International Journal of Food Microbiology 135 (2009) 165–170

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International Journal of Food Microbiology

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Efficacy of Lippia alba (Mill.) N.E. Brown essential oil and its monoterpenealdehyde constituents against fungi isolated from some edible legume seedsand aflatoxin B1 production

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

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

0168-1605/$ – see front matter © 2009 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2009.08.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 February 2009Received in revised form 30 June 2009Accepted 1 August 2009

Keywords:Lippia albaEssential oilGeranialNeralAntifungal activity

The present study deals with evaluation of antifungal properties of Lippia alba essential oil (EO) and two ofits monoterpene aldehyde constituents against legume-contaminating fungi. Seventeen different fungalspecies were isolated from 11 varieties of legumes, and aflatoxigenic isolates of Aspergillus flavus wereidentified. Hydrodistillation method was used to extract the EO from fresh leaves. The GC and GC–MSanalysis of EO revealed the monoterpene aldehydes viz. geranial (22.2%) and neral (14.2%) as the majorcomponents. The antifungal activity of EO, geranial and neral was evaluated by contact assay on Czapek's-dox agar. The EO (0.25–1 μL/mL) and its two constituents (1 μL/mL) showed remarkable antifungal effectsagainst all the fungal isolates (growth inhibition range 32.1–100%). Their minimal inhibitory (MIC) andfungicidal (MFC) concentrations for A. flavus were lower than those of the systemic fungicide Bavistin.Aflatoxin B1 (AFB1) production by three isolates of A. flavus was strongly inhibited even at the lowerfungistatic concentration of EO and its constituents. There was no adverse effect of treatments on seedgermination, and rather, there was enhanced seedling growth in the EO-treated seeds. It is concluded thatL. alba EO and two of its constituents could be safely used as effective preservative for food legumes againstfungal infections and mycotoxins.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Legumes represent the richest source of proteins and amino acidsfor human and animal health. The recent trends indicate that legumesshare nutraceutical properties which not only contribute to a balanceddiet but can also prevent the widely diffused diseases including type IIdiabetes, cardiovascular diseases, digestive tract diseases and obesity(Duranti, 2006).

Fungal contamination in stored seeds and grains is a seriousproblem in the tropical and sub-tropical countries as the prevailingwarm/humid climate is the ideal condition for fungal growth/activity(Weinberg et al., 2008). Their invasion may initiate at any stage fromthe standing crop through to harvest and post-harvest handlings untilthey reach the consumer. The infected seeds have decreasednutritional value, loss in germinability, discolouration, increase infree fatty acids (FFA), and more importantly the production ofmycotoxins (El-Nagerabi and Elshafie, 2000; Dhingra, et al., 2001;Embaby and Abdel-Galil, 2006).

+91 5422368174.ey).

l rights reserved.

Aflatoxin is one of the most common and hazardous mycotoxinsproduced by Aspergillus flavus Link ex. Fries. In developing countries,about 4.5 billion people are systematically exposed to excesses ofaflatoxin (Williams et al., 2004). Aflatoxicosis induces depressed feedefficiency, abnormal liver chemistry, depressed immune response,carcinogenesis, and even death (Pier, 1992). In plants, aflatoxinsinhibit seed germination, seedling growth, root elongation, synthesisof photopigments, proteins, nucleic acids, and some vital enzymes(Jones et al., 1980).

Management of fungal contamination of harvested seeds/grains isbased on physical (aeration, cooling and rapid drying) and chemicaltreatments with ammonia, food preservatives or even with pesticides.Since most of these control strategies require expensive chemicalsand technical expertise to monitor physical parameters (temperatureand pressure), they are not affordable by rural subsistence farmers(Atanda et al., 2007). Also, the widespread and indiscriminate use ofchemical preservatives or pesticides has significant drawbacks in notbeing economical, handling hazards, toxic residues on the grains andmore importantly the emergence of resistant food-borne microorgan-isms (Ishii, 2006).

These risks have increased public awareness for safer alternativesto chemical preservatives that are accessible, simple in application,non-toxic to humans and plants, and have sustained broad-spectrumfungitoxicity. Essential oils (EOs) have been classified as GRAS

166 R. Shukla et al. / International Journal of Food Microbiology 135 (2009) 165–170

(Generally Recognized as Safe) (Burt, 2004) and have been recom-mended as preservatives for food commodities based on theirantimicrobial and anti-mycotoxigenic effects (Mishra and Dubey,1994; Varma and Dubey, 2001; Kumar et al., 2007; Kumar et al., 2008;Rasooli et al., 2008; Razzaghi-Abyaneh et al., 2008; Tatsadjieu et al.,2009).

Lippia alba (Miller) N.E. Brown, the aromatic shrub (family:Verbenaceae) is a potential source of EO in India. Ethnopharmacolo-gical studies revealed that leaves can be used as an infusion againststates of excitement, hypertension, digestive troubles, nausea andcold, to heal wounds locally and as syrup against cough andbronchitis. In addition, the antimicrobial, analgesic, anti-inflammato-ry and antioxidative potential of EOs have also been ascertained(Oliveira et al., 2006; Hennebelle et al., 2008).

In the present study, legume seeds were screened for incidence ofmycoflora and the isolation of aflatoxigenic strains of A. flavus. EO ofLippia alba and two of its major components were evaluated forfungitoxicity and anti-aflatoxigenicity against selected fungal isolates.

2. Materials and methods

2.1. Legume seeds and mycoflora analysis

Eleven varieties of edible legume seeds viz. peanut (Arachishypogaea L.), pigeon pea (Cajanus cajan L.), chick pea (Cicerarietinum L.), soya bean (Glycine max L.), lentil (Lens culinarisMedikus),red bean (Phaseolus vulgaris L.), white pea (Pisum sativum L.), moth bean(Vigna aconitifolia (Jacq.) Marechal), black gram (Vigna mungo (L.)Hepper), mung bean Vigna radiata (L.) R. Wilczek and cow pea (Vignaunguiculata (L.) Walp) were purchased locally.

The seed mycoflora was examined using the blotter test and theagar plate method as recommended by International Seed TestingAssociationInternational Seed Testing Association, 1999 InternationalSeed Testing Association, 1999. International Rules for Seed Testing.Seed Science and Technology 27 (Suppl.). Seeds were surface-sterilized (1% solution of sodium hypochlorite) and rinsed in threechanges of sterile distilled water. Seeds were placed in Petri platescontaining blotter pads and potato dextrose agar (PDA) mediumincubated for 7 days (28±2 °C). The developing fungal colonies wereisolated, identified (Burnett and Hunter, 1999) and routinely main-tained on PDA (4 °C). The incidence of fungi was determined based onthe occurrence of a particular species in samples of 10 seeds.

2.2. Aflatoxigenic isolates of A. flavus

A. flavus isolates from each variety of legume seed were screenedfor the production of aflatoxin B1 (AFB1) following Sinha et al. (1993).The isolates were cultured separately in 25 mL SMKY broth (sucrose200 g; MgSO4·7H2O, 0.5 g; KNO3, 0.3 g and yeast extract, 7 g; 1 Ldistilled water) in 100 mL flask for 10 days. The content of each flaskwas filtered and extracted with 20 mL chloroform in a separatingfunnel. The extract was evaporated to dryness on water bath andredissolved in 1 mL chloroform. AFB1 was detected by thin layerchromatography. Fifty micro liter chloroform extract was spotted onTLC plates and developed in the solvent system comprising toluene/isoamyl alcohol/methanol (90:32:2; v/v/v). The plate was air driedand the intensity of AFB1 observed in UV-transilluminator (360 nm).

2.3. Plant material and oil constituents

Lippia alba, growing wild in the premises of Banaras HinduUniversity, Varanasi was identified by morphological features withthe help of Flora of BHU Campus (Dubey, 2004) and the voucherspecimen (LHP/Ver-21/2008) was deposited at the Laboratory ofHerbal Pesticides, Banaras Hindu University, Varanasi. Geranial and

neral (monoterpene aldehydes) were purchased from GenuineChemical Co., Mumbai, India.

2.4. Extraction of essential oil

Fresh plant leaves (200 g) were collected in the month of June andsubjected to hydro-distillation (4 h) using a Clevenger-type apparatus(Kumar et al., 2007). The yield (mL/kg) of EO was averaged over fourexperiments and calculated based on plant material fresh weight. EOwas stored airtight and subjected to gas chromatography-massspectrometry (GC–MS).

2.5. Oil analysis

The EO was analyzed by gas chromatography (PerkinElmer Auto XLGC, MA, USA) equipped with a flame ionization detector and the GCconditions were: EQUITY-5 column (60 m×0.32 mm×0.25 µm); H2

was the carrier gas; column Head pressure 10 psi; oven temperatureprogram isotherm 2 min at 70 ºC, 3 ºC/min gradient to 250 ºC, isotherm10min; injection temperature, 250 ºC; detector temperature 280 ºC.

GC–MS analysis was performed using PerkinElmer Turbomass GC–MS. The GC column was EQUITY-5 (60 m×0.32 mm×0.25 µm) fusedsilica capillary column. The GC conditions were: Injection tempera-ture, 250 ºC; column temperature, isothermal at 70 ºC for 2 min, thenprogrammed to 250 ºC at 37 ºC/min and held at this temperature for10 min; ion source temperature, 250 ºC. Helium was the carrier gas.The effluent of the GC column was introduced directly into the sourceof MS and spectra obtained in the EI mode with 70 ev ionizationenergy. The sector mass analyzer was set to scan from 40 to 500 amufor 2 s. The identification of individual compounds is based on theirretention times relative to those of authentic samples and matchingspectral peaks available with Wiley, NIST and NBS mass spectrallibraries or with the published data (Adams, 2007).

2.6. Antifungal assay

The antifungal activity of EO and its two constituents was testedagainst fungal isolates by contact assay based on hyphal growthinhibition (Chang et al., 2008) using Czapek's-dox agar (CDA)medium (NaNO3, 2 g; K2HPO4, 1 g; MgSO4, 0.5 g; KCl, 0.5 g; FeSO4,0.01 g; sucrose, 30 g; agar, 15 g; 1 L distilled water, pH 6.8±0.2; SiscoResearch Lab., Mumbai). The requisite amount of EO was dissolved in0.5 mL acetone, and added to 9.5 mL molten CDA in different Petriplates to achieve final concentrations (0.25, 0.50, 0.75 and 1 μL/mL).Geranial and neral were treated at 1 μL/mL. CDA plates containingacetone (0.5 mL) only, served as negative control. In addition, CDAplates treated with Bavistin (50% carbendazim; BASF India Ltd.,Mumbai) at 4 mg/mL were used as positive control. A 5 mm disc oftest fungus was placed upside down on the center of the plate withfungal species in contact with growth medium. Cultures wereincubated in the dark at 28±2 °C (7 days). Antifungal index wascalculated as the following—

Antifungal indexð%Þ = 1−DaDb

� �× 100

where Da: the diameter of growth zone in the test plate; Db: thediameter of growth zone in the control plate.

2.7. Determination of MIC and MFC

The minimal inhibitory concentration (MIC) and minimal fungi-cidal concentration (MFC) for A. flavus (the most prevalent fungus)were determined by broth dilution method as reported earlier(Shukla et al., 2008). Different concentrations of the EO andconstituents were dissolved in 0.5 mL acetone and incorporated to

Table 1Fungi isolated from edible legume seeds.

Legumes Common name Fungi isolated

Arachis hypogaea L.Cajanus cajan L.Cicer arietinum L.Glycine max L.Lens culinaris MedikusPhaseolus vulgaris L.Pisum sativum L.Vigna aconitifolia (Jacq.) MarechalVigna mungo (L.) HepperVigna radiata (L.) R. WilczekVigna unguiculata (L.) Walp

PeanutPigeon peaChick peaSoya beanLentilRed beanWhite peaMoth beanBlack gramMung beanCow pea

⁎Aspergillus flavus (8), Aspergillus niger (4), Fusarium oxysporum (3)A. flavus (6), Alternaria alternata (5), Aspergillus glaucus (3)A. flavus (10), A. niger (4), A. alternata (4)A. flavus (8), Aspergillus shydowi (5)A. flavus (4), A. alternata (7), Rhizopus stolonifer (4)A. flavus (8), Trichoderma sp. (3)A. flavus (3), Aspergillus terreus (6), Penicillium italicum (2), Fusarium graminearum (2)A. flavus (7), Curvularia lunata (4), Fusarium sp. (2)A. flavus (6), A. niger (5), Rhizoctonia solani (3)‡A. flavus (5), A. niger (7), Fusarium nivale (3)†A. flavus (8), Aspergillus fumigatus (3), Cladosporium cladosporioides (3)

Aflatoxigenic isolates: ⁎ (NKD-235), ‡ (NKD-116), † (NKD-122).Values in parentheses are incidence of a particular species in samples of 10 seeds.

Table 2Chemical composition of Lippia alba essential oil.

No. Compounds Percentage Rt

123456789101112131415161718192021222324252627282930313233343536373839404142434445

Hexenyl acetateMethyl acetyl acetoneα-Thujeneα-Pinene1-Hepten-3-olMethyl heptenoneβ-Myrcene3-Octanol2,3,4 Trimethyl pentanem-CymeneDL-Limonene(Z)- β -ocimene(E)- β -ocimeneβ-CitronellolCyclopentylacetoneLinaloolPerilleneE-ChrysanthemalNeolyratolMyrtenalLinalyl acetateβ-CitronellolNerolNerolidolNeralGeraniol formateGeranialGeranyl acetate, 2,3-epoxy-Myrtenyl acetateGeranyl acetateGermacrene-Dβ-ElemeneMethyleugenolE-Caryophylleneγ-Cadineneβ-Ferneseneα-HumuleneE-Caryophylleneγ-CadineneGermacrene-Dα-CopaeneNerolidol(−) Elema-1,3,11(13)-Trien-12-ol1,5-Diphenylhex-3-enePhytolTotal

0.07tr0.331.024.462.847.170.110.470.400.250.500.080.050.071.590.060.760.240.751.560.891.171.54

14.201.86

22.211.952.463.09trtr0.286.170.581.460.040.240.751.480.751.014.281.420.44

91.05

7.178.429.259.52

10.9011.2011.4011.5012.3212.8213.0013.2213.7013.9515.5715.9516.0718.4018.9520.7021.8221.9722.0722.2722.6723.2224.0024.5526.7229.2229.8229.9530.2031.3031.7032.6032.8233.1533.7234.0035.4737.2238.5241.1057.40

tr: traces.

167R. Shukla et al. / International Journal of Food Microbiology 135 (2009) 165–170

9.5 mL CD broth tubes containing 106spores/mL. The tubes wereincubated at 30 °C for a week. The lowest concentration that did notpermit any visible growth was taken as MIC. Cells from the tubesshowing no growth were sub-cultured on treatment-free CDA platesto determine if the inhibition was reversible. MFC is the lowestconcentration at which no growth occurred on the plates.

2.8. Efficacy of the essential oil and constituents on aflatoxin B1 synthesis

Requisite amount of EO and constituents were dissolved in 0.5 mLacetone, and added to 24.5 mL SMKY to achieve the variousconcentrations up to MIC. The medium was inoculated with toxigenicisolates of A. flavus to give 106spores/mL and incubated at 28±2 °C(10 days). The mediumwas filtered and fungal mat was dried at 80 °C(12 h) to determine the net mycelial dry weight. The filtrate was usedfor aflatoxin extraction as described above. For quantitative estima-tions, fluorescent spot of AFB1 on TLC plate was scrapped, dissolved in5 ml cold methanol, and centrifuged (3000 rpm, 5 min). Opticaldensity of the supernatant recorded at 360 nm and the AFB1 amountcalculated according to Sinha et al. (1993):

Aflatoxin B1 contentðμg= LÞ = D × ME × l

× 1000

where, D=absorbance, M=molecular weight of aflatoxin (312),E=molar extinction coefficient of aflatoxin B1 (21,800) and l=pathlength (1 cm cell was used).

2.9. Phytotoxicity assay

The phytotoxicity of the EO and compounds in terms of seedgermination and seedling growth of chick pea was assayed withrespect to control sets following Kordali et al. (2008). Two layers offilter paper were placed on the bottom of each Petri plate (9 cm) and 5seeds were placed equidistantly on the filter paper moistened with10 mL of distilled water. Ten microliters of the EO and compoundswere dripped on Whatman No. 1 filter paper strip placed on the lidusing a micropipette. Petri plates were sealed with parafilm toprevent escaping of volatile compounds and kept at 23±2 °C in agrowth chamber. Percent germination of seeds of control and treatedsets was recorded .The length of radicle and plumule was monitoredat 24, 48, 72, 96, 120 and 144 h interval.

2.10. Statistical analysis

Antifungal, antiaflatoxigenic and phytotoxicity experiments wereperformed in triplicate and data analyzed are mean±SE subjected toone way ANOVA. Means are separated by Tukey's multiple range testswhen ANOVA was significant (pb0.05) (SPSS 10.0; Chicago, IL, USA).

3. Results

Seed samples from eleven legume varieties were tested for theinherent mycoflora wherein a total of 17 different fungal species wereisolated (Table 1). Aspergillus (6 species) was the most prevalentfollowed by Fusarium (4 species). All legumes were found positive for

Table 3Antifungal activities of Lippia alba essential oil and its components against different fungal isolates.

Fungal isolates Antifungal index (%)

‡Essential oil ‡Geranial ‡Neral †Bavistin

0.25 0.5 0.75 1.0 1.0 1.0

Alternaria alternata,Aspergillus flavus,Aspergillus fumigatus,Aspergillus glaucusAspergillus niger,Aspergillus shydowi,Aspergillus terreus,Cladosporium cladosporioides Curvularia lunata,Fusarium graminearumFusarium nivaleFusarium oxysporum,Fusarium sp.Penicillium italicum,Rhizoctonia solaniRhizopus stoloniferTrichoderma spp.

59.2±1.953.5±2.257.0±3.163.7±1.740.4±3.860.0±5.051.6±1.632.1±1.550.7±1.563.5±1.745.0±0.066.1±1.177.4±1.166.0±3.077.0±1.041.6±1.652.0±1.5

73.4±1.663.0±1.578.3±1.669.8±0.954.2±2.982.9±2.473.0±3.644.8±2.862.0±1.574.5±2.350.2±2.868.5±1.780.3±0.368.3±1.686.8±3.491.6±1.677.5±3.2

82.5±2.767.0±0.790.0±1.092.7±3.667.3±2.310079.3±0.659.6±0.373.4±3.285.3±2.670.9±4.380.2±4.880.6±4.174.0±3.010010089.1±0.6

97.3±2.672.5±1.010010077.4±1.610089.5±1.277.6±1.396.0±4.010091.6±2.010095.1±2.8100100100100

10096.6±1.610010094.6±2.910010087.8±1.510010096.6±3.3100100100100100100

79.4±2.168.7±3.190.9±0.583.1±2.171.9±1.595.5±4.483.5±1.558.3±4.476.2±1.186.7±1.782.8±1.484.6±3.396.7±1.695.9±2.110010073.0±3.7

24.4±2.819.5±0.436.7±1.640.8±2.816.5±2.019.7±0.546.9±1.826.2±1.239.9±0.656.9±1.751.0±2.045.2±0.259.3±2.939.2±2.267.0±1.576.6±1.639.7±2.3

‡μL/mL.†4 mg/mL.Values are mean (n=3)±SE.

168 R. Shukla et al. / International Journal of Food Microbiology 135 (2009) 165–170

A. flavus. Three AFB1 producing isolates of A. flavus viz. NKD-116, NKD-122 and NKD-235 were detected from seeds of Vigna radiata,V. unguiculata and Arachis hypogaea, respectively.

The hydro-distillation of L. alba leaves yielded a pale yellow coloredoil (yield: 0.8 mL/kg). GC–MS analysis of EO revealed 45 differentcomponents, representing 91.05% of the total compounds present. Theconstituents identifiedbyGC–MSandother parameters are summarisedin Table 2. The most abundant constituents were monoterpenealdehydes viz. geranial (22.21%) and neral (14.20%). The other maincomponents of the oil were monoterpene hydrocarbon, β-myrcene(7.17%); sesqueterpene hydrocarbon, E-caryophyllene (6.41%) and1-hepten-3-ol (4.46%), (−) elema-1,3,11(13)-trien-12-ol (4.28%),geranyl acetate (3.09%), methyl heptenone (2.84%), myrtenyl acetate(2.46%) etc.

The EO of L. alba exhibited moderate to high antifungal activityagainst all the 17 fungal species tested. The antifungal index of the EOincreased against each fungus with the rise in concentration from 0.25

Table 4Effect of different concentrations of Lippia alba essential oil and its components on mycelia

Treatments Con. Toxigenic isolates

A. flavus (NKD-116)

MDW AFB1

Essential oil 0.00.20.40.60.81.0

430±2.1a

400±4.0ab

374±5.3bc

297±4.3de

217±7.5hi

133±7.0k

305.8±9.4a

192.7±4.8cd

119.5±1.8f

82.9±4.7g

0.0±0.0h

0.0±0.0h

Geranial 0.20.40.60.81.0

314±3.7d

265±4.7 fg

187±4.5ij

102±4.6k

0±0l

168.1±4.1de

83.2±5.6g

0.0±0.0h

0.0±0.0h

0.0±0.0h

Neral 0.20.40.60.81.0

405±3.2a

356±6.0c

282±11.6ef

239±6.6gh

168±8.7j

237.8±6.0b

197.1±3.6c

146.8±4.4e

83.1±9.3g

21.6±2.4h

Con.=Concentration (μL/mL); MDW=Mycelial dry weight (mg); AFB1=Aflatoxin B1 contValues are mean (n=3)±SE.The means followed by same letter in the same column are not significantly different accor

to 1 μL/mL onwards (Table 3). Geranial (1 μL/mL), the primecomponent of the EO caused 100% inhibition of 13 fungi out of 17tested; whereas, at the same concentration EO and neral arrested100% mycelial growth of 9 and 2 fungal species, respectively.C. cladosporioides was found to be highly resistant against geranialand neral (1 μL/mL) as well as at lower concentration of EO (0.25 to0.75 μL/mL) showing least antifungal index amongst the fungi tested.However, A. flavus was found highly resistant against EO at 1 μL/mL.Bavistin, the commercial benzimidazole fungicide was found to beleast effective amongst all the treatments with antifungal activityranging from 16.5 to 76.6%. None of the fungal species was inhibitedabsolutely at 4 mg/mL of Bavistin.

The MICs of EO, geranial and neral were 1.26, 1.03 and 1.58 μL/mL,respectively against A. flavus. MFC of geranial (1.13 μL/mL) was lowerthan that of EO (1.26 μL/mL) or neral (2.1 μL/mL). All the three MICsandMFCs are significant (pb0.05) and lower than the positive control(Bavistin, N5 mg/mL).

l biomass and Aflatoxin B1 production in SMKY medium.

A. flavus (NKD-122) A. flavus (NKD-235)

MDW AFB1 MDW AFB1

402±4.3a

383±3.6a

303±4.0c

282±2.6c

200±5.7e

94±5.3g

143.6±7.7a

104.7±3.8b

47.1±4.2c

0.0±0.0d

0.0±0.0d

0.0±0.0d

388±5.7a

344±3.7b

324±5.6b

250±4.6c

177±5.3e

98±2.9 fg

321.8±4.1a

212.5±7.2b

151.5±6.8c

87.0±5.8d

13.0±6.6 fg

0.0±0.0g

307±4.6c

238±6.0d

123±12.0f

52±2.3h

0±0i

55.6±4.8c

13.5±6.8d

0.0±0.0d

0.0±0.0d

0.0±0.0d

271±4.7c

210±4.6d

154±3.3e

84±3.2g

14±14.6h

176.0±4.1c

94.6±6.3d

29.3±8.0ef

0.0±0.0g

0.0±0.0g

391±1.6a

335±5.0b

292±4.0c

221±6.6de

142±3.7f

108.6±6.6b

93.0±5.5b

49.6±2.0c

0.0±0.0d

0.0±0.0d

385±5.0a

324±3.7b

253±3.6c

175±4.6e

117±2.4f

233.8±9.8b

172.7±5.4c

96.5±2.8d

48.2±3.1e

8.1±4.6 fg

ent (μg/L).

ding to ANOVA and Tukey's multiple comparison tests.

169R. Shukla et al. / International Journal of Food Microbiology 135 (2009) 165–170

Table 4 summarizes the inhibitory effects of EO and two of its majorcomponents on AFB1 production and mycelial dry weight of threetoxigenic isolates of A. flavus (NKD-116, NKD-122 and NKD-235). Thequalitative TLC of EO, geranial and neral treated cultures showed thatthese compounds strongly inhibited AFB1 production in a dose-dependent manner. Based on the quantitative estimation, AFB1

inhibition was at 0.2 μL/mL and onward concentrations of EO, geranialor neral against all the three isolates. In the negative control ofNKD-116,NKD-122 and NKD-235 isolates, AFB1 production was 305.8, 143.6 and321.8 μg/L, respectively. However, AFB1 production by NKD-116, NKD-122 and NKD-235 was inhibited completely at 0.8, 0.6, 1.0 μL/mL of EOand 0.6, 0.6, 0.8 μL/mL of geranial, respectively. On the other hand, only21.6, 0 and 8.1 μg/L AFB1 were produced by NKD-116, NKD-122 andNKD-235 isolates, respectively at 1 μL/mL dose of neral in SMKYmedium.Mycelial dryweight decreasedwith increasing concentrationsof treatments.

A 100% germination of seeds was recorded following 48 h in all thefour sets, including control. Seedling emergence was significantly(pb0.05) not different in all the sets until 48 h of incubation, but thesize of radicles in EO treated seeds was higher than the control,geranial and neral for the rest of the incubations. The mean length ofradicle was a maximum of 133.7 mm in EO treatment compared to102 mm (neral), 100 mm (control) and 95.7 mm (geranial) at 144 hof exposure. Plumules originated after 96 h, and no significantdifference in their length was observed at 144 h in control (23 mm),EO (23.3 mm), geranial (19.3 mm) and neral (21.6 mm) treated seedsaccording to ANOVA and Tukey's multiple comparison tests.

4. Discussion

The present investigation revealed that all legume seeds tested,were contaminated with various fungi and A. flavus dominated inalmost all the seed samples. These results confirm the earlierobservations where Aspergillus spp. were one of the most predomi-nant fungi and aflatoxin producers in some of the stored pulses (El-Nagerabi and Elshafie, 2000; Embaby and Abdel-Galil, 2006).

The essential oil of L. alba has been standardized on GC–MS data. Inthe case of L. alba, a number of chemotypes have been reported,abundant in γ-terpinene (Gomes et al., 1993), limonene (Pino et al.,1997), carvone (Matos et al., 1996), linalool (Siani et al., 2002), citral-myrcene (Matos, 1996) or 1,8-cineol-camphor (Dellacassa et al.,1990). In our study, GC–MS data depicted geranial and neral as majorcomponents of EO. The chemical composition of L. alba EO is differentfrom earlier reports and a novel biological property of the oil in termsof inhibition of aflatoxin and food borne fungi is being reported. Theantifungal activity of EO was also compared with its majorcomponents, geranial and neral to conclude, whether, the EO or itscomponents may be recommended as food preservative against foodcontaminating fungi and aflatoxins.

A perusal of the literature reveals the antifungal activity of Lippiaberlandieri, L. sidoides and L. rugosa against yeast, dermatophytes andother filamentous fungi (Portillo-Ruiz et al., 2005; Fontenelle, et al.,2007; Tatsadjieu et al., 2008); however, fungitoxocity of L. alba is notwell-explored except by Rao et al. (2000) who reported antifungalactivity of oil vapours against sugarcane pathogens. Lee et al. (2008)and Park et al. (2007) have also reported bioactivity of geranial andneral against phytopathogens and dermatophytes. To the best of ourknowledge, there has not been a relevant study on the effectiveness ofL. alba leaf oil and its constituents against aflatoxin production byfungi. In the present study, L. alba EO and two of its components wereinhibitory to all the storage fungi although to a varying degree. AFB1

production was significantly inhibited at lower than fungistaticconcentrations of oil, geranial or neral.

The superiority of EO and its components over the commonly usedsynthetic fungicide Bavistin (50% carbendazim) at the lowest levels ofMICs, further favours their exploitation as the alternative fungitox-

icant. Interestingly, there was no adverse effect of treatments on seedgermination and, rather, the enhanced seedling growth was recordedin EO treated seeds compared to control, geranial or neral. Itcollectively suggests that the synergistic effect of constituents in EOmay be responsible for the enhanced seed germination.

EOs are the potentially useful additives for food preservation toprolong shelf life and improve the quality of stored food products. Afew EO-based preservatives are already commercially available. ‘DMCBase Natural’ in this category, comprises 50% EO from rosemary, sageand citrus and 50% glycerol (Mendoza-Yepes et al., 1997). Limitationsof employing EOs and their components as preservatives includealteration of organoleptic properties of food commodities. However,recent encapsulation techniques using various surfactants seem toovercome such problems (Gaysinsky et al., 2005).

Geranial andneral are the trans- andcis-isomersof citral, respectivelyand with the characteristic lemon odor. Citral, a widely used naturalingredient, is added to foods, soft drinks and cosmetics as the flavoringand fragrance agent. Hence, there would be no chance of their negativeeffects on sensory quality, although detailed investigations on organo-leptic parameters are needed before final recommendation.

A food preservative may be antimicrobial or antioxidant. Theantimicrobial and aflatoxin inhibitory activity of L. alba EO is beingreported in our study whereas antioxidant activity has been earlierreported (Hennebelle et al., 2008). Hence, L alba EO may berecommended as pant based food additive for complete preventionagainst quantitative losses from food borne fungi, qualitative losses dueto aflatoxins and as free radical scavenger due to antioxidant property.

In conclusion, our findings suggest that L. alba EO and two of itscomponents are highly effective against the isolated storage fungi andAFB1 production by A. flavus. Hence, the oil could be potentiallyapplied in food preservation, alternatives to synthetic fungicides toimprove the storage life of staple foods, especially grains and legumes.

Acknowledgements

This work was financially supported by Council of Scientific andIndustrial Research (CSIR), New Delhi, India.

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