International Journal of Food Microbiology 142 (2010) 114–119

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International Journal of Food Microbiology

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Efficacy of chemically characterized Piper betle L. essential oil against fungal andaflatoxin contamination of some edible commodities and its antioxidant activity

Bhanu Prakash, Ravindra Shukla, Priyanka Singh, Ashok Kumar,Prashant Kumar Mishra, Nawal Kishore Dubey ⁎Laboratory of Herbal Pesticides, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India

⁎ Corresponding author. Tel.: +91 9415295765.E-mail address: nkdubey2@rediffmail.com (N.K. Dub

0168-1605/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.ijfoodmicro.2010.06.011

a b s t r a c t

a r t i c l e i n f o

Article history:Received 12 January 2010Received in revised form 20 May 2010Accepted 15 June 2010

Keywords:Aflatoxin B1

AntifungalAntioxidantEssential oilPiper betle

The study investigates fungal contamination in some dry fruits, spices and areca nut and evaluation of theessential oil (EO) of Piper betle var. magahi for its antifungal, antiaflatoxigenic and antioxidant properties. Atotal of 1651 fungal isolates belonging to 14 species were isolated from the samples and Aspergillus wasrecorded as the dominant genus with 6 species. Eleven aflatoxin B1 (AFB1) producing strains of A. flavuswererecorded from the samples. Eugenol (63.39%) and acetyleugenol (14.05%) were the major components of 32constituents identified from the Piper betle EO through GC and GC–MS analysis. The minimum inhibitoryconcentration (MIC) of P. betle EO was found 0.7 μl/ml against A.flavus. The EO reduced AFB1 production in adose dependent manner and completely inhibited at 0.6 μl/ml. This is the first report on efficacy of P. betle EOas aflatoxin suppressor. EO also exhibited strong antioxidant potential as its IC50 value (3.6 μg/ml) was closeto that of ascorbic acid (3.2 μg/ml) and lower than that of the synthetic antioxidants such as butylatedhydroxytouene (BHT) (7.4 μg/ml) and butylated hydroxyanisole (BHA) (4.5 μg/ml). P. betle EO thus exhibitedspecial merits possessing antifungal, aflatoxin suppressive and antioxidant characters which are desirable foran ideal preservative. Hence, its application as a plant based food additive in protection and enhancement ofshelf life of edible commodities during storage and processing is strongly recommended in view of thetoxicological implications by synthetic preservatives.

ey).

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1. Introduction

Microbial contamination is a major problem of food and feedstuffsduring storage. Among microorganisms, moulds have potent capabilityto spoil the food itemsby producinghydrolytic enzymes. Different typesof mycotoxins have been reported in mould contaminated ediblecommodities from diverse meteorological regions of the world(Tatsadjieu et al., 2009).However, in tropical and sub-tropical countries,improper and traditional storage conditions provide conducive condi-tions for the growth and proliferation of moulds. There are reports ofsevere cases of mycotoxicoses in humans and livestock due toconsumption of such contaminated commodities (Bhatnagar andGarcia, 2001). Aflatoxins produced by toxigenic strains of A. flavus,have received significant attention throughout the world because oftheir hepatocarcinogenic, teratogenic, mutagenic and immunosuppres-sive properties (Leontopoulos et al., 2003). About 5 billion people areexposed to aflatoxins in developing countries and aflatoxicosis is ranked6th among the 10 most important health risks identified by WHO(Williamset al., 2004). Despite suchahigh level of toxigenicity, aflatoxin

contamination in edible commodities has attracted less attention thanthe bacterial contamination.

Several synthetic additives and preservatives are effectively usedinmanagement of post harvest losses but their continuous applicationmay cause the development of fungal resistance as well as residualtoxicity (Brent and Hollomon, 1998). Synthetic preservatives are alsoresponsible for the origin of partially reduced form of oxygen such assuperoxide (O2

−) hydrogen peroxide (H2O2) and hydroxyl radicals(OH−) which are highly reactive molecules causing oxidative diseasesby damaging the proteins, lipids and DNA (Halliwell, 1997) and alsoresponsible for the stimulation of aflatoxin biosynthesis (Jayashreeand Subramanyam 2000).

To overcome these problems someplant based preservatives such asazadirachtin, carvone, allyl isothiocynate fromAzadirachta indica,Carumcarvi and mustard oil, respectively have been developed as safe andnovel antimicrobials and are used on large scale as food additives(Chacon et al., 2006; de Carvalho and da Fonseca, 2006; Gopal et al.,2007). Amongnatural products, essential oils (EOs) of higher plants andtheir components are gaining interest as food additives and widelyaccepted by consumers because of their relatively high volatility,ephemeral nature and biodegradability. Carvacrol, cinnamaldehyde,citral, thymol and limonene are some major bioactive compounds ofsome essential oils which are recommended as food additives byEuropean commission with no harm to human health (Burt, 2004).

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Piper betle L. (family: Piperaceae) is an indigenous climber of theIndo-Malaya region. Its ethno-medicinal application has been wellknown for a long time. It is used traditionally in skin and eye diseases(Farnsworth and Bunyapraphatsara, 1992). Carminative, aphrodisiacand anticancerous properties of P. betle EO have also been reported(Manosroi et al., 2006; Bissa et al., 2007).

The present study was performed to investigate fungal contamina-tion in some dry fruits, spices and areca nut. In addition, the EO of P.betle var. magahi was evaluated for its antifungal, antiaflatoxigenicand antioxidant properties in order to assess its efficacy as a foodadditive.

2. Materials and methods

2.1. Chemicals and equipments

Chemicals and equipment viz. chloroform, methanol, sodiumsulphate, tween-80, toluene, isoamyl alcohol, PDA (potato, 200 g;dextrose, 20 g; agar, 18 g and distilled water 1000ml) and SMKYmedium (sucrose, 200 g; MgSO4·7H2O, 0.5 g; KNO3, 0.3 g; yeastextract, 7.0 g; distilled water, 1000 ml), ascorbic acid, butylatedhydroxytoluene (BHT), butylated hydroxyanisole (BHA) and, 2,2-diphenyl-1-picrylhydrazil (DPPH) were procured from HiMedia Labo-ratories Pvt. Ltd., Mumbai, India. Eugenol and Nystatin were procuredfrom Genuine Chemical Company, Mumbai and Wettasul-80 fromSulphur Mills Ltd., Mumbai, India. The major equipment used werehydro-distillation apparatus (Merck Specialities Pvt. Ltd., Mumbai,India), centrifuge, UV transilluminator (Zenith Engineers, Agra, India)and spectrophotometer (Systronics India Ltd., Mumbai, India).

2.2. Edible commodities

A total of 70 samples of edible commodities viz. dry fruits (Anacardiumoccidentale L., Prunus amygdalus Batsch., Arachis hypogea L.), spices (Pipernigrum L., Piper longum L., C. sativum L.) and a nut (Areca catechu L.) werecollected from retail outlets located in Varanasi, India. The collectedsamples were stored in sterilized polythene bags to prevent furthercontamination and were stored at 10 °C until analysis.

2.3. Moisture content and pH

Fifty grams of each sample was dried at 100 °C in hot air oven for24 h and moisture content was calculated based on difference withthe fresh weight (Mandeel 2005). One gram of each material wasfinely ground using mortar-pestle and 1:10 (sample: distilled water)suspension of each sample was prepared and stirred for 24 h. The pHof the suspension was recorded using electronic pH meter.

2.4. Mycological analysis

Mycological analysis of selected edible commodities was carriedout according to Aziz et al., (1998). Ten grams of each powdered samplewas homogenized in 90 ml sterile distilled water in an Erlenmeyerflask (250 ml). Five fold serial dilutions were prepared and 1 ml ofaliquot (10−4) of each sample was inoculated on a Petri dish containing10 ml freshly prepared PDA medium. Three replicates of each samplewere prepared and incubated (27±2 °C) for seven days. Differentfungal colonies were counted and species were identified followingRaper and Fennel (1977), Pitt (1979) and Domsch et al., (1980). Thepercent relative density of different fungi and their occurrencefrequency on each sample was determined following Singh et al.,(2008). The cultures of fungal isolates were maintained on PDA.

Relative density %ð Þ =No: of colony of fungus

total no: of colony of all fungal species× 100

The occurrence frequency of isolated fungi was determinefollowing Mandeel (2005)

Occurrence frequency %ð Þ =No: of fungal isolates on each sample

total no: of fungal isolate on all samples× 100

2.5. Detection of aflatoxigenic isolates

Randomly selected isolates of A. flavus from each sample werescreened for their aflatoxin B1 (AFB1) producing potential by thin layerchromatography (TLC) following Kumar et al., (2007). A. flavus isolateswere aseptically inoculated in 25 ml SMKY medium and incubated for10 days (27±2 °C). The content of each flaskwas filtered and extractedwith 20 ml chloroform using separating funnel. The extract wasevaporated to dryness on water bath and was redissolved in 1 mlchloroform. Fifty microliter of chloroform extract was spotted on TLCplates and developed in toluene:isoamyl alcohol:methanol (90:32:2; v/v/v). The plate was air dried and AFB1 was observed in UV-transilluminator (360 nm). The intensity of the blue fluorescent spotin the UV transilluminator varies with different aflatoxigenic strainsfrom light blue to deep blue. The toxigenic A. flavus (LHPac-3), isolatedfrom A. catechu produced maximum blue fluorescence under UV lightand was, therefore, selected for further investigations.

2.6. Isolation of essential oil

Leaves of P. betle L.var. magahi were purchased from the localmarket of Varanasi and subjected to hydro-distillation using Cleven-ger's apparatus (Prasad et al., 2009). The essential oil (EO) wasseparated and collected in sterilized glass vial. Water traces wereremoved using anhydrous sodium sulphate and EO was stored at 4 °Cfor the experimental processes.

2.7. GC and GC–MS analysis of P. betle EO

P. betle EOwas subjected to gas chromatography (PerkinElmer AutoXL GC, MA, USA) equipped with a flame ionization detector and the GCcondition were: EQUITY-5 column (60 m×0.32mm×0.25 μm); H2 wasthe carrier gas; column head pressure 10 psi; oven temperatureprogram isotherm 2 min at 70 °C, 3 °C/min gradient to 250 °C,isotherm10 min; injection temperature, 250 °C; detector temperature280 °C. GC–MS analysis was performed using PerkinElmer TurbomassGC–MS. The GC column was EQUITY-5 (60 m×0.32 mm×0.25 μm)fused silica capillary column. The GC conditions were: injectiontemperature, 250 °C; column temperature, isothermal at 70 °C for2 min, then programmed to 250 °C at 37 °C/min and held at thistemperature for 10 min; ion source temperature, 250 °C. Helium wasthe carrier gas. The effluent of the GC column was introduced directlyinto the source of MS and spectra obtained in the EI mode with 70 eVionization energy. The sector mass analyzer was set to scan from 40 to500 amu for 2 s. The identification of individual compounds is based ontheir retention times relative to those of authentic samples andmatching spectral peaks available with Wiley, NIST and NBS massspectral libraries or with the published data (Adams, 2007).

2.8. Fungitoxic investigation of P. betle EO

The minimum inhibitory concentrations (MICs) of EO againstdifferent fungal isolates were determined using Potato dextrosebroth (PDB) medium following Shukla et al., (2008). Differentconcentrations of essential oil (0.1 to 2.0 μl/ml) were dissolved in0.5 ml acetone and then incorporated with 9.5 ml PDB in test tubes.Fungal spore suspension (106spores/ml) in 0.1% Tween-80 wasinoculated to each tube and incubated for a week. PDB withoutessential oil was served as control. The lowest concentration of theoil that did not permit any visible fungal growth was recorded as

116 B. Prakash et al. / International Journal of Food Microbiology 142 (2010) 114–119

MIC. The tubes showing no visible fungal growth were sub-culturedon EO-free PDA plates to determine if the inhibition was reversible.The fungitoxic spectrum of P. betle EO against different fungalisolates was observed at its MIC in PDB. The MICs of two prevalentfungicides viz. Nystatin and Wettasul-80 were also determinedagainst A. flavus.

2.9. Efficacy of P. betle EO and eugenol in checkingaflatoxin B1 production

Requisite amount of P. betle EO and eugenol were dissolvedseparately in 0.5 ml acetone and added to 24.5 ml SMKY to achieve thevarious concentrations from 0.1 to 0.7 μl/ml. The medium inoculatedwith 1 ml spore suspension (106 spores) of toxigenic isolate of A. flavus(LHPac-3) was incubated for ten days at (27±2 °C). The medium wasfiltered and mycelium was dried at 80 °C (12 h). AFB1 was detected bythin layer chromatography as mentioned in Section 2.5. The developedblue spots on TLC plate were scratched, dissolved in methanol (5 ml)and centrifuged at 3000 rpm (5 min). Absorbance of the supernatantwas recorded at 360 nm and AFB1 was calculated following AOAC(1984) and Kumar et al., (2007).

AFB1content μg= lð ÞD × ME × l

× 1000:

D = absorbance, M = molecular weight (312), E = molarextinction coefficient AFB1 (21800), l = path length (1 cm).

2.10. Antioxidant activity of P. betle EO

The antioxidant activity of the EO was measured by DPPH radicalscavenging assay on TLC and measuring the free radical scavengingactivity through spectrophotometer following Tepe et al., (2005).

2.10.1. DPPH radical scavenging assay on TLCTo determine the antioxidant activity of EO, 5 μl (1:10 dilution in

methanol) was applied on TLC plate and developed in ethyl acetateandmethanol (1:1). The platewas sprayedwith 0.2% DPPH solution inmethanol (2, 2-diphenyl-1-picrylhydrazil) and left at room temper-ature for 30 min. Yellow spot formed due to bleaching of purple colorof DPPH reagent was recorded as positive antioxidant activity of EO.

2.10.2. Free radical scavenging activityFree radical scavenging activity of the P. betle EO was measured by

recording the extent of bleaching of the purple-colored DPPH solutionto yellow. Different concentrations (1.25 to 10.00 μg/ml) of thesamples were added to 0.004% DPPH solution in methanol (5 ml).After a 30 min of incubation at room temperature, the absorbance wastaken against a blank at 517 nm using spectrophotometer. Scavengingof DPPH free radical with reduction in absorbance of the sample wastaken as a measure of their antioxidant activity following Sharififaret al., (2007). Butylated hydroxytoluene (BHT), Butylated hydro-xyanisole (BHA) and ascorbic acid were used as positive control. IC50,which represented the concentration of the essential oil that caused50% neutralization of DPPH radicals, was calculated from the graphplotting between percentage inhibition and concentration.

I% = Ablank–Asample = Ablank

� �x100

where, Ablank is the absorbance of the control (without testcompound), and Asample is the absorbance of the test compound.

2.11. Statistical analysis

Antifungal and antioxidant experiments were performed intriplicate and data analyzed are mean±SE subjected to one way

ANOVA. Means are separated by the Tukey's multiple range test whenANOVA was significant (pb0.05) (SPSS 10.0; Chicago, IL, USA).

3. Results

The moisture content of the commodities varied significantly. Thehighestmoisture content (25.90%)was recorded inA. hypogea followedbyP. amygdalus (21.11%) and the lowest (11.36%) was in the case ofC. sativum followed by the A. catechu (13.45%). The magnitude of pHwasfound inacidic range. The lowestpH(4.7)was recorded inA. catechuwhilethe highest (6.45) in case of P. nigrum (Table 1). A total of 1651 fungalisolates belonging to 14 species were recorded from the samples.Aspergillus was recorded as the dominant genus. Aspergillus flavus andAspergillus niger were found in all the investigated samples. Some fungiviz.Nigrospora sp., Mycelia sterilia, Aspergillus terreuswere found only in P.amygdalus, Anacardium occidentale, A. hypogea, respectively. The highestpercent relative density was recordedwith A. flavus (40.69%) followed byA. niger (24.10%) and C. cladosporioides (11.81%). The lowest relativedensity was recorded with mucorales (0.72%) followed by Nigrospora sp.(0.90%). Highest frequency of occurrence was recorded in A. catechu(20.65%), whereas, minimum (9.87%) in C. sativum and Piper longum.

During the investigations on toxigenicity of A. flavus isolates fromthe selected commodities, 11 isolates out of 24 were foundaflatoxigenic with blue spots on TLC plates. The toxigenic A. flavus(LHPac-3), isolated from A. catechu was used for antiaflatoxigenicbioassay as it produced maximum blue fluorescence under UV light.

The yield of EOwas 4.0 ml/kg through hydro-distillation. Chemicalcompositions of EO were identified by the GC–MS analysis and 32different components were identified. Their retention time and areapercentage are summarized in Table 2. Major components of EO wereeugenol (63.39%) and acetyleugenol (14.05%).

MIC of P. betle EO against A. flavus was found at 0.7 μl/ml. The oilexhibited pronounced fungitoxicity against all the fungal isolates. Thelowest MIC (0.3 μl/ml) of the oil was recorded against M. sterilia andthe highest was observed against A. niger as 0.73 μl/ml (Table 3). Thefungicides viz. Nystatin and Wettasul-80 inhibited A. flavus at 1.85 μl/ml and 2.78 mg/ml, respectively, and thus found to be less efficaciousthan the P. betle EO.

The P. betle EO inhibited AFB1 production in a dose dependentmanner. At the lowest concentration of 0.1 μl/ml, enhanced AFB1

production (1165.93 μg/l) was recorded even higher than the controlset (978.93 μg/l). However, the P. betle EO inhibited AFB1 productionon higher concentrations and completely inhibited at 0.6 μl/ml(Table 4). Eugenol, the major component of the P. betle EO wasfound to bemore efficacious than the oil. It inhibited the growth of thetoxigenic strain LHPac-3 of A. flavus and the aflatoxin production at0.4 μl/ml and 0.1 μl/ml, respectively.

The appearance of yellow spot due to bleaching the purple color ofthe DPPH confirmed the positive antioxidant activity of EO. Percentinhibition and IC50 values of EO and synthetic antioxidant aresummarized in Fig 1. The oil showed strong free radical scavengingactivity as its IC50 value (3.6 μg/ml) was found close to ascorbic acid(3.2 μg/ml) and lower than BHT (7.4 μg/ml), BHA (4.5 μg/ml).

4. Discussion

The results of the present investigation indicate that all the selectededible commodities were heavily contaminated with the differentmould species. The samples were also found associated with toxigenicstrains of A. flavus. Hence, the biodeterioration of the samples wasqualitative as well as quantitative in nature.

Moisture content and pHare twomain abiotic factors responsible forthe growth and proliferation of moulds. In all the samples, pH andmoisture content ranged between 4.7 to 6.4 and 11 to 25%, respectively,which are favorable limits for the growth of moulds. High moisturecontent of most of the samples may be one of the factors for their

Table 1Mycoflora analysis of selected edible commodities.

Commoditiesname

Fungal species pH Moisturecontent

Totalisolates

Totalspecies

Occurencefrequency

A.f. A.n. A.fu. A.s. A.c. A.t. P.i. F.o. C.c. C.l. A.a. M.s. N.i. M.

Anacardiumoccidentale

110 49 32 8 10 – 18 – 40 – 20 – 4 5.93±0.23ab

16.06±1.11b

291 9 17.62

Prunusamygdalus

40 30 10 – 40 – – 10 20 – – – 15 3 5.56±0.18bc

21.11±1.13c

168 8 10.18

Arachishypogea

140 39 12 – – 18 7 – 35 – 12 – – – 6.44±0.08a

25.90±1.09d

263 7 15.93

Pipernigrum

125 55 9 9 30 – 4 – 30 – – – – – 6.45±0.03a

15.11±0.93ab

262 7 15.86

Piperlongum

70 40 – 10 – – 17 – 20 – 6 – – – 5.93±0.04ab

14.87±0.60ab

163 6 9.87

Coriandrumsativum

68 75 – – 8 – – 10 – – – – – 2 5.12±0.05cd

11.36±0.39a

163 5 9.87

Arecacatechu

118 110 16 – – – 10 – 50 17 17 – – 3 4.70±0.06d

13.45±0.63ab

341 8 20.65

Totalisolates

671 398 79 27 88 18 56 20 195 17 35 20 15 12 1651

Relativedensity

40.69 24.10 4.78 1.60 5.33 1.09 3.39 1.21 11.81 1.03 2.12 1.21 0.90 0.72

A.f. Aspergillus flavus, A.n. Aspergillus niger, A.fu. Aspergillus fumigatus, A.s. Aspergillus sydowi, A.c. Aspergillus candidus, A.t. Aspergillus terreus, P.i. Penicillium italicum, F.o. Fusariumoxysporum, C.c. Cladosporium cladosporoides, C.l. Curvularia lunata, A.a. Alternaria alternata, M.s. Mycelia sterlia, N.i. Nigrospora sp., M. Mucor sp.The means followed by same letter in the same column are not significantly different according to ANOVA and Tukey's multiple comparison tests.

117B. Prakash et al. / International Journal of Food Microbiology 142 (2010) 114–119

biodeterioration. However, a critical observation on the mycologicalanalysis of the samples clearly showed that neither moisture contentnor pH of the samples individually influenced the fungal distribution. A.catechu having comparatively lowermoisture content (13.45%) showedthe highest occurrence frequency and diversity of moulds as well asaflatoxin content indicating that chemical profile of substrate may alsobe a deciding factor for the growth of moulds strengthening the earlierhypothesis of Singh et al., (2008).

GC and GC–MS analysis of EO revealed 32 different componentswhich constitute 97% of the oil. In the present investigation, eugenol

Table 2Chemical composition of P. betle essential oil.

Sn. Compound Rt. (min.) %

1 α-pinene 9.6 0.092 Camphene 10.150 0.093 β-myrcene 11.425 0.124 L-limonene 13.100 0.285 Cis-ocimene 13.300 0.206 Phenyl acetylaldehyde 13.650 0.137 t-ocimene 13.800 0.668 Linalyl acetate 16.050 0.209 Decanal 20.975 0.1810 Chavicol 23.275 0.5511 Cyclohexene,4-methyl- 27.476 0.1512 Chavicol 27.701 0.5513 Eugenol 28.851 63.3914 β-elemene 30.176 0.2415 Methyl-eugenol 30.426 0.2116 Undecanal 30.576 0.4317 t-caryophyllene 31.501 4.2218 Bicyclo(4.1.0)hept-3-en- 31.876 0.1219 α -humulene 33.01 0.6820 γ-muurolene 33.926 1.2721 Germacrene D 34.251 2.8522 Germacrene B 34.876 0.8123 Acetyleugenol 35.826 14.0524 Aluminum sulphate 38.651 0.3425 Ledene 39.001 0.1826 Globulol 40.126 0.1227 4-allyl-1,2-diacetoxybenzene 40.676 0.1328 γ-cadinene 40.926 3.8529 γ-muurolene 41.426 0.1530 t-caryophyllene 41.551 0.5331 Aluminum sulphate 42.151 0.1032 γ-ionene 42.751 0.13

Rt: retention time.

(63.39%) and its ester derivative acetyleugenol (14.05%) were recordedas major components of oil. However, some earlier workers havereported phenolics like chavibetol (53.1%) and chavibetol acetate(15.5%) (Rimando et al., 1986), safrol (48.69%) (Arambewela et al.,2005) and 4-allyl-2-methoxy-phenolacetate (31.47%), 3-allyl-6-meth-oxyphenol (25.96%) (Apiwat et al., 2006) as prime components of P.betel EO. Such chemotypic variations have been reported in most of theEOs due to ecological and geographical conditions, age of the plant andtime of harvesting (Bagamboula et al., 2004). The apparent variation inthe chemical profile the oils may influence their antimicrobial activity.Hence, it is advisable that thepercentage of themajor components of theEOs should be mentioned if applied as food additive.

The literature is so far silent about the antifungal efficacy of P. betleEO against storage fungi. Hence, detailed investigationswere performedto record its efficacy as fungitoxicant, aflatoxin suppressor andantioxidant to evaluate it as a novel plant based antimicrobial andfood additive. The efficacy of EO against the moulds is either due to theeffect of major component or by the synergistic effect of overallcomponents (Burt, 2004). However, in the present investigation,eugenol, the major component of the P. betle EO was more efficaciousas fungal growth inhibitor and aflatoxin suppressor than the EO. Itappears that the remaining components of the oil synergistically actedin negative direction and reduce the activity of eugenol. It is also

Table 3Minimum inhibitory concentration (MIC) of P. betle essential oil against fungal isolates.

Fungal isolates MIC (μl/ml)

Aspergillus flavus 0.70±0.000ab

Aspergillus niger 0.73±0.016a

Aspergillus fumigatus 0.40±0.000fg

Aspergillus terreus 0.60±0.000bcde

Aspergillus sydowi 0.63±0.033abcd

Apergillus candidus 0.57±0.033cde

Penicillium italicum 0.40±0.000 fg

Fusarium oxysporum 0.50±0.000ef

Alternaria alternata 0.53±0.033de

Cladosporium cladosporoides 0.67±0.033abc

Curvularia lunata 0.50±0.000ef

Mucor sp. 0.37±0.033g

Nigrospora sp 0.53±0.033de

Mycelia sterilia 0.30±0.000g

Values are mean (n=3)±SE.The means followed by same letter in the same column are not significantly differentaccording to ANOVA and Tukey's multiple comparison tests.

Table 4Effect of different concentrations of P. betle essential oil and eugenol on mycelial weightand Aflatoxin B1 production in SMKY medium.

Conc. Piper betle EO Eugenol

(μl/ml) MDW AfB1 content MDW AfB1 content

CNT 532.33±08.95a 978.93±11.91b 532.33±08.95a 978.93±11.91a

0.1 484.00±08.32ab 1165.93±24.37a 494.33±07.31ab 0.0b

0.2 480.00±10.39bc 832.00±28.87c 466.33±06.00b 0.0b

0.3 433.67±13.02cd 614.60±28.49d 378.33±12.25c 0.0b

0.4 390.00±12.58de 249.98±39.43e 0.0d 0.0b

0.5 366.67±09.53e 150.70±14.90 e 0.0d 0.0b

0.6 118.33±09.13f 0.0f 0.0d 0.0b

0.7 0.0g 0.0f 0.0d 0.0b

Conc.= concentration (μl/ml); MDW = mycelial dry weight (mg).Values are mean (n=3)±SE. Themeans followed by same letter in the same column arenot significantly different according to ANOVA and Tukey's multiple comparison tests.

118 B. Prakash et al. / International Journal of Food Microbiology 142 (2010) 114–119

apparent from the present investigation that the eugenol has tremen-dous capacity as aflatoxin inhibitor than the growth suppressor. Thepresence of OH group in eugenol may be able to form hydrogen bondswith the active site of the target enzymes and increases the activity bydenaturing the enzyme, responsible for toxin secretion as emphasizedby Bluma et al., (2008).The oil exhibited remarkable fungitoxicityagainst all the fungal isolates infestingdifferent edible commodities. TheEO was also found more efficacious than the two commonly usedfungicides viz. Nystatin andWettasul-80. TheMIC of EO against A. flavuswas found to be lower than some earlier reported EOs viz. Ocimumgrattissimum, Ocimum basilicum, Cymbopoga citrates, Thymus vulgare,and Monodora myristica (Nguefack et al., 2004). Hence, the EO of the P.betle may be recommended for complete protection of food commod-ities from the fungal infestation at low concentration.

At low concentration of EO (0.1 μl/ml), AFB1 production by thetoxigenic strain ofA.flavuswas increased than the control. However, theaflatoxin inhibitory efficacy of the oil enhanced with higher concentra-tions and at 0.6 μl/ml, it completely checked aflatoxin production by thetoxigenic isolate. It shows that the low fungicide doses create somestress condition which was responsible for the production of moresecondary metabolites as a defense mechanism by the fungus. Someearlierworkers have also reported that low fungicide doses to stimulatethe toxin production (Magan et al., 2002).

Free radical scavenging activity of the P. betle EO was found to beconcentration dependent. The IC50 value of the EOwas very close to thatof ascorbic acid and lower than that of BHT and BHA, thus, reflecting itssuperiority as better preservative over the synthetic antioxidants. TheIC50 of P. betle EO was also found quite lower than that of some earlier

Fig. 1. Radical scavenging activity of P. betle essential oil.

reported EOs viz. Zataria multiflora and Thymus caramanicuswhose IC50values were 22.4 and 263.09 μg/ml, respectively (Sharififar et al., 2007;Safaei-Ghomi et al., 2009). Free Radical scavenging activity of EOs maybedue to thepresenceof thephenolic compoundsor synergistic effect ofoverall compounds (Sharififar et al., 2007). Because of free radicalscavenging activity, the oil may be recommended as a plant basedantioxidant in enhancement of shelf life of food commodities, thus,retarding oxidative rancidity of lipids. In addition, its use as preservativeof edible commodities would protect the human being from oxidativediseases.

EOs, being plant based product and biodegradable in nature may beused as alternatives of synthetic preservatives and fumigants againstbiodeterioration of food items. Many of the antimicrobial formulationscontaining the EOs and their constituents are actually exempted fromtoxicity data requirements by the EPA (Burt, 2004; Holley and Patel,2005). Essential oils of many edible and medicinal plants are used indifferent pharmaceutical preparations which minimize questionsregarding their safe use. Essential oils from aromatic and medicinalplants are potentially useful as antimicrobial agents and their uses asmedicines have long been recognized (Kim et al., 1995). The attractionof modern society towards herbal products (Smid and Gorris, 1999)desiring fewer synthetic ingredients in foods and recommendation ofherbal products as ‘generally recognized as safe’ (GRAS) as foodadditives may lead scientific interest in the exploitation of essentialoils as plant based food additives. A few EO based preservatives arealready commercially available (Mendoza-Yepes et al., 1997).

In conclusion, the present study explores the efficacy of P. betle EO asantifungal, antiaflatoxigenic and antioxidant agent. Our study is the firstreport on antiaflatoxigenic activity of P. betle EO to the best of ourknowledge. The leaves of the plant are chewed by most of the Indiansbecause of its stimulating qualities (Bissa et al., 2007). Hence, therewouldbeno chance of off-flavour and adverse of organoleptic taste of the treatededibles if the P. betle EO is recommended as plant based antimicrobial.Moreover, the P. betle EO oil would be cheaper in formulation because ofavailability of sufficient amount of raw material and high yield of the oilduring hydro-distillation. Based on the findings of the present investiga-tion, it appears that P. betle EO has special merit possessing antifungal,aflatoxin suppressive andantioxidant characterswhich are desirable of anideal preservative. Therefore, its application in protection and enhance-ment of shelf life of edible commodities during the storage andprocessingis strongly recommended as a botanical food additive.

Acknowledgement

Authors are thankful to Council of Scientific and IndustrialResearch (CSIR), New Delhi, India for financial assistance.

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