Efficacy of chemically characterized Piper betle L. essential oil against fungal and aflatoxin 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 abstractarticle info Article history: Received 12 January 2010 Received in revised form 20 May 2010 Accepted 15 June 2010 Keywords: Aflatoxin B 1 Antifungal Antioxidant Essential oil Piper betle The study investigates fungal contamination in some dry fruits, spices and areca nut and evaluation of the essential oil (EO) of Piper betle var. magahi for its antifungal, antiaflatoxigenic and antioxidant properties. A total of 1651 fungal isolates belonging to 14 species were isolated from the samples and Aspergillus was recorded as the dominant genus with 6 species. Eleven aflatoxin B 1 (AFB 1 ) producing strains of A. flavus were recorded from the samples. Eugenol (63.39%) and acetyleugenol (14.05%) were the major components of 32 constituents identified from the Piper betle EO through GC and GC–MS analysis. The minimum inhibitory concentration (MIC) of P. betle EO was found 0.7 μl/ml against A.flavus. The EO reduced AFB 1 production in a dose dependent manner and completely inhibited at 0.6 μl/ml. This is the first report on efficacy of P. betle EO as aflatoxin suppressor. EO also exhibited strong antioxidant potent ial as its IC 50 value (3.6 μg/ml) was close to that of ascorbic acid (3.2 μg/ml) and lower than that of the synthetic antioxidants such as butylated hydroxytouene (BHT) (7.4 μg/ml) and butylated hydroxyanisole (BHA) (4.5 μg/ml). P. betle EO thus exhibited special merits possessing antifungal, aflatoxin suppressive and antioxidant characters which are desirable for an ideal preservative. Hence, its application as a plant based food additive in protection and enhancement of shelf life of edible commodities during storage and processing is strongly recommended in view of the toxicological implications by synthetic preservatives. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Microbial contamination is a major problem of food and feedstuffs during storage. Among microorganisms, moulds have potent capability to spoil the food items by producing hydrolytic enzymes. Different types of mycotoxins have been reported in mould contaminated edible commodities 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 of severe cases of mycotoxicoses in humans and livestock due to consumption of such contaminated commodities (Bhatnagar and Garcia, 2001). Aflatoxins produced by toxigenic strains of A. flavus, have received significant attention throughout the world because of their hepatocarcinogenic, teratogenic, mutagenic and immunosuppres- sive properties (Leontopoulos et al., 2003). About 5 billion people are exposed to aflatoxins in developing countries and aflatoxicosis is ranked 6th among the 10 most important health risks identified by WHO (Williams et al.,2004). Despite such a high level of toxigenicity, aflatoxin contamination in edible commodities has attracted less attention than the bacterial contamination. Several synthetic additives and preservatives are effectively used in management of post harvest losses but their continuous application may cause the development of fungal resistance as well as residual toxicity (Brent and Hollomon, 1998). Synthetic preservatives are also responsible for the origin of partially reduced form of oxygen such as superoxide (O 2 − ) hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals (OH − ) which are highly reactive molecules causing oxidative diseases by damaging the proteins, lipids and DNA (Halliwell, 1997) and also responsible for the stimulation of aflatoxin biosynthesis (Jayashree and Subramanyam 2000). To overcome these problems some plant based preservatives such as azadirachtin, carvone, allyl isothiocynate from Azadirachta indica, Carum carvi and mustard oil, respectively have been developed as safe and novel antimicrobials and are used on large scale as food additives (Chacon et al., 2006; de Carvalho and da Fonseca, 2006; Gopal et al., 2007). Among natural products, essential oils (EOs) of higher plants and their components are gaining interest as food additives and widely accepted by consumers because of their relatively high volatility, ephemeral nature and bi odegradability. Carvacrol, cinnamaldehyde, citral, thymol and limonene are some major bioactive compounds of some essential oils which are recommended as food additives by European commission with no harm to human health (Burt, 2004). International Journal of Food Microbiology 142 (2010) 114–119 ⁎ Corresponding author. Tel.: +91 9415295765. E-mail address: nkdubey2@rediffmail.com (N.K. Dubey). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.06.011 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro Piper betle L. (family: Piperaceae) is an indigenous climber of the Indo-Malaya region. Its ethno-medicinal application has been well known for a long time. It is used traditionally in skin and eye diseases (Farnsworth and Bunyapraphatsara, 1992). Carminative, aphrodisiac and 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, antiaflatoxigenic and antioxidant properties in order to assess its efficacy as a food additive. 2. Materials and methods 2.1. Chemicals and equipments Chemicals and equipment viz. chloroform, methanol, sodium sulphate, tween-80, toluene, isoamyl alcohol, PDA (potato, 200 g; dextrose, 20 g; agar, 18 g and distilled water 1000 ml) and SMKY medium (sucrose, 200 g; MgSO4·7H2O, 0.5 g; KNO3, 0.3 g; yeast extract, 7.0 g; distilled water, 1000 ml), ascorbic acid, butylated hydroxytoluene (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 procured from Genuine Chemical Company, Mumbai and Wettasul-80 from Sulphur Mills Ltd., Mumbai, India. The major equipment used were hydro-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 (Anacardium occidentale L., Prunus amygdalus Batsch., Arachis hypogea L.), spices ( Piper nigrum L., Piper longum L., C. sativum L.)andanut(Areca catechu L.) were collected fro m retail outlets located i n Varanasi, India. The collected samples w ere stored in sterilized polythene b ags to prevent further contaminat ion a nd wer e store d at 10 °C until an alysis. 2.3. Moisture content and pH Fifty grams of each sample was dried at 100 °C in hot air oven for 24 h and moisture content was calculated based on difference with the fresh weight (Mandeel 2005). One gram of each material was finely ground using mortar-pestle and 1:10 (sample: distilled water) suspension of each sample was prepared and stirred for 24 h. The pH of the suspension was recorded using electronic pH meter. 2.4. Mycological analysis Mycological analysis of selected edible commodities was carried out according to Aziz et al., (1998). Ten grams of each powdered sample was homogenized in 90 ml sterile distilled water in an Erlenmeyer flask (250 ml). Five fold serial dilutions were prepared and 1 ml of aliquot (10 −4 ) of each sample was inoculated on a Petri dish containing 10 ml freshly prepared PDA medium. Three replicates of each sample were prepared and incubated (27±2 °C) for seven days. Different fungal colonies were counted and species were identified following Raper and Fennel (1977), Pitt (1979) and Domsch et al., (1980).The percent relative density of different fungi and their occurrence frequency on each sample was determined following Singh et al ., (2008). The cultures of fungal isolates were maintained on PDA. Relative density %ðÞ= No: of colony of fungus total no: of colony of all fungal species ×100 The occurrence frequency of isolated fungi was determine following Mandeel (2005) Occurrence frequency %ðÞ= 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 were screened for their aflatoxin B 1 (AFB 1 ) producing potential by thin layer chromatography (TLC) following Kumar et al., (2007). A. flavus isolates were aseptically inoculated in 25 ml SMKY medium and incubated for 10 days (27±2 °C). The content of each flask was filtered and extracted with 20 ml chloroform using separating funnel. The extract was evaporated to dryness on water bath and was redissolved in 1 ml chloroform. Fifty microliter of chloroform extract was spotted on TLC plates and developed in toluene:isoamyl alcohol:methanol (90:32:2; v/ v/v). The plate was air dried and AFB 1 was observed in UV- transilluminator (360 nm). The intensity of the blue fluorescent spot in the UV transilluminator varies with different aflatoxigenic strains from light blue to deep blue. The toxigenic A. flavus (LHPac-3), isolated from A. catechu produced maximum blue fluorescence under UV light and was, therefore, selected for further investigations. 2.6. Isolation of essential oil Leaves of P. betle L.var. magahi were purchased from the local market of Varanasi and subjected to hydro-distillation using Cleven- ger's apparatus (Prasad et al., 2009). The essential oil (EO) was separated and collected in sterilized glass vial. Water traces were removed using anhydrous sodium sulphate and EO was stored at 4 °C for the experimental processes. 2.7. GC and GC–MS analysis of P. betle EO P. betle EO was subjected to gas chromatography (PerkinElmer Auto XL GC, MA, USA) equipped with a flame ionization detector and the GC condition were: EQUITY-5 column (60 m ×0.32mm×0.25 μm); H 2 was the carrier gas; column head pressure 10 psi; oven temperature prog ram isother m 2 min at 70 °C, 3 °C/min gradient to 250 °C, isotherm10 min; injection temperature, 250 °C; detector temperature 280 °C. GC–MS analysis was performed using PerkinElmer Turbomass GC–MS. The GC column was EQUITY-5 (60 m×0.32 mm×0.25 μm) fused silica capillary column. The GC conditions were: injection temperature, 250 °C; column temperature, isothermal at 70 °C for 2 min, then programmed to 250 °C at 37 °C/min and held at this temperature for 10 min; ion source temperature, 250 °C. Helium was the carrier gas. The effluent of the GC column was introduced directly into the source of MS and spectra obtained in the EI mode with 70 eV ionization energy. The sector mass analyzer was set to scan from 40 to 500 amu for 2 s. The identification of individual compounds is based on their retention times relative to those of authentic samples and matching spectral peaks available with Wiley, NIST and NBS mass spectral libraries or with the published data (Adams, 2007). 2.8. Fungitoxic investigation of P. betle EO The mini mum inhibitory concentrations ( MICs) of EO agains t dif ferent fungal isolates were determined using Potato dextrose bro th (PDB) medium following Shukla et al., (2008). Different concentrat ions of e ssential oil (0.1 to 2.0 μl/ml) were dissolved in 0.5 ml acetone and then incorporated with 9.5 ml PDB in test t ubes. Fun gal spore suspension (10 6 spores/ml) in 0.1 % Tween-80 was inoculated to each tube and incubated for a week. PDB without essential oil was served as control. The lowes t concentration of the oil that did not permit any visible fungal growt h was recorded as 115B. Prakash et al. / International Journal of Food Microbiology 142 (2010) 114–119 MIC. The tubes showing no visible fungal growth were sub-cultured on EO-free PDA p lates to determine if the inhibition was reversible. The fungitoxic spectrum of P. betle EO against different fungal isolates was observed at its MIC in PDB. The MI Cs of two prevale nt fun gicides viz. Nystatin and Wettasul-80 were also determined against A. flavus. 2.9. Efficacy of P. betle EO and eugenol in checking aflatoxin B 1 production Requisite amount of P. betle EO and eugenol were dissolved separately in 0.5 ml acetone and added to 24.5 ml SMKY to achieve the various concentrations from 0.1 to 0.7 μl/ml. The medium inoculated with 1 ml spore suspension (10 6 spores) of toxigenic isolate of A. flavus (LHPac-3) was incubated for ten days at (27±2 °C). The medium was filtered and mycelium was dried at 80 °C (12 h). AFB 1 was detected by thin layer chromatography as mentioned in Section 2.5. The developed blue spots on TLC plate were scratched, dissolved in methanol (5 ml) and centrifuged at 3000 rpm (5 min). Absorbance of the supernatant was recorded at 360 nm and AFB 1 was calculated following AOAC (1984) and Kumar et al., (2007). AFB 1 content μg = lðÞ D×M E×l ×1000: D = absorbance, M = molecular weight (312), E = molar extinction coefficient AFB 1 (21800), l = path length (1 cm). 2.10. Antioxidant activity of P. betle EO The antioxidant activity of the EO was measured by DPPH radical scavenging assay on TLC and measuring the free radical scavenging activity through spectrophotometer following Tepe et al., (2005). 2.10.1. DPPH radical scavenging assay on TLC To determine the antioxidant activity of EO, 5 μl (1:10 dilution in methanol) was applied on TLC plate and developed in ethyl acetate and methanol (1:1). The plate was sprayed with 0.2% DPPH solution in methanol (2, 2-diphenyl-1-picrylhydrazil) and left at room temper- ature for 30 min. Yellow spot formed due to bleaching of purple color of DPPH reagent was recorded as positive antioxidant activity of EO. 2.10.2. Free radical scavenging activity Free radical scavenging activity of the P. betle EO was measured by recording the extent of bleaching of the purple-colored DPPH solution to yellow. Different concentrations (1.25 to 10.00 μg/ml) of the samples were added to 0.004% DPPH solution in methanol (5 ml). After a 30 min of incubation at room temperature, the absorbance was taken against a blank at 517 nm using spectrophotometer. Scavenging of DPPH free radical with reduction in absorbance of the sample was taken as a measure of their antioxidant activity following Sharififar et al., (2007). Butylated hydroxytoluene (BHT), Butylated hydro- xyanisole (BHA) and ascorbic acid were used as positive control. IC 50, which represented the concentration of the essential oil that caused 50% neutralization of DPPH radicals, was calculated from the graph plotting between percentage inhibition and concentration. I% = A blank –A sample = A blank  x100 where, A blank is the abso rbance of the control (without test compound), and A sample is the absorbance of the test compound. 2.11. Statistical analysis Antifungal and antioxidant experiments were performed in triplicate and data analyzed are mean ±SE subjected to one way ANOVA. Means are separated by the Tukey's multiple range test when ANOVA was significant (p b 0.05) (SPSS 10.0; Chicago, IL, USA). 3. Results Themoisturecontentofthecommoditiesvariedsignificantly. The highest moisture content (25.90%) was recorded in A. hypogea followed by P. amygdalus (21. 11%) and the lowest ( 11.36%) was in the case of C. sati vum followed by the A. catechu (13.45%) . The magnitude of pH was found i n acidic range. The lowest pH (4.7) was recorded in A. catechu while the highest (6.45) in case of P. nigrum (Table 1). A total of 1651 fungal isolates bel ongi ng to 14 species were recorded from the samples. Aspergillus was recorded as the dominant genus. Aspergillus flavus and Aspergillus niger were found in all the investigated samples. Some fungi viz. Nigrospora sp., Mycelia sterilia, Aspergillus terreus were found only in P. amygdalus, Anacardium occidentale, A. hypogea, respectively. The highest percent relative density was recorded with A. flavus (40.69%) followed by A. niger (24.10%) and C. cladosporioides (11.81%). The lowest relative density 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 from the selected commodities, 11 isolates out of 24 were found aflatoxigenic with blue spots on TLC plates. The toxigenic A. flavus (LHPac-3), isolated from A. catechu was used for antiaflatoxigenic bioassay as it produced maximum blue fluorescence under UV light. The yield of EO was 4.0 ml/kg through hydro-distillation. Chemical compositions of EO were identified by the GC–MS analysis and 32 different components were identified. Their retention time and area percentage are summarized in Table 2. Major components of EO were eugenol (63.39%) and acetyleugenol (14.05%). MIC of P. betle EO against A. flavus was found at 0.7 μl/ml. The oil exhibited pronounced fungitoxicity against all the fungal isolates. The lowest MIC (0.3 μl/ml) of the oil was recorded against M. sterilia and the highest was observed against A. niger as 0.73 μl/ml (Table 3). The fungicides 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 efficacious than the P. betle EO. The P. betle EO inhibited AFB 1 production in a dose dependent manner. At the lowest concentration of 0.1 μl/ml, enhanced AFB 1 production (1165.93 μg/l) was recorded even higher than the control set (978.93 μg/l). However, the P. betle EO inhibited AFB 1 production on higher concentrations and completely inhibited at 0.6 μl/ml (Table 4 ). Eugenol, the major component of the P. betle EO was found to be more efficacious than the oil. It inhibited the growth of the toxigenic strain LHPac-3 of A. flavus and the aflatoxin production at 0.4 μl/ml and 0.1 μl/ml, respectively. The appearance of yellow spot due to bleaching the purple color of the DPPH confirmed the positive antioxidant activity of EO. Percent inhibition and IC 50 values of EO and synthetic antioxidant are summarized in Fig 1. The oil showed strong free radical scavenging activity as its IC 50 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 selected edible commodities were heavily contaminated with the different mould species. The samples were also found associated with toxigenic strains of A. flavus. Hence, the biodeterioration of the samples was qualitative as well as quantitative in nature. Moisture content and pH are two main abiotic factors responsible for the growth and proliferation of moulds. In all the samples, pH and moisture content ranged between 4.7 to 6.4 and 11 to 25%, respectively, which are favorable limits for the growth of moulds. High moisture content of most of the samples may be one of the factors for their 116 B. Prakash et al. / International Journal of Food Microbiology 142 (2010) 114–119 biodeterioration. However, a critical observation on the mycological analysis of the samples clearly showed that neither moisture content nor pH of the samples individually influenced the fungal distribution. A. catechu having comparatively lower moisture content (13.45%) showed the highest occurrence frequency and diversity of moulds as well as aflatoxin content indicating that chemical profile of substrate may also be a deciding factor for the growth of moulds strengthening the earlier hypothesis of Singh et al., (2008). GC and GC–MS analysis of EO revealed 32 different components which constitute 97% of the oil. In the present investigation, eugenol (63.39%) and its ester derivative acetyleugenol (14.05%) were recorded as major components of oil. However, some earlier workers have reported 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 the EOs due to ecological and geographical conditions, age of the plant and time of harvesting (Bagamboula et al., 2004). The apparent variation in the chemical profile the oils may influence their antimicrobial activity. Hence, it is advisable that the percentage of the major components of the EOs should be mentioned if applied as food additive. The literature is so far silent about the antifungal efficacy of P. betle EO against storage fungi. Hence, detailed investigations were performed to record its efficacy as fungitoxicant, aflatoxin suppressor and antioxidant to evaluate it as a novel plant based antimicrobial and food additive. The efficacy of EO against the moulds is either due to the effect of major component or by the synergistic effect of overall components (Burt, 2004). However, in the present investigation, eugenol, the major component of the P. betle EO was more efficacious as fungal growth inhibitor and aflatoxin suppressor than the EO. It appears that the remaining components of the oil synergistically acted in negative direction and reduce the activity of eugenol. It is also Table 1 Mycoflora analysis of selected edible commodities. Commodities name Fungal species pH Moisture content Total isolates Total species Occurence frequency 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. Anacardium occidentale 110 49 32 8 10 – 18 – 40 – 20 – 4 5.93± 0.23 ab 16.06± 1.11 b 291 9 17.62 Prunus amygdalus 40 30 10 – 40 ––10 20 –––15 3 5.56 ± 0.18 bc 21.11± 1.13 c 168 8 10.18 Arachis hypogea 140 39 12 ––18 7 – 35 – 12 –––6.44± 0.08 a 25.90± 1.09 d 263 7 15.93 Piper nigrum 125 55 9 9 30 – 4 – 30 –––––6.45± 0.03 a 15.11± 0.93 ab 262 7 15.86 Piper longum 70 40 – 10 ––17 – 20 – 6 –––5.93± 0.04 ab 14.87± 0.60 ab 163 6 9.87 Coriandrum sativum 68 75 –– 8 ––10 – ––––2 5.12± 0.05 cd 11.36± 0.39 a 163 5 9.87 Areca catechu 118 110 16 –––10 – 50 17 17 ––3 4.70 ± 0.06 d 13.45± 0.63 ab 341 8 20.65 Total isolates 671 398 79 27 88 18 56 20 195 17 35 20 15 12 1651 Relative density 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. Fusarium oxysporum, 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. Table 2 Chemical composition of P. betle essential oil. Sn. Compound Rt. (min.) % 1 α-pinene 9.6 0.09 2 Camphene 10.150 0.09 3 β-myrcene 11.425 0.12 4 L-limonene 13.100 0.28 5 Cis-ocimene 13.300 0.20 6 Phenyl acetylaldehyde 13.650 0.13 7 t-ocimene 13.800 0.66 8 Linalyl acetate 16.050 0.20 9 Decanal 20.975 0.18 10 Chavicol 23.275 0.55 11 Cyclohexene,4-methyl- 27.476 0.15 12 Chavicol 27.701 0.55 13 Eugenol 28.851 63.39 14 β-elemene 30.176 0.24 15 Methyl-eugenol 30.426 0.21 16 Undecanal 30.576 0.43 17 t-caryophyllene 31.501 4.22 18 Bicyclo(4.1.0)hept-3-en- 31.876 0.12 19 α -humulene 33.01 0.68 20 γ-muurolene 33.926 1.27 21 Germacrene D 34.251 2.85 22 Germacrene B 34.876 0.81 23 Acetyleugenol 35.826 14.05 24 Aluminum sulphate 38.651 0.34 25 Ledene 39.001 0.18 26 Globulol 40.126 0.12 27 4-allyl-1,2-diacetoxybenzene 40.676 0.13 28 γ-cadinene 40.926 3.85 29 γ-muurolene 41.426 0.15 30 t-caryophyllene 41.551 0.53 31 Aluminum sulphate 42.151 0.10 32 γ-ionene 42.751 0.13 Rt: retention time. Table 3 Minimum inhibitory concentration (MIC) of P. betle essential oil against fungal isolates. Fungal isolates MIC (μl/ml) Aspergillus flavus 0.70± 0.000 ab Aspergillus niger 0.73± 0.016 a Aspergillus fumigatus 0.40± 0.000 fg Aspergillus terreus 0.60± 0.000 bcde Aspergillus sydowi 0.63± 0.033 abcd Apergillus candidus 0.57± 0.033 cde Penicillium italicum 0.40± 0.000 fg Fusarium oxysporum 0.50± 0.000 ef Alternaria alternata 0.53 ±0.033 de Cladosporium cladosporoides 0.67± 0.033 abc Curvularia lunata 0.50± 0.000 ef Mucor sp. 0.37± 0.033 g Nigrospora sp 0.53± 0.033 de Mycelia sterilia 0.30± 0.000 g Values are mean (n=3) ±SE. 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 apparent from the present investigation that the eugenol has tremen- dous capacity as aflatoxin inhibitor than the growth suppressor. The presence of OH group in eugenol may be able to form hydrogen bonds with the active site of the target enzymes and increases the activity by denaturing the enzyme, responsible for toxin secretion as emphasized by Bluma et al., (2008).The oil exhibited remarkable fungitoxicity against all the fungal isolates infesting different edible commodities. The EO was also found more efficacious than the two commonly used fungicides viz. Nystatin and Wettasul-80. The MIC of EO against A. flavus was found to be lower than some earlier reported EOs viz. Ocimum grattissimum, 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), AFB 1 production by the toxigenic strain of A. flavus was increased than the control. However, the aflatoxin inhibitory efficacy of the oil enhanced with higher concentra- tions and at 0.6 μl/ml, it completely checked aflatoxin production by the toxigenic isolate. It shows that the low fungicide doses create some stress condition which was responsible for the production of more secondary metabolites as a defense mechanism by the fungus. Some earlier workers have also reported that low fungicide doses to stimulate the toxin production (Magan et al., 2002). Free radical scavenging activity of the P. betle EO was found to be concentration dependent. The IC 50 value of the EO was very close to that of ascorbic acid and lower than that of BHT and BHA, thus, reflecting its superiority as better preservative over the synthetic antioxidants. The IC 50 of P. betle EO was also found quite lower than that of some earlier reported EOs viz. Zataria multiflora and Thymus caramanicus whose IC 50 values were 22.4 and 263.09 μg/ml, respectively (Sharififar et al., 2007; Safaei-Ghomi et al., 2009). Free Radical scavenging activity of EOs may be due to the presence of thephenolic compounds or synergistic effect of overall compounds (Sharififar et al., 2007). Because of free radical scavenging activity, the oil may be recommended as a plant based antioxidant in enhancement of shelf life of food commodities, thus, retarding oxidative rancidity of lipids. In addition, its use as preservative of edible commodities would protect the human being from oxidative diseases. EOs, being plant based product and biodegradable in nature may be used as alternatives of synthetic preservatives and fumigants against biodeterioration of food items. Many of the antimicrobial formulations containing the EOs and their constituents are actually exempted from toxicity data requirements by the EPA (Burt, 2004; Holley and Patel, 2005). Essential oils of many edible and medicinal plants are used in different pharmaceutical preparations which minimize questions regarding their safe use. Essential oils from aromatic and medicinal plants are potentially useful as antimicrobial agents and their uses as medicines have long been recognized (Kim et al., 1995). The attraction of modern society towards herbal products (Smid and Gorris, 1999) desiring fewer synthetic ingredients in foods and recommendation of herbal products as ‘generally recognized as safe’ (GRAS) as food additives may lead scientific interest in the exploitation of essential oils as plant based food additives. A few EO based preservatives are already commercially available (Mendoza-Yepes et al., 1997). In conclusion, the present study explores the effica cy of P. betle EO as antifungal, antiaflat oxigenic and antioxidan t agent. Our study is the first report on antiaflatoxigenic activity of P. betle EO to the b est of o ur knowledge. The leaves of the plant are chewed by most of the Indians because of its stimulating qualities (Bissa et al., 2007). Hence, t her e would be no chance of off-flavour and adverse of organoleptic taste of the treated edibles if the P. betle EO is recommended as plant based antimicrobial. Moreover, the P. betle EO oil would be cheaper in f ormula tion because of availability of sufficient amount of raw material and high yield of the oil during hydro-distillation. Based on the findings of the present investiga- tion, it appears that P. betle EO has special merit possessing antifungal, aflatox in suppressive and antioxidant characters which are de sirabl e of an ideal preservative. Therefore, its application in protection and enhance- ment of shelf life of edible commodities during the storage and processing is strongly recommended as a botanical food additive. 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IC 50 value (3.6 μg/ml) was close to that of ascorbic acid (3.2 μg/ml) and lower than that of the synthetic antioxidants such as butylated hydroxytouene (BHT) (7.4 μg/ml) and butylated hydroxyanisole. 1998). Synthetic preservatives are also responsible for the origin of partially reduced form of oxygen such as superoxide (O 2 − ) hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals (OH − ) which. way ANOVA. Means are separated by the Tukey's multiple range test when ANOVA was significant (p b 0.05) (SPSS 10.0; Chicago, IL, USA). 3. Results Themoisturecontentofthecommoditiesvariedsignificantly.