCC 6538 and KCTC 1916),L. monocytogenes (ATCC 19116, ATCC 19118 and ATCC 19166), B. subtilisATCC 6633, P. aeruginosa KCTC 2004 and E. coli ATCC 8739, with thediameter of inhibition zones ranging from 16.1~21.4 mm, as compared withstandard drug streptomycin. On the other hand, hexane, chloroform and ethylacetate subfractions showed interesting antibacterial effect, with inhibitionzones in the range of 10.1~20.2 mm. In this study, in some cases, the oil,methanol extract and organic subfractions (chloroform and ethyl acetate)exhibited higher antibacterial activity compared with streptomycin, whiletetracycline showed higher activity in some other cases than the essential oiland solvent fractions. Hexane fraction displayed a moderate inhibitory effect.However, the residual methanol subfraction did not show any activity againstall the bacterial strains tested (data not shown). The blind control did notinhibit the growth of the bacteria tested. No inhibitory effect was observedagainst E. coli 0157 (human), S. enteritidis KCTC 12021 and S. typhimuriumKCTC 2515, in all cases.

MIC

As shown in Table 3, the MIC values for the oil were found moresusceptible to S. aureus ATCC 6538, L. monocytogenes ATCC 19116 andB. subtilis ATCC 6633 (62.5 mg/mL for each) than those of S. aureus KCTC1916, L. monocytogenes ATCC 19118, P. aeruginosa KCTC 2004 and E. coliATCC 8739 (125 mg/mL for each). On the other hand, MIC values of themethanol extract and its derived subfractions of hexane, chloroform and ethylacetate against the tested bacteria were found in the range of 62.5~500 mg/mL(Table 3). Methanol extract and its chloroform fraction showed higher


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DISCUSSION

Since ancient times, the aromatic plant extracts have been used for manypurposes, such as food, drugs and perfumery. Historically, many plant oils andextracts have been reported to have antimicrobial properties (Hoffman 1987).Also, the renewal of interest in food industry and increasing consumerdemands for effective, safe and natural products means that quantitative dataon plant oils and extracts are required. A number of publications documentedthe antimicrobial activity of essential oil constituents and plant extracts(Morris et al. 1979; Nasar-Abbas and Halkman 2004). In recent years, severalresearchers have also reported that mono- and sesquiterpene hydrocarbons andtheir oxygenated derivatives are the major components of essential oils from

TABLE 3.MINIMUM INHIBITORY CONCENTRATION OF ESSENTIAL OIL, MeOH EXTRACT AND

SUBFRACTIONS OF MeOH EXTRACT OF ERIGERON RAMOSUS (WALT.) B.S.P. AGAINSTFOODBORNE PATHOGENS AND SPOILING BACTERIA

Microorganism MIC*

Essentialoil

MeOHextract

Subfractions of MeOH extract

Hexane CHCl3 EtOAc

Staphylococcus aureus ATCC 6538 62.5 62.5 250 62.5 125S. aureus KCTC 1916 125 62.5 250 125 125Listeria monocytogenes ATCC 19116 62.5 125 250 125 125L. monocytogenes ATCC 19118 125 125 500 125 250L. monocytogenes ATCC 19166 250 250 500 250 250L. monocytogenes ATCC 15313 250 250 500 250 500Bacillus subtilis ATCC 6633 62.5 62.5 125 62.5 125Pseudomonas aeruginosa KCTC 2004 125 125 500 125 125Enterobacter aerogenes KCTC 2190 250 250 500 250 500Escherichia coli ATCC 8739 125 250 nd 250 500E. coli 0157:H7 ATCC 43888 500 500 nd 500 ndE. coli 0157 (human) nd nd nd nd ndSalmonella enteritidis KCTC 12021 nd nd nd nd ndS. typhimurium KCTC 2515 nd nd nd nd nd

* Minimum inhibitory concentration (values in mg/mL).nd, not detected.


plant origin, which have enormous potential to inhibit microbial pathogens(Gudzic et al. 2002; Filipowicz et al. 2003). Most of the studies on the mecha-nism of phenolic compounds have focused on their effects on cellular mem-branes. Actually, phenolics not only attack cell wall and cell membranes,thereby affecting their permeability and release of intracellular constituents(ribose, Na glutamate, etc.), but also interfere with membrane function, such aselectron transport, nutrient uptake, protein and nucleic acid synthesis, andenzyme activity. Thus, active phenolic compounds might have several invasivetargets, which could lead to their inhibition of bacteria.

In this study, the essential oil and methanolic extracts of E. ramosus(Walt.) B.S.P. showed remarkable antibacterial effects against most of thebacteria, such as S. aureus (ATCC 6538 and KCTC 1916), L. monocytogenes(ATCC 19116, ATCC 19118, ATCC 19166 and ATCC 15313), B. subtilisATCC 6633, P. aeruginosa KCTC 2004, E. aerogenes KCTC 2190, E. coliATCC 8739 and E. coli 0157:H7 ATCC 43888. This activity could beattributed to the presence of phenolic compounds and oxygenated mono-and sesquiterpene hydrocarbons, and these finding are in agreement withthe previous reports (Guillen and Manzanos 1998; Shunying et al. 2005).E. ramosus (Walt.) B.S.P.-mediated oil also contained high percentage ofb-caryophyllene (24.0%), a-humulene(14.5%), 1,8-cineole (9.0%), eugenol(7.2%), globulol (7.1%), caryophyllene oxide (5.2%), d-cadinene (5.0%) anda-copaene (4.9%), as earlier reported the major components of the variousessential oils, which had potential antimicrobial properties (Porter and Wilkins1999; Azaz et al. 2002; Oumzil et al. 2002; Filipowicz et al. 2003; Burt 2004;Erdemgil et al. 2007; Salamci et al. 2007). Those claims are further supportedby our findings, indicating high contents of b-caryophyllene, a-humulene,1,8-cineole, eugenol, globulol, caryophyllene oxide, d-cadinene anda-copaene, comprising 76.9% of the oil (Table 1). On the other hand, thecomponents in lower amount such as geraniol (1.9%), spathulenol (1.4%),nerolidol (0.8%), trans-b-farnesene (0.6%), ledol (0.6%), b-selinene (0.5%)and n-nonanal (0.5%) also contributed to antibacterial activity of the oil(Demetzos et al. 1997; Bisignano et al. 2001; Bougatsos et al. 2004; Burt2004; Cha et al. 2005). It is also possible that the minor components might beinvolved in some type of synergism with the other active compounds (Marinoet al. 2001).

When comparing the data obtained in different studies, most publicationsprovide generalization about whether or not a plant oil or extract possessesactivity against gram-positive and gram-negative bacteria. However, fewprovide details about the extent or the spectrum of this activity. Deans et al.(1995) observed that the susceptibility of gram-positive and gram-negativebacteria to plant volatile oils had little influence on growth inhibition.However, some oils appeared more specific, exerting a greater inhibitory


activity against gram-positive bacteria. It is often reported that gram-negativebacteria are more resistant to the plant-based essential oils (Reynolds 1996).The hydrophilic cell wall structure of gram-negative bacteria is constitutedessentially of a lipopolysaccharide (LPS) that blocks the penetration of hydro-phobic oil and avoids the accumulation of essential oils in target cell mem-brane (Bezic et al. 2003). This is the reason that gram-positive bacteria werefound to be more sensitive to the essential oil, methanol extract and variousmethanol-derived subfractions of E. ramosus (Walt.) B.S.P. than those ofgram-negative bacteria.

In this study, we found that essential oil, methanol extract and variousmethanol-derived subfractions of E. ramosus (Walt.) B.S.P. severely inhibitedthe growth of foodborne pathogens and spoiling bacteria. Therefore, essentialoils and plant extracts are being considered as potential alternatives tosynthetic bactericides or as leading compounds for new classes of naturalbactericides.

On the basis of the results of this study, E. ramosus (Walt.) B.S.P. may actas an alternative to synthetic bactericides for use in food industries, wherebacterial pathogens cause severe destruction. At present, food safety isundoubtedly an important public health problem, and there is a need to developnew methods for eliminating foodborne pathogens and spoiling bacteria. Thus,the essential oil and extracts of E. ramosus (Walt.) B.S.P. might be a valuablefood additive. However, if plant oils and extracts are to be used for foodpreservation or medicinal purposes, issues of safety and toxicity will alwaysneed to be addressed.

ACKNOWLEDGMENT

This work was supported by a grant from RIC Program of MOCIE,Republic of Korea.

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inhibition of foodborne pathogens and spoiling bacteria by essential oil and extracts of erigeron...

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