In vitro inhibition of foodborne pathogens by volatile oil and organic extracts of Poncirus trifoliata Rafin. seeds

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Research ArticleReceived: 16 October 2008 Revised: 26 December 2008 Accepted: 26 December 2008 Published online in Wiley Interscience: 17 February 2009

(www.interscience.wiley.com) DOI 10.1002/jsfa.3527

In vitro inhibition of foodborne pathogens byvolatile oil and organic extracts of Poncirustrifoliata Rafin. seedsAtiqur Rahman,a,b Sharif M Al-Reza,a,b Jung In Yoona and Sun Chul Kanga∗

Abstract

BACKGROUND: Poncirus trifoliata Rafin. is widely used in oriental medicine because of its beneficiary effects on health. In thisstudy, we examined the chemical compositions of the volatile oil isolated from the seeds of P. trifoliata by hydrodistillation, andtested the efficacy of the oil and various organic extracts as an antibacterial potential against a panel of foodborne pathogens.

RESULTS: The hydrodistilled volatile oil was analysed by GC–MS. Thirty-six compounds representing 90.67% of the total oil wereidentified, of which veridiflorol, spathulenol, α-humulene, α-cadinol, δ-cadinene, T-muurolol, hexadecanoic acid, germacreneD, bergaptene and aromadendrene were the major volatile compounds. The oil and the organic extracts of chloroform, ethylacetate and methanol revealed a remarkable antibacterial effect against the tested pathogens such as Staphylococcus aureus,Bacillus subtilis, Listeria monocytogenes, Pseudomonas aeruginosa, Enterobacter aerogenes, Salmonella typhimurium, S. enteritidisand Escherichia coli. Also, the oil had strong detrimental effect on the viable count of the tested bacteria.

CONCLUSION: Our findings demonstrate that the oil and organic extracts derived from P. trifoliata seeds might be a potentialsource of a preservative for the food or pharmaceutical industries.c© 2009 Society of Chemical Industry

Keywords: Poncirus trifoliata Rafin.; volatile oil; veridiflorol; foodborne pathogens; antibacterial activity; GC–MS

INTRODUCTIONFoodborne illnesses associated with Staphylococcus aureus, Listeriamonocytogenes, Escherichia coli O157:H7 and Salmonella enteritidispresent a major public health concern throughout the world.1

Also, the presence and growth of micro-organisms in food maycause spoilage and as a result destroy their quality and quantity.Further, microbial contamination of food still poses importantpublic health and economic concerns for the human society.Plant secondary metabolites, such as essential oils, are studiedfor their antimicrobial activities and most of the plant-derivedextracts and essential oils are known to possess antimicrobialactivities.2 Therefore, they are screened intensely and applied inthe fields of food microbiology, pharmacology, pharmaceuticals,phytopathology and food preservation. In addition to passivetransfer of pathogens to food, active growth of a pathogen mayalso occur in foods, for instance because of improper storage,which leads to marked increases in microbial load.3

With the increase of bacterial resistance to antibiotics, interesthas been generated to investigate the antimicrobial effects ofessential oils and different extracts against a range of bacteria,to develop other classes of natural antimicrobials useful for theinfection control or for the preservation of food. The Gram-positivebacterium Staphylococcus aureus is mainly responsible for post-operative wound infections, toxic shock syndrome, endocarditis,osteomyelitis and food poisoning.4 Listeria monocytogenes isresponsible for the severe foodborne illness listeriosis which hasbeen recognised as one of the emerging zoonotic diseases duringthe last two decades.5 The Gram-negative bacterium Escherichia

coli is present in human intestines and causes urinary tractinfection, coleocystitis or septicaemia.6 There is, therefore, stilla need for new methods of reducing or eliminating food spoilageand foodborne pathogens to ensure public health. Thus plantessential oils and extracts are promising natural antimicrobialagents with potential applications in pharmaceutical or foodindustries for controlling pathogenic bacteria.

Poncirus trifoliata Rafin. (Rutaceae), also known as trifoliateorange, is a close relative to the Citrus trees. It is a decidious orsemi-decidious shrub, a native of China and Korea, and is alsoknown as the Korean bitter orange. Traditionally, trifoliata oranges(P. trifoliata) have been widely used in folk medicine as a remedy forgastritis, dysentery, inflammation, digestive ulcers, etc. A scientificinvestigation into the health-maintaining properties of trifoliataorange fruit has revealed its anti-inflammatory, antibacterial andanti-anaphylactic activities.7 In Korea, fruit extracts of P. trifoliataare used in some over-the-counter drugs for the treatment ofa variety of gastrointestinal (GI) disorders.8 Yi and co-authors9

∗ Correspondence to: Professor Sun Chul Kang, Department of Biotechnol-ogy, Daegu University, College of Engineering, Kyoungsan, Kyoungbook712-714, Korea. E-mail: [email protected]

a Department of Biotechnology, Daegu University, Kyoungsan, Kyoungbook712-714, Korea

b Department of Applied Chemistry and Chemical Technology, Islamic University,Kushtia 7003, Bangladesh

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Antibacterial effects of Poncirus trifoliata www.soci.org

reported that poncirus fruit is a potent antileukaemic agent bypromoting apoptosis of cancer cells. Several compounds suchas poncirin, coumarins, auraptine, hesperidin and naringin havebeen identified from poncirus fruits.10 However, there is no reportavailable in the literature on the analyses of volatile oil fromP. trifoliata seeds and its antibacterial properties. Hence, effortshave been made to investigate the role of volatile oil and variousextracts of P. trifoliata as an antibacterial potential.

In this study, we examined the chemical composition of thevolatile seed oil of P. trifoliata by gas chromatography–massspectrometry (GC–MS), and tested the efficacy of the oil andorganic seed extracts of P. trifoliata for controlling the growthof a range of bacteria comprising foodborne pathogens, with anemphasis upon the possible future use as alternative antibacterialcompounds.

MATERIALS AND METHODSPlant materialThe fruits of P. trifoliata were collected from the local area ofKyoungsan, Republic of Korea, in September and October 2007and identified by morphological features and by consulting a bookkept in the library at the Department of Biotechnology. A voucherspecimen number has been deposited in the herbarium of Collegeof Engineering, Department of Biotechnology, Daegu University,Republic of Korea.

Isolation of the volatile seed oilThe seeds were separated from fruits, washed with distilled waterand air dried at room temperature for 5 days. The dried seedswere then pulverised to a powder by using a grinding machine.The dried seed powder (200 g) of P. trifoliata was subjected tohydrodistillation using a Clevenger-type apparatus and extractedwith 2 L of water for 3 h (until no more volatile oil was obtained).The volatile oil was dried over anhydrous Na2SO4 and preservedin a sealed vial at 4 ◦C until further analysis.

Preparation of various organic seed extractsThe dried seed powder (50 g) of P. trifoliata was extracted withhexane, chloroform, ethyl acetate and 70% methanol, separatelyin a 1000 mL Erlenmeyer flask at room temperature for 7 days. Theprocess was repeated three times to ensure complete extraction.The solvents from the combined extracts were then filtered byWhatman filter paper no.1, and the filtrates were evaporated byvacuum rotary evaporator (EYELA N1000, SB-1000; Tokyo RikakikaiCo. Ltd, Tokyo, Japan). The extraction process yielded hexane(3.6 g), chloroform (4.2 g), ethyl acetate (6.2 g) and methanol(6.1 g) extracts.

Gas chromatography–mass spectrometry analysisGC–MS analysis of the volatile oil was performed using a ShimadzuGC–MS (GC-17A; Shimadzu, Kyoto, Japan) equipped with a ZB-1 MS fused silica capillary column (30 m × 0.25 i.d., film thickness0.25 µm). For GC–MS detection, an electron ionisation systemwith ionisation energy of 70 eV was used. Helium gas was usedas a carrier gas at a constant flow rate of 1 mL min−1. Injectorand mass transfer line temperature were set at 220 and 290 ◦C,respectively. The oven temperature was programmed from 50 ◦Cto 150 ◦C at 3 ◦C min−1, then held isothermally for 10 min andfinally raised to 250 ◦C at 10 ◦C min−1. Diluted samples (1/100, v/v,in methanol) of 1 µL were injected manually in the splitless mode.

The relative percentage of the oil constituents was expressed aspercentage by peak area normalisation.

The identity of the components of the volatile oil was assignedby comparison of their retention indices (RIs), relative to a series ofn-alkane indices on the ZB-1 capillary column and GC–MS spectrafrom the Wiley 6.0 MS data and literature data.11 The relativeamounts (RAs) of individual components of the oil were expressedas percentages of the peak area relative to the total peak area.

Micro-organismsThe following food spoiling and foodborne bacteria wereused in the antibacterial test: Staphylococcus aureus ATCC6538, S. aureus KCTC 1916, Bacillus subtilis ATCC 6633, Listeriamonocytogenes ATCC 19166, Pseudomonas aeruginosa KCTC 2004,Enterobacter aerogenes KCTC 2190, Salmonella typhimurium KCTC2515, S. enteritidis KCTC 12 021, Escherichia coli O157-Human, E.coli ATCC 8739 and E. coli O157:H7 ATCC 43888. The strains wereobtained from the Korea Food and Drug Administration (KFDA),Daegu, Republic of Korea. Active cultures for experimental usewere prepared by transferring a loopful of cells from stock culturesto flasks and inoculated in Luria–Bertani (LB) broth medium at37 ◦C for 24 h. Cultures of each bacterial strains were maintainedon LB agar medium at 4 ◦C.

Antibacterial activity assayThe dried organic extracts were dissolved in the same solventused for their extraction to a final concentration of 40 mg mL−1

and sterilised by filtration through 0.45 µm sterile Milliporefilters. The antibacterial test was then carried out by agardisc diffusion method using 100 µL of standardised inoculumsuspension containing 106 to 108 CFU mL−1 of bacteria.12 Thevolatile oil was diluted 1 : 5 (v/v) with methanol and aliquots of10 µL were spotted onto the filter paper discs; while 10 µL of 40 mgmL−1 of each organic extract (400 µg disc−1) was applied on thefilter paper discs (6 mm diameter) and placed on the inoculatedagar. Negative controls were prepared using the same solventsemployed to dissolve the samples. A standard reference antibiotic,streptomycin (20 µg disc−1, from Sigma–Aldrich Co., St Louis, MO,USA), was used as a positive control for the tested bacteria. Theplates were then sealed with parafilm and incubated at 37 ◦Cfor 24 h. Antibacterial activity was evaluated by measuring thediameter of the zones of inhibition against the tested bacteria.Each assay in this experiment was replicated three times.

Minimum inhibitory concentrationMinimum inhibitory concentration (MIC) of the oil and organicextracts was tested by two-fold serial dilution method.13 The testsamples of oil and various extracts were first dissolved in methanol,and incorporated into LB broth medium to obtain a concentrationof 2000 µg mL−1 and serially diluted to achieve 1000, 500, 250125, 62.5 and 31.25 µg mL−1, respectively. The final concentrationof methanol in the culture medium was maintained at 0.1% (v/v).A 10 µL standardised suspension of each tested organism (106 to108 CFU mL−1) was transferred to each tube. The control tubescontained only bacterial suspension, were incubated at 37 ◦C for24 h. The lowest concentration of the test samples, which did notshow any growth of tested organism after macroscopic evaluation,was determined as MIC.

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Effect of volatile oil on viable counts of bacteriaFor viable counts, each of the tubes containing bacterialsuspension (approximately 106 to 108 CFU mL−1) of S. aureusATCC 6538, B. subtilis ATCC 6633, L. monocytogenes ATCC 19166and P. aeruginosa KCTC 2004 in LB broth medium was inoculatedwith the minimum inhibitory concentration of the volatile oil in10 mL LB broth, and kept at 37 ◦C.14 Samples for viable cell countswere taken out at 0, 20, 40, 60, 80 and 100 min time intervals.Enumeration of viable counts on LB plates weas monitored asfollows: after incubation, 1 mL of the resuspended culture wasdiluted into 9 mL buffer peptone water, thereby diluting 10-fold.In total, a 0.1 mL sample of each treatment was diluted and spreadon the surface of LB agar. The colonies were counted after 24 hof incubation at 37 ◦C. The controls were inoculated without oilfor each bacterial strain with the same experimental condition asmentioned above.

Statistical analysisThe volatile oil and various organic extracts were assayed forantibacterial activity. Each experiment was carried out in triplicate,and mean values were calculated. A Student’s t-test was computedfor the statistical significance of the results.

RESULTSChemical composition of the volatile seed oilThe hydrodistillation of the seeds of P. trifoliata gave a yellow-ish oil with a yield of 0.34% (w/w). GC–MS analyses of theoil led to the identification of 36 different compounds, repre-senting 90.67% of the total oil. The identified compounds arelisted in Table 1 according to their elution order on a ZB-1 cap-illary column. The major compounds detected in the oil wereveridiflorol (17.34%), spathulenol (14.21%), α-humulene (12.32%),α-cadinol (7.24%), δ-cadinene (5.63%), T-muurolol (6.23%), hex-adecanoic acid (4.54%), germacrene D (2.28%), bergaptene (2.13%)and aromadendrene (1.32%) as shown in Table 1. Sesquiterpenehydrocarbons and oxygenated sesquiterpenes were the predom-inant portions of the seed oil of P. trifoliata. Isocaryophyllene(0.87%), pentadecanoic acid (0.82%), β-selinene (0.81%), elemicin(0.78%), trans-β-farnesene (0.76%), β-bisabolene (0.73%), andγ -muurolene (0.65%) were also found to be the minor componentsof P. trifoliata seeds oil (Table 1).

In vitro antibacterial activityThe in vitro antibacterial activities of volatile oil and various organicextracts of P. trifoliata seeds against the bacteria used werequalitatively assessed by the presence or absence of inhibition

Table 1. Chemical composition of the volatile seed oil of Poncirustrifoliata

Compound RIa %RAb

Hydrocarbons

Aldehydes and ketones

Tetradecanal 1609 0.32

Z-7-Hexadecenal 1808 1.11

Hexahydrofarnesyl acetone 1816 0.83

Aromatic

1,2-Dihydro-1,1,6-trimethyl-naphthalene 1224 0.58

Table 1. (Continued)

Compound RIa %RAb

Terpenoids

Sesquiterpene hydrocarbons

Isoladene 1372 0.58

β-Elemene 1390 0.51

Isocaryophyllene 1402 0.87

Aromadendrene 1428 1.32

γ -Elemene 1430 0.79

trans-α-Bergamotene 1432 0.71

α-Humulene (α-Caryophyllene) 1447 12.32

allo-Aromadendrene 1454 0.62

trans-β-Farnesene 1455 0.76

γ -Muurolene 1460 0.65

β-Charmigrene 1470 0.61

cis-β-Guaiene 1480 0.28

Germacrene D 1490 2.28

β-Selinene 1492 0.81

α-Muurolene 1494 0.62

β-Himachalene 1495 0.83

δ-Cadinene 1469 5.63

β-Bisabolene 1513 0.73

α-Calacorene 1538 0.58

Oxygen-containing sesquiterpenes

(−)-Spathulenol (Spathulenol) 1550 14.21

Veridifloral 1568 17.34

δ-Cadinol 1619 0.85

α-Cadinol 1626 7.24

T-Muurolol 1641 6.23

Aromadendrene oxide(II) 1664 0.62

Fatty acids

Tridecanoic acid 1660 0.31

Pentadecanoic acid 1832 0.82

Hexadecanoic acid 1968 4.54

Octadecanoic acid 2180 0.57

Arylpropanoid

Elemicin 1552 0.78

Furanocoumarins

Bergaptene 2055 2.13

Isopimpeinellin 2216 0.69

TOTAL 90.67

a Retention index relative to n-alkanes on a ZB-1 capillary column.b Relative area (peak area relative to the total peak area).

zones. According to the results given in Table 2, a total of 11food spoilage and foodborne bacterial strains, including fourGram-positive and seven Gram-negative bacteria were tested. Theoil exhibited antibacterial activity against all four Gram-positiveand five Gram-negative bacteria. The oil (10 µL disc−1 of 1 : 5 (v/v)dilution with methanol) exhibited a potent inhibitory effect againstS. aureus ATCC 6538, S. aureus KCTC 1916, L. monocytogenes ATCC19166, B. subtilis ATCC 6633, P. aeruginosa KCTC 2004, E. coliATCC 8739 and S. enteritidis KCTC with diameter of inhibitionzones ranging from 14.0 to 22.0 mm, as shown in Table 2. Organicextracts prepared in chloroform, ethyl acetate and methanol alsosignificantly inhibited the growth of all the bacteria tested, atthe concentrations of 400 µg disc−1 (Table 2). The ethyl acetate

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Table 2. Antibacterial activity of volatile oil and various organic extract of Poncirus trifoliata seeds against foodborne pathogenic bacteria

Zones of inhibition (mm)∗

Organic extract†

Micro-organism Volatile oil CHCl3 EtOAc MeOH Antibiotic

S. aureus ATCC 6538 22.0 ± 1.2a 14.1 ± 1.1ab 18.1 ± 1.5b 17.0 ± 1.2a 13.4 ± 0.7b

S. aureus KCTC 1916 20.2 ± 1.0b 13.1 ± 0.5bc 16.2 ± 1.0c 15.2 ± 0.5b 14.0 ± 1.1b

B. subtilis ATCC 6633 21.2 ± 1.2ab 145.3 ± 0.5a 18.0 ± 1.2c 15.0 ± 0.5b 20.2 ± 1.4a

L. monocytogenes ATCC 19166 18.2 ± 0.7c 15.0 ± 1.2a 20.3 ± 1.6a 17.5 ± 1.1a 14.3 ± 1.2b

P. aeruginosa KCTC 2004 15.3 ± 1.1d 13.0 ± 1.0ab 15.2 ± 0.5cd 15.1 ± 1.2b 13.0 ± 0.6b

E. aerogenes KCTC 2190 13.2 ± 0.6e 12.2 ± 0.9cd 15.2 ± 0.6cd 14.2 ± 0.7b 13.2 ± 1.2b

E. coli ATCC 8739 14.0 ± 1.2de 11.5 ± 0.7d 13.2 ± 0.6ef 12.1 ± 0.6c 13.1 ± 0.6b

E. coli O157 : H7 ATCC 43888 13.2 ± 0.5e 11.1 ± 0.5d 12.2 ± 0.5f 12.1 ± 0.6c 12.0 ± 1.2b

E. coli O157 (human) ND 12.1 ± 0.5cd 13.2 ± 0.6ef 12.1 ± 0.6c 14.2 ± 1.2b

S. enteritidis KCTC 12021 15.2 ± 1.1d 11.3 ± 0.5d 14.2 ± 0.6de 12.5 ± 0.5c 14.2 ± 0.5b

S. typhimurium KCTC 2515 ND 11.2 ± 0.7d 13.2 ± 0.6ef 11.2 ± 0.7c 13.4 ± 1.2b

∗ Diameter of inhibition zones (mm) around the discs (6 mm) impregnated with 10 µL oil of 1 : 5 (v/v) dilution with MeOH.† Various organic extracts (400 µg disc−1).The standard antibiotic was streptomycin (20 µg disc−1).ND, not detected. Values are given as the mean ± SD (n = 3).Values in the same column with different superscripts are significantly different (P = 0.05) by Duncan’s test.

extract showed the strongest antibacterial effect against allfour Gram-positive bacteria and three Gram-negative bacteria(P. aeruginosa KCTC 2004, E. aerogenes KCTC 2190 and S. enteritidisKCTC 12021) in the diameter of inhibition zones of 14.2–20.3 mm.On the other hand, chloroform and methanol extracts exhibitedsignificant antibacterial effect with inhibition zones in the range11.1–17.5 mm. In this study, in most of the cases, the oil andorganic extracts exhibited higher or similar types of antibacterialactivity compared to streptomycin. However, the hexane extractdid not show any activity against the bacterial strains tested(data not shown). The blind control did not inhibit the growth ofthe bacteria tested. The extracts exhibited a moderate inhibitoryeffect against S. typhimurium KCTC 2515, E. coli O157-Human, E. coliATCC 8739 and E. coli O157:H7 ATCC 43888 with diameter zonesof inhibition in the range 11.1–12.5 mm, while the oil had noinhibitory effect against E. coli O157-Human and S. typhimuriumKCTC 2515.

Minimum inhibitory concentrationAs shown in Table 3, the MIC values for the oil were found tobe lower for S. aureus (ATCC 6538 and KCTC 1916), B. subtilisATCC 6633, L. monocytogenes ATCC 19166 and P. aeruginosaKCTC 2004 (62.5–125 µg mL−1) than for E. aerogenes KCTC 2190,S. enteritidis KCTC 12021, E. coli ATCC 8739 and E. coli O157:H7 ATCC43888 (250–500 µg mL−1). On the other hand, MIC values of theorganic extracts of chloroform, ethyl acetate and methanol againstthe bacteria tested were found in the range 62.5–500 µg mL−1

(Table 3). The ethyl acetate extract showed higher antibacterialactivity compared with chloroform and methanol extracts. Inthis study, the Gram-positive bacteria were found to be moresusceptible to the volatile oil and various organic extracts thanGram-negative bacteria.

Effect of volatile oil on viable counts of bacteriaBased on the susceptibility, further, elaborative study carried outon S. aureus ATCC 6538, B. subtilis ATCC 6633, L. monocytogenes

Table 3. Minimum inhibitory concentration of volatile oil andvarious organic extracts of Poncirus trifoliata seeds against foodbornepathogenic bacteria

Minimum inhibitory concentration(µg mL−1)

Organic extract

Micro-organism Volatile oil CHCl3 EtOAc MeOH

S. aureus ATCC 6538 62.5 125 125 125

S. aureus KCTC 1916 62.5 250 125 125

B. subtilis ATCC 6633 62.5 125 62.5 125

L. monocytogenes ATCC 19166 125 125 62.5 125

P. aeruginosa KCTC 2004 125 250 125 125

E. aerogenes KCTC 2190 250 250 125 250

E. coli ATCC 8739 250 500 250 500

E. coli O157 : H7 ATCC 43888 500 500 500 500

E. coli O157 (human) ND 500 250 500

S. enteritidis KCTC 12021 250 500 250 500

S. typhimurium KCTC 2515 ND 500 500 500

ND, not detected.

ATCC19166 and P. aeruginosa KCTC 2004, displayed differentsensitivities of the volatile oil. The effects of volatile oil on thegrowth of all the bacterial strains tested demonstrated the reducedviability of the bacteria at MIC values of the oil. Complete inhibitionof both strains of S. aureus ATCC 6538 and B. subtilis ATCC 6633was observed at an MIC of the oil at 20 and 40 min exposure,respectively. Also, the steep decline in CFU numbers was observedat 40 min exposure against L. monocytogenes ATCC19166 andP. aeruginosa KCTC 2004. Exposure of 80 min of the oil MIC revealedcomplete inhibition of CFU numbers against all the bacterial strainstested (Fig. 1).


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Figure 1. Effect of the volatile oil from Poncirus trifoliata seeds (MICconcentration) on the viability of the bacteria tested. CT, control withouttreatment.

DISCUSSIONFood contamination is the cause of many health and economicproblems especially when psychrotrophic pathogens and spoilagemicro-organisms are present. The use of natural substances asbacteriocins or other biologically derived antimicrobials appearsto be an important requirement in food packaging methodologyfor microbial control.15 Fruits, vegetables, grains and foodconstituents can be contaminated by various micro-organisms andtheir hazardous toxic metabolites. Moreover, micro-organisms arealso associated with food spoilage, which causing financial loss.Plant extracts and essential oils have constituted a natural sourceof antimicrobial mixtures for centuries. They are used as naturalantimicrobials in food systems, as well as preventing the growthof foodborne bacteria and moulds, resulting in an extended shelflife of processed foods.16

The results of the antibacterial screening showed that volatileoil and organic extracts of chloroform, ethyl acetate and methanolhave potential activity against most of the bacterial strains. Thisactivity could be attributed to the presence of oxygen-containingsesquiterpenes and sesquiterpene hydrocarbons, and thesefindings are in agreement with previous reports.17 In our opinion,major components of P. trifoliata oil, veridiflorol (17.34%),spathulenol (14.21%), α-humulene (12.32%), α-cadinol (7.24%), δ-cadinene (5.63%), T-muurolol (6.23%), hexadecanoic acid (4.54%),germacrene D (2.28%), bergaptene (2.13%) and aromadendrene(1.32%) have key roles for their antibacterial activities.18 – 20

The antibacterial activity of individual components of essentialoils such as spathulenol or α-humulene has been reportedpreviously.21,22 On the other hand, components in lower amounts,such as isocaryophyllene, pentadecanoic acid, β-selinene,elemicin, trans-β-farnesene, β-bisabolene and γ -muurolene, alsocontributed to antibacterial activity of the oil.23,24 It is also possiblethat the minor components might be involved in some type ofsynergism with the other active compounds.25

Also, the results from the viable count assay revealed thatexposure to the MIC of the oil had a severe effect on the cell viabilityof the tested bacteria. S. aureus ATCC 6538 and B. subtilis ATCC 6633were found to be more sensitive to the oil. The oil also exerted itsmaximum bacterial activity against L. monocytogenes ATCC19166and P. aeruginosa KCTC 2004, as is evident by the significant

reduction in microbial counts at 40 min exposure and completeinhibition of cell viability at 80 min exposure to the volatile oil.Similar to our findings, one of the n-6 essential oils also exertedan inhibitory effect against P. aeruginosa.26 A direct effect of n-6essential oils on bacterial cells is a result of peroxidation ending infree radicals, capable of attacking bacterial outer membrane andfacilitating the action of antimicrobials.

Deans et al.27 reported that the susceptibility of Gram-positiveand Gram-negative bacteria to plant volatile oils had littleinfluence on growth inhibition. However, some oils appearedmore specific, exerting a greater inhibitory activity against Gram-positive bacteria. It is often reported that Gram-negative bacteriaare more resistant to plant-based volatile oils.28 This resistance hasbeen attributed to the presence of cell wall lipopolysaccharides,which can screen out the essential oils; the lipids are thus preventedfrom accumulating on the transporting cell membrane, and fromentering the cells.29 This is the reason why Gram-positive bacteriawere found to be more sensitive to the volatile oil and variousorganic extracts of P. trifoliata seeds than those of Gram-negativebacteria.

CONCLUSIONThe results of our study suggest the possibility of using theseed extract or oil of P. trifoliata as natural antimicrobials in thefood and/or pharmaceutical industry because the oil or extractpossesses strong antibacterial activities. Hayouni et al.30 reportedthe essential oils of Salvia officinalis L. and Schinus molle L. hada preservative effect against Salmonella inoculated in mincedbeef. Such findings would also be confirmed in further studies toestablish the real application of P. trifoliata seed oil or extracts infoods and/or pharmaceuticals.

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In vitro inhibition of foodborne pathogens by volatile oil and organic extracts of Poncirus trifoliata Rafin. seeds