Effect of Cymbopogon martinii, Foeniculum vulgare, and ...

Research ArticleEffect of Cymbopogon martinii Foeniculum vulgareand Trachyspermum ammi Essential Oils on the Growthand Mycotoxins Production by Aspergillus Species

Negero Gemeda1 Yimtubezinash Woldeamanuel2 Daniel Asrat2 and Asfaw Debella1

1 Traditional amp Modern Medicine Research Directorate Ethiopian Health amp Nutrition Research InstitutePO Box 1242 Addis Ababa Ethiopia

2Department of Microbiology Immunology amp Parasitology Addis Ababa University PO Box 9086 Addis Ababa Ethiopia

Correspondence should be addressed to Negero Gemeda 4negerogmailcom

Received 5 February 2014 Revised 10 April 2014 Accepted 30 May 2014 Published 23 June 2014

Academic Editor Rong Di

Copyright copy 2014 Negero Gemeda et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study was performed to investigate effect of essential oils onAspergillus spore germination growth andmycotoxin productionIn vitro antifungal and antiaflatoxigenic activities ofCymbopogonmartinii Foeniculum vulgare and Trachyspermum ammi essentialoils were carried out on toxigenic strains ofAspergillus species Plant materials were hydrodistilled for 4-5 h in Clevenger apparatus025 120583LmL 05 120583LmL 1 120583LmL 2 120583LmL and 4 120583LmL concentrations of each essential oil were prepared in 01 Tween 80 (VV)T ammi oil showed highest antifungal activity Absolutemycelial inhibitionwas recorded at 1 120583LmLby essential oils ofT ammiTheoil also showed complete inhibition of spore germination at a concentration of 2 120583LmL In addition T ammi oil showed significantantiaflatoxigenic potency by totally inhibiting toxin production from A niger and A flavus at 05 and 075 120583LmL respectively Cmartinii F vulgare and T ammi oils as antifungals were found superior over synthetic preservative Moreover a concentration of5336297 120583Lkg body weight was recorded for LC50 on mice indicating the low mammalian toxicity In conclusion the essentialoils from T ammi can be a potential source of safe natural food preservative for food commodities contamination by Aspergillusspecies

1 Introduction

Food borne diseases caused by fungal pathogens are still themajor public health problems in this era of advancement infood production technologies [1] Quarter of the worldrsquos foodcommodities have been wasted due to the contamination bytoxic fungi or by fungal metabolic products [2] Improperstorage condition offers favorable environment for the growthof Aspergillus species [2] and production of mycotoxins [2]Consumptions of such contaminated food lead to a seriouscases of illness and mycotoxicoses [3 4] Among theseaflatoxicosis canmanifest both acute (hemorrhage acute liverdamage edema and death) and chronic toxicological effectsof cancer mutagenicity immune suppression birth defectsestrogenic gastrointestinal urogenital vascular kidney andnervous system disorder [1 2 5] In Africa particularly inparts of sub-SaharanAfrica about 250000-hepatocarcinoma

related deaths occur annually due to aflatoxin ingestion alone[1 4]

Current consumer desires safe and uncontaminated foodcommodities throughout the supply chain (from ldquofarmto platerdquo) [2] Several synthetic preservatives have beeneffectively used in management of food contamination byAspergillus species but their continuous application has ledto the development of fungal resistance [6] number of envi-ronmental and health problems [7ndash9] hormonal imbalanceand spermatotoxicity [10] and also some individuals produceallergic reactions to these substances [11] However naturalproducts could potentially serve as effective alternatives ofsynthetic chemicals for the control of food contaminationby Aspergillus species [12 13] Among natural productsessential oils (EOs) of aromatic plants are gaining interestas food additives and widely accepted by consumers becauseof their relatively low toxicity highly volatile transient and

Hindawi Publishing CorporationInternational Journal of Food ScienceVolume 2014 Article ID 874135 9 pageshttpdxdoiorg1011552014874135

2 International Journal of Food Science

biodegradability nature [14 15] European Union allowedthe use of essential oils in food and aromatherapy [16] SoEOs with antimicrobial activity are possible candidates forthe preservativation of food commodities against Aspergillusspecies [17]

Cymbopogon martinii L Foeniculum vulgare Miller andTrachyspermum ammi L Sprague ex Turrillare medicinalaromatic plants of Ethiopia They are used traditionally asfood additives and also are used for the treatment of variousdiseases [18] However as far as our knowledge is concernedthere is no reliable evidence that indicated that these plantsessential oils have fungitoxic and antiaflatoxigenic potentialsagainst aflatoxigenic Aspergillus species in Ethiopia The aimof this study was to evaluate the effect of essential oilson growth spore and mycotoxin production of Aspergillusspecies that could alternate synthetic chemical preservatives

2 Material and Method

21 Chemicals and Media Our media chemicals and sol-vents used in the study were obtained from differentcompanies in different countries Potato Dextrose Agarwas obtained from HiMedia Laboratories Pvt Ltd IndiaSabouraud dextrose agar from Oxoid Ltd EnglandSabouraud dextrose broth from Difco Ltd England sucroseand yeast extract from Labort fine Chem Pvt Ltd IndiaAflatoxin Mix Kit-M from Supelco USA anhydrous Sodiumsulphate MgSO

4sdot7H2O Potassium hydroxide Vanillin and

Silicagel60 thin layer chromatography plate 02mm fromMerck-Schuchardt Germany potasium nitrate from RhonePoulenc US sodium benzoate from Codex FarmacopiaItaly thymol Chloroform Ethyl acetate sulphuric acidTween 20 and Tween 80 from Sigma-Aldrich ChemieGermany acetone from Labort Fine Chem Pvt Ltd Indiaethanol absolute and Methanol from Finken India andtoluene from AnalaR England

22 Plant Materials Cymbopogon martinii (aerial part) wascollected from the botanical garden of TMMRD EHNRIEthiopia Foeniculum vulgare (leaf lamina and leaf sheath)from Shashamane Ethiopia and Trachyspermum ammi(fruits) from Tepi Ethiopia Following collection identifi-cation of plants was carried out in Traditional and ModernDrug Research Department of the Ethiopian Health andNutrition Research Institute (EHNRI) Fresh areal partsof C martinii F vulgare and dried fruits of T ammi(250 g) were placed in a 5 L round-bottom distillation flaskand the plant material was wetted with 3 L distilled waterThe essential oils were obtained by hydrodistillation usingClevenger-type apparatus for continuous 3 h The volatile oilwas taken from the upper layer The excess aqueous layerwas further portioned using dichloromethane to extract andenrich the essential oil from the water layer The organiclayer (dichloromethane extract) was filtered and dried withanhydrous sodium sulfate and concentrated using rotaryevaporator to give the crude essential oil

23 Phytochemical Analysis of the Essential Oils

231 TLC Analysis Following the extraction of essentialoils that are intended for biological assay a portion ofessential oils were subjected to thin-layer chromatography(pre coated Silica gel G60F254) finger print analyses Wagnerand Bladtsrsquo procedure for plant drug analysis was used forthe developing of chromatogram and for the identificationof major secondary metabolites responsible for biologicalactivity [19]

232 GC-Analysis Gas chromatographic analysis of the oilof T ammi was performed on a Shimadzu GC-2010 systemwith split mode The column used was a ZB-1MS equivalentto OV-1 fused silica capillary column 30m times 025mm idfilm thickness 025120583m coated with 5 diphenyl-95 poly-dimethylsiloxane and operated with the following oven tem-perature programs 50∘C held for 2min rising at 3∘Cminto 210∘C Injection temperature and volume were 250∘C and10 120583L respectively injection mode was split split ratio was10 1 carrier gas was nitrogen at 652 cms linear velocityand inlet pressure was 100KPa detector temperature was270∘C nitrogen flow rate was 521mLmin air flow rate was400mLmin make-up 32 (H2air) flow rate was 40mLminand sampling rate was 40ms

24 Fungal Strains Toxigenic strains of Aspergillus flavus(AF001 AF0061015840 AF009 AF019 AF027 and AF037) andAspergillus niger (AN002) were selected for this study Thestrains were isolated from food commodities that werecollected from different local markets in Addis Ababa priorto the study In addition standard strains of Aspergillusflavus (ATCC 13697) and Aspergillus niger (ATCC 10535)were used in the study as a control The standard strainswere obtained fromMicrobiology laboratory Traditional andModern Medicine Research Directorate Ethiopian Healthand Nutrition Research Institute

25 Antifungal Activity

251 Determination of Sporicidal Activity Sporicidal activi-ties of C martinii F vulgare and T ammi were conductedusing spore germination assay according to standard ref-erence method [20] The test organisms were grown onPDA medium for sporulation and spores were harvestedafter 10 days of incubation Spores were collected by adding5mL of sterile water containing 01 (vv) Tween 80 toeach Petri dish and rubbing the surface with a sterile L-shaped spreader (3 times) The suspension was collectedand then centrifuged at room temperature at 2000 rpm for5minThe supernatant was discarded and recentrifuged until1mL of highly concentrated spore solution remained Ahaemocytometer slide was used to count spore production tohave approximately 108 sporemL [21]

Concentrations of essential oils were prepared in 5mLof sabouraud dextrose broth in 100mL flask and then 1mLof the spore suspension was added to each flask The flaskswere then incubated for 24 h at 25∘C on a rotary shaker

International Journal of Food Science 3

(60 rpm) as to evenly disperse the oil throughout the brothAt the end of the incubation period germinated spores wereobserved using a light microscope at 400x magnificationExperiment was performed in triplicate and the extent ofspore germinationwas assessed by looking for the presence ofgerm tubes Results were expressed in terms of the percentageof spores germinated as compared to the control fromthe average of the triplicates Percentage spore germinationinhibition is calculated according to the following formula

Spore Germination inhibition = (sc minus st)(sc)times 100 (1)

where sc is the average number of spore germinated in controlset and st is the average number of spore germinated in testset

252 Determination of Minimum Inhibitory ConcentrationThe minimum inhibitory concentrations (MICs) of C mar-tinii F vulgare and T ammi essential oils were determinedusing agar dilution methods [20 22] Two-fold serial dilu-tions of essential oils in sabouraud dextrose agar were madeto have a final concentration of 025120583LmL 05 120583LmL1 120583LmL 2 120583LmL 4 120583LmL and 8 120583LmL The experimentswere performed in triplicates Control plates containing noessential oils were run simultaneously The agar surface ofthe plates containing the dilution of essential oils and thecontrol plate are inoculated by five millimeter discs of thetest fungi taken from advancing edge of 7-day-old culturesThe plate containing the lowest concentration of essential oilswas seeded first Control plates were seeded last to insurethat viable organismswere present throughout the procedureIncubate the inoculated plates at 26plusmn2∘C for seven days beforebeing read End-points for each EOs are best determined byplacing plates on a dark background and observing the lowestconcentration that inhibits visible growth which is recordedas the MIC The MIC of essential oil was usually recorded inmicroliter per milliliter

253 Determination of Mycelial Dry Weight The effectof essential oils on mycelial dry weight was evaluated insabouraud dextrose broth [20] Two-fold dilution of T ammiessential oil in sabouraud dextrose broth wasmade by addingone part of oil into each nine part of sabouraud dextrosebroth in a 100mL flask to have a final concentration of025 120583LmL 05120583LmL 1 120583LmL 2 120583LmL and 4 120583LmLTheexperiments were performed in triplicates The flasks wereaseptically inoculated with 036mL spore suspensions (asymp106sporemL) The flask containing the lowest concentration ofEOswas seeded first Control plates were seeded last to insurethat viable organismswere present throughout the procedureIncubate the inoculated plates at 26plusmn2∘C for seven days beforebeing read Flasks containing mycelia were filtered throughWhatman filter number 1 and thenwere washedwith distilledwater The mycelia were placed on preweighed Petri platesand were allowed to dry at 60∘C for 6 h and then at 40∘Covernight The flasks containing dry mycelia were weighed

Percentage of growth inhibition on the basis of dry weightwas calculated as

mycelial dry weight

=control weight minus sample weight

control weighttimes 100

(2)

26 Efficacy of T ammi Oil in Arresting Aflatoxin ElaborationThe methods that have been adopted by Kumar et al [23]were used to determine antiaflatoxigenic efficacy of EOs withlowestMIConA flavususing SMKYbrothmedium (Sucrose200 g MgSO

4sdot7H2O 05 g KNO

3 03 g yeast extract 70 g

distilled water 1000mL and pH 56 plusmn 02) Different con-centrations of the oil (01 02 03 04 05 06 07 08 09and 10 120583LmL) were prepared separately by dissolving theirrequisite amount in 05mL 5 Tween 20 and then mixing itwith 245mL of SMKY medium in 100mL Erlenmeyer flask[23] The control sets were kept parallel to the treatment setswithout essential oil Then flasks were inoculated asepticallywith 1mL spore suspension (asymp106 sporesmL) prepared in01 Tween 80 and incubated at 27 plusmn 2∘C for 10 days Thecontent of each flask was filtered (Whatman filter papernumber 1) The filtrate was extracted with 20mL chloroformin a separating funnel and the extracts were passed throughanhydrous sodium sulphate kept in Whatman filter papernumber 42The extractswere evaporated till dryness onwaterbath at 70∘C Dry residues were dissolved in 1mL chloroformand 50120583L of chloroform extract spotted on TLC plate (20 times20 cm2 of Silica gel-G60F254) then developed in chloroformacetone (9 1 vv) The intensity of aflatoxin was observed inUltraviolate Fluorescence Analysis Cabinet at an excitationwavelength of 366 nm

27 Animal Trials to Determine Safety Limit of the OilsThe safety limit of the best fungitoxic essential oils wasdetermined by recording LC50 value on mice following theprotocol of Kumar and his collaborator [23] Mice with anaverage weight and age (35 g 3 months) were selected as testanimals for the mammalian toxicity experiments Requisiteamount of essential oils was mixed properly with Tween80 to prepare different solutions containing desired dose ofessential oilsThemicewere administeredwith 05mLof eachsolution of essential oils orally separately through a gavagesyringe to each set containing 10 mice (equal proportionfor gender) In control sets equal volume of Tween 80 wasgiven to mice After 72 h the mortality of the animals wasrecorded and LC50 was calculated in terms of per kg bodyweight of mice using SPSS version 200 and Minitab version16 computer softwarersquos by Probit analysis

28 Statistical Analysis All the measurements were repli-cated three times for each treatment and data were enteredinto excel spreadsheet and are presented as mean plusmn SESDSignificant differences between strains aflatoxin producerand non-producer between treatment and control wereanalyzed using statistical software (SPSS 200 Chicago ILUSA) at 95 level of confidence by Chi-square analysis Data


Table 1 TLC finger print of essential oils of aromatic plants

Plant Spot (bands) Rf value and corresponding colors1st 2nd 3rd 4th 5th 6th 7th 8th

Thy 051 (violet re) mdash mdash mdash mdash mdash mdash mdashTa 052 (violet red) 045 (brown) 035 (gray) 023 (gray) mdash mdash mdash mdashCm 096 (t violet) 078 (blue) 073 (gray) 065 (blue) 054 (violet) 049 (blue) 039 (blue) 03 (blue)Fv 095 (blue violet) 088 (violet) 069 (gray) 056 (violet) mdash mdash mdash mdashThy thymol standard Cm Cymbopogon martinii Fv Foeniculum vulgare and Ta Trachyspermum ammi

(i) (ii) (iii) (iv)

(a) (b)

Figure 1 TLC fingerprint (a b = before and after heating at 110∘C for 5 minute resp) of thymol standard and essential oils of Trachyspermumammi Cymbopogon martinii and Foeniculum vulgare from (i) to (iv) respectively

were first tested for normality and then subjected to one-way analysis of variance (ANOVA) using statistical software(SPSS 200 Chicago IL USA and Minitab 160 England)Significant differences betweenmean values were determinedusing Tukeyrsquos and Dunkenrsquos multiple range tests followingone-way ANOVA and 119875 values lt005 were considered assignificant

3 Results

TLC fingerprint was used for screening of essential oilsas bioactive compounds were separated in a sequence ofdifferent zones and characterized by the value of retentionfactors (Rf) in Toluene ethyl acetate (93 07) solvent systemand the color of zone they produce after being treated withdetection reagent (vanillin-sulfuric acid) Each plant essentialoil has shown quite different thin layer chromatographyfinger print (Table 1 and Figure 1) TLC screening indicatedthe presence of many terpenoids in the essential oil testedwhich was confirmed by the presence of different coloredspots The highest number of spots was obtained in the chro-matogram of C martinii essential oil that showed distinctive8 spotbands while F vulgare and T ammi essential oils wereseparated in four different spotbandswhen visually observedafter treatment with vanillin-sulphuric acid reagent

A total of 14 components were shown on our GC analysisof T ammi essential oil accounting for 100 of the total

Table 2 Chemical constituents of fruit essential oil of Trachysper-mum ammi

Constituents Retention time(min)

Percentagecomposition ()

Thymol 22305 515122Carvacrol 25505 285048Unidentified 41259 03734120572-terpinolene 42972 18346Unidentified 43546 00273120574-terpinene 44691 51631Unidentified 45047 03331120588-cymene 45721 61766Unidentified 50211 00976Unidentified 50543 00230120573-pinene 54103 50505Unidentified 54597 00273Unidentified 55978 06719Unidentified 56431 02045

amount (Table 2) Thymol (515) was the major constituentof T ammi essential oil followed by Carvacrol (285) para-cymene (62) gamma-terpinene (52) and beta-pinene(51) main constituents


Table 3 Percent of spore germination inhibition of toxigenic Aspergillus flavus by essential oils using fungal spore germination assay

Concentration (120583LmL) C martinii F vulgare T ammi025 4957 plusmn 002c 3257 plusmn 120d 8814 plusmn 034c

05 7514 plusmn 001b 4571 plusmn 193c 9486 plusmn 034b

1 9857 plusmn 000a 8114 plusmn 150b 10000 plusmn 000a

2 10000 plusmn 000a 9980 plusmn 000a 10000 plusmn 000a


Spore percent inhibition values are expressed as mean plusmn SD Mean value with different letters in the same column are significantly different (P lt 005)

Table 4 Antifungal activity of essential oils against the toxigenic strain of Aspergillus flavus using agar dilution technique

Concentration (120583LmL) C martinii F vulgare T ammi025 2305 plusmn 047a 2309 plusmn 042a 2271 plusmn 045a

05 2119 plusmn 066a 2162 plusmn 046a 1728 plusmn 034b

1 1852 plusmn 049b 1910 plusmn 038b 000 plusmn 000c


4 000 plusmn 000c 000 plusmn 000b 000 plusmn 000c

8 000 plusmn 000c 000 plusmn 000c 000 plusmn 000c

Values are expressed as mean plusmn SD Mean value with different letters in the same column are significantly different (P lt 005)

Each tested concentration of essential oils showed notableinhibition of Aspergillus flavus spore germination T ammiessential oils have pronounced spore germination inhibitioneffect on tested organisms Absolute spore germination wasrecorded for essential oils of T ammi C martinii and Fvulgare at a concentration of 1 120583LmL 2120583LmL and 4 120583LmLrespectively Moreover the amount of essential oils tested hassignificant spore inhibition effect (119875 lt 005) (Table 3)

The mean score for antifungal activity of essential oilswere revealed by Table 4 A remarkable antifungal activityagainst the growth of aflatoxigenic strains ofAspergillus flavuswas recorded after the treatment with different concentra-tions of T ammi essential oil followed by C martinii essentialoil and F vulgare essential oil Increased mycelial expansionswere observed at lower concentration of essential oils whileabsolute inhibitions of mycelial expansion were observed athigher concentration indicating dose-dependent activitiesInterestingly the minimum inhibitory concentration wasrecorded at 1 120583LmL for T ammi and 4 120583LmL for both Cmertinii and F vulgare At these points the plant essential oilcompletely inhibited the growth of all isolated fungal strainsA flavus (Table 4)

All the three essential oils were documented to have betterfungitoxic activity against aflatoxigenic Aspergillus specieswhen compared to synthetic chemical preservative sodiumbenzoate (Table 5) Statistical results showed that both thekind and the concentration of essential oil have significanteffect (119875 lt 005) The most promising MIC was recorded byT ammi at 1 120583LmL against A flavus and A niger

The effect of T ammi essential oil on the dry mycelialweight of Aspergillus species in sabouraud dextrose broth ispresented inTable 6 Results of statistical analysis showed thateach tested concentration of essential oils has significantlydifferent mycelial dry weight inhibition (119875 lt 005) It can beclearly seen that a complete inhibition of mycelial dry weightis at a concentration of 1 2 and 4 120583LmL At least a 12 43 and

Table 5 Comparative antifungal activity of essential oils withsodium benzoate

Aspergillus species MICs (mgmL)Cm Fv Ta Sb

AFST 400 800 100 gt1600AF001 400 800 100 gt1600AF0061015840 400 800 100 gt1600AF009 400 800 100 gt1600AF019 400 800 100 gt1600AF027 400 800 100 gt1600AF037 400 800 100 gt1600ANST 200 800 100 1600AN002 200 800 100 1600AFST Aspergillus flavus (ATCC 13697) AF Aspergillus flavus ANSTAspergillus niger (ATCC 10535) AN Aspergillus niger Cm Cymbopogonmartinii Fv Foeniculum vulgare Sb Sodium Benzoate and Ta Trachysper-mum ammi

71 of dry mycelia biomass weight suppression was recordedat 025 05 and 075 120583LmL respectively against Aspergillusspecies A dose-dependent suppression of mycelial growth ofAspergillus species was observed as the higher concentrationof the T ammi essential oil inhibited hundred percent of themycelial growth

Table 7 shows antiaflatoxigenic potentials of T ammiessential oil against aflatoxigenic strains ofAspergillus species(A flavus and A niger) It has been documented fromthis studyrsquos chromatogram that the aflatoxin production inSMKY liquid medium was reduced by the essential oils ofT ammi in dose-dependent manner Aflatoxin productionwas completely inhibited at a concentration of 050 120583LmLfor strains of A niger and at the concentration of 075120583LmLfor A flavus These concentrations were less than thosewhich are recorded for minimum inhibitory concentration


Table 6 Percentage of growth inhibition of Aspergillus species on the basis of dry weight after treatments with different concentrations ofTrachyspermum ammi essential oils

Aspergillus species Percentage of growth inhibition025 120583LmL 05 120583LmL 075 120583LmL 1 120583LmL

AFST 1193 plusmn 000a 4307 plusmn 034b 7118 plusmn 001c 10000 plusmn 000d

AF001 2423 plusmn 120a 5106 plusmn 134b 7305 plusmn 150c 10000 plusmn 000d






ANST 2934 plusmn 102a 7262 plusmn 234b 9185 plusmn 001c 10000 plusmn 000d

AN002 2637 plusmn 002a 7566 plusmn 057b 9248 plusmn 034c 10000 plusmn 000d

AFST Aspergillus flavus (ATCC 13697) AF Aspergillus flavus ANST Aspergillus niger (ATCC 10535) and AN Aspergillus niger Values are expressed as meanplusmn SD Mean value with different letters in the same raw are significantly different (P lt 005)

Table 7 Antiaflatoxigenic activity of Trachyspermum ammi essential oils at a concentration of 000 025 05 075 1 and 2120583LmL againsttoxigenic Aspergillus species

Aspergillus Aflatoxin production (fluorescence under UV 366120582)species Clowast 025 120583LmL 05 120583LmL 075 120583LmL 1 120583LmL 2 120583LmLAFST + + + ND ND NDAF001 + + + ND ND NDAF0061015840 + + + ND ND NDAF009 + + + ND ND NDAF019 + + + ND ND NDAF027 + + + ND ND NDAF037 + + + ND ND NDAFST Aspergillus flavus (ATCC 13697)AFAspergillus flavus + aflatoxin production detected ND aflatoxin production not detected and Clowast Tween 20 (5)

and absolute mycelial dry weight inhibiting concentration(1 120583LmL) In all untreated control (Tween 20 5) highlevel of aflatoxin production was observed in all aflatoxigenicAspergillus species

During the safety limit tests all the test animals wereobserved closely for up to 14 days symptoms of toxicityrecovery and death were noted No sign of toxicity wasrecorded for the mice in groups of control 3000 3500 and4000 120583Lkg body weights The majority of mice from groups4500 5000 and 5500 have showed hypoactivity (decreasedmotility debiting effect) and decreased feed intake Micefrom groups 6000 and 7000120583Lkg have shown the sign ofprostration anaesthesia andmuscle spasmwithin 30minutesand were followed by deaths within the 24 hour The micethat have shown hypoactivity and decreased feed intake haverecovered within 24 hours

As it can be observed from Figure 2 that there wasno mortality at the log concentration of 3000 through4000 120583Lkg while there was one death at 4500 two mortal-ities at a dose of 5000 four mortalities at 5500 and sevenmortalities at 6000 and 10 mortalities from 7000 The LC50was determined by drawing a vertical line on the 119883-axisfrom the point of the straight line the 50 mortality taken

385380375370365360

99

95908070605040302010

5

1

()

Mean 372724StDev 00522390Median 372724IQR 00704693

Table of statistics

Probability plot for mortality

Probit data-ML estimatesNormal

Inverse log

Log concentration (120583Lkg body)

concentration = LC50= 5336297

Figure 2 Probit transformed responses of mice treated with differ-ent concentrations ofT ammi essential oils after oral administration


(Figure 2) and by calculating the inverse log of the valuefound on 119883-axis The LC50 of the essential oils was thus5336297 120583Lkg body weight

4 Discussion

In this millennium infection from Aspergillus speciesbecomes the major public health problem of modern mycol-ogy [24]They have a capability to cause directly infection andindirectly mycotoxicosis especially upon the consumption offood contaminated with Aspergillus species Many chemicalpreservatives have been used for the control of Aspergillusfood contamination [25] The widespread use of chemicalpreservative has significant drawbacks including increasedcost handling hazards concern about pesticide residueson food and threat to human health and environment[10] Public awareness of these risks has increased interestin finding safer alternatives natural products to replacecurrently used synthetic chemical preservatives to controlAspergillus food contamination One such alternative is theuse of essential oils with antifungal and antiaflatoxigenicactivities since they tend to have low mammalian toxicityless environmental effects and wide public acceptance [17]C martinii Foeniculum vulgare and T ammi are commoneconomic food spices in Ethiopia and thus it is an advantageto develop safe botanical food preservative against toxigenicAspergillus species that have strong affinity to colonize variousfood commodities due to its secretion of hydrolytic enzymes[18]

Our TLC analysis confirmed the presence of variouscomponents of essential oils which were characterized bythe distance they travel in a particular TLC system andtheir appearance (color) after visualization of the spotsEssential oils are very complex natural mixtures which cancontain about 20ndash60 components at quite different con-centrations [26] They are characterized by two or threemajor components at fairly high concentrations (20ndash70)compared to other components present in trace amounts[26] This chromatogram developed from essential oils withthe distinctive spot Rf and color were due to the presence ofmajor component of essential oils (i) alcohols geraniol in Cmartini (ii) phenols thymol and carvacrol in T ammi (iii)aldehydes anisaldchyde in F vulgare (iv) ketones fenchonein F vulgare (v) esters geranyl acetate in C martinii and (vi)phenylpropanoids anethole in F vulgare essential oils [19]And in our GC analysis we confirmed the presence of thymol(515) and carvacrol (285) as major components TLCand GC finger prints are the most common chromatographictechniques widely available for phytochemical analysis ofplant essential oils They are used in standardizing theconstituents of essential oils that are classified as generallyrecognized as safe by FDA for their use as food additives incontrolling food spoilage [17]

Remarkable sporicidal activities against toxigenic strainsof organisms tested were recorded in our study Hundredpercent inhibitions of spore germination were recorded at4 120583LmL for essential oil of F vulgare and at 2 120583LmL foressential oils of C martinii and T ammi Similarly previousstudies reported mycosporicidal activity of essential oils

from C mertinii [27] antimicrobial activity of T ammiessential oils [24 28] and antifungal effects of F vulgareessential oils [29] The impacts of essential oil on sporulationmay be due to denaturation of the enzymes responsiblefor spore germination or interference with the amino acidinvolved in germination [30] The vapor action exerted byvolatile constituents of this essential oil on surface mycelialdevelopment andor the transduction of signals involvedin the switch from vegetative to reproductive developmentcould also be responsible for the spore germination inhibitionactivity [30 31]

Essential oils had a clear dose-dependent antifungalactivity on A flavus at the concentration tested in our agardilution assay to determine MICs Our finding suggeststhat the increment of dose to 2 4 and 8 120583LmL have asignificant effect on the inhibition of fungal in reduction offungal growth while all tested doses have significant effect ininhibition of spore germination Absolute inhibition of fungalgrowth were seen at a concentration of 2 120583LmL 4 120583LmLand 8 120583LmL for the essential oils of T ammi C martiniiand F vulgare respectively and the concentrations wererecorded as MICs Volatile constituents of the essential oilsmight create vapor action which could be responsible for theactivities [26] Previous study reported antifungal propertyof essential oils certainly differences in major and minorconstituents of the oils that are responsible for their biologicalactivity by geographical location and seasons of collectionand the difference in test organisms used in the study couldcontribute to the difference in MIC of C martinii [32] Fvulgare [33] and T ammi [34]

All essential oils have better fungitoxic activity than syn-thetic preservative used in our studywhichwas in accordancewith the finding of previous study [26] The inferior activitycould be due to nature of the chemicals since it was reportedthat sodium benzoate was highly active at pH 35 weak acidcompounds are more lipophilic in their nondissociated formwhich enables them to cross the cell membrane that ledto pH lowering of cytoplamic cell with rupture of certainmetabolic reactions of the microorganism permeabilizationof the cytoplasmic membrane and cell death Other authorsdemonstrated that benzoic acid has membrane-perturingpotentials In addition these acids induce loss of mitochon-drial function and one possibility that we entertained wasthat this could be the result of mitochondrial autophagy [35]Moreover essential oils are complex mixtures of numerousmolecules and one might wonder if their biological effectsare the result of a synergism of all molecules or reflect onlythose of the main molecules present at the highest levels Inthe literature in most cases only the main constituents ofcertain essential oils like thymol carvacrol carvone geraniolwere analyzed [26]Thus synergistic functions of the variousmolecules contained in an essential oil in comparison tothe action of one or two main components of the oil seemquestionable However it is possible that the activity of themain components is modulated by other minor molecules[26] Moreover it is more likely that several components ofthe essential oils play a role in cell penetration lipophilicor hydrophilic attraction and fixation on cell walls andmembranes and cellular distribution [26]


Essential oil of T ammi has pronounced activity in reduc-ing mycelial biomass The effects are dose-dependent as upon the increment of the concentration of the oils the myceliadry weights of the tested Aspergillus species were recordedThe reduction in fungal mycelia biomass may be due to thepresence of phenolic compounds in the essential oils [26]In addition lypophilic nature of the essential oils helps tocross cell membrane of the fungal cell interacting with theenzymes and proteins of themembrane thus producing a fluxof protons towards the cell exterior which induces changes inthe cells and ultimately leading to death [26] Similar findingswere reported by previous study that showed T ammi oilshave the ability to reduce mycelial dry weight [36] At lowdose phenols affected enzyme activity especially of thoseenzymes associated with energy production while at greaterconcentrations caused protein denaturation [36]

Another crucial finding of this study was that essential oilof T ammi has antiaflatoxicogenic activity Aflatoxin can beproduced byAspergillus species but the fungusmay no longerbe present in the food hence preservative used for the controlof aflatoxin should act on both fungus and the mycotoxinthey produce Interestingly the productions of aflatoxinwere inhibited by T ammi essential oil at concentrationslower than recorded for MIC spore germination inhibitionand mycelial dry weight Thus the inhibition of aflatoxinproduction cannot be completely attributed to reduced fungalgrowth but may be because of inhibition of carbohydratecatabolism in Aspergillus species by acting on some keyenzymes reducing its ability to produce aflatoxins Similarfinding was reported from previous study [37] Reductionof mycelial growth and spore germination inhibition andaflatoxin production inhibition could be due to the presenceof thymol and carvacrol (phenolic OH group) that formhydrogen bonds with target enzyme active site [38]

Results from safety limit of T ammi essential on miceshowed that it is unlikely to present acute toxicity supportingthe consumption of this plant seeds as spice in Ethiopiancommunity All preservatives used against food spoilagemoulds should not be harmful for human beings uponconsumption The safety limit of the T ammi essential oilwas also determined through its oral administration (acuteoral toxicity) on mice and its LC50 value was found tobe 5336297 120583Lkg body weight and classified using WHOrecommended classification in the U group [39] The highvalue of LC50 is a symbol of the nonmammalian toxicity ofthe T ammi essential oil and hence it may be recommendedas a safe preservative of food as 500000120583L of the testsubstancekg body weight is the practical upper limit for theamount of test material that can be administered in one oralgavage dose to a rodent Nowadays using essential oils as foodadditives is common throughout the world [17]

5 Conclusions

Result of current study indicated that essential oils of plantspossess antifungal property against toxigenic strains ofAspergillus speciesWe found that essential oils ofCmartiniiF vulgare and T ammi have fungitoxic potential Moreover

essential oils of T ammi have antiaflatoxigenic potentials andno mammalian toxicity on mice Therefore essential oils ofT ammi could be recommended as best safe botanical foodpreservative as it has antifungal as well as antiaflatoxigenicactivity superiority over synthetic fungicides and nonmam-malian toxicity In addition this essential oil has practicalapplicability as fumigant of food commodities due to theiraromatic volatility nature Moreover we can also minimizethe residual effect of this plant in food commodities by dryingfood staffs using sun light before consumption As a result ofthese finding and opportunities we suggest T ammi essentialoil as a potential source of safe botanical food preservativethat inhibits Aspergillus spore germination growth andmycotoxin production inhibition However further studiesshould be conducted to explore large scale utilization and alsoexploring the efficacy of T ammi essential oils using othertoxigenic organism that contaminate food commodities

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Financial supports for this study were provided by AddisAbaba University and by Ministry of Finance and Eco-nomic Development through EHNRI (through Project no342020401012) The authors are thankful for the staff ofthe Traditional and Modern Medicine Directorate EHNRIfor their help and support during their studyThey would liketo thank Mr Awoke Kebede for his generous permission touse his aflatoxin standard (AflatoxinMix Kit-M)The authorsare grateful to theDepartment ofMicrobiology Immunologyand Parasitology (DMIP) College of Health Science AAUand EHNRI for their facility and financial support

References

[1] C S DeWaal and N Robert Global and Local Food SafetyAround the World Center for Science in the Public InterestWashington DC USA 2005

[2] J F Leslie R Bandyopadhyay and A Visconti MycotoxinsDetectionMethodsManagement Public Health andAgriculturalTrade CAB International London UK 2008

[3] C P Wild and Y Y Gong ldquoMycotoxins and human disease alargely ignored global health issuerdquo Carcinogenesis vol 31 no1 Article ID bgp264 pp 71ndash82 2010

[4] J M Wagacha and J W Muthomi ldquoMycotoxin problem inAfrica current status implications to food safety and health andpossible management strategiesrdquo International Journal of FoodMicrobiology vol 124 no 1 pp 1ndash12 2008

[5] M E Zain ldquoImpact of mycotoxins on humans and animalsrdquoJournal of Saudi Chemical Society vol 15 no 2 pp 129ndash1442011

[6] S Brul and P Coote ldquoPreservative agents in foods modeof action and microbial resistance mechanismsrdquo InternationalJournal of Food Microbiology vol 50 no 1-2 pp 1ndash17 1999


[7] C A Damalas and I G Eleftherohorinos ldquoPesticide exposuresafety issues and risk assessment indicatorsrdquo InternationalJournal of Environmental Research and Public Health vol 8 no5 pp 1402ndash1419 2011

[8] A L Rubin ldquoMammalian toxicity of microbial pest controlagentsrdquo in Handbook of Pesticide Toxicology Principle R IKrieger and W C Krieger Eds pp 859ndash871 Acadamic PressNew York NY USA 2nd edition 2001

[9] A H Havelaar S Brul A de Jong R de Jonge M HZwietering and B H ter Kuile ldquoFuture challenges to microbialfood safetyrdquo International Journal of FoodMicrobiology vol 139supplement pp S79ndashS94 2010

[10] LM L Nollet andH S RathoreHandbook of Pesticides Meth-ods of Pesticide Residues Analysis Taylor amp Francis Boca RatonFla USA 2010

[11] F Cardinale F Mangini M Berardi et al ldquoIntolerance to foodadditives an updaterdquo Minerva Pediatrica vol 60 no 6 pp1401ndash1409 2008

[12] A Gonzalez-Coloma M Reina C M Diaz and B M FragaldquoNatural product-based biopesticides for insect controlrdquo inComprehensive Natural Products vol 3 pp 237ndash268 2010

[13] M Hyldgaard T Mygind and R L Meyer ldquoEssential oils infood preservation mode of action synergies and interactionswith foodmatrix componentsrdquo Frontiers in Microbiology vol 3article 12 2012

[14] A Gupta S Sharma and S N Naik ldquoBiopesticidal value ofselected essential oils against pathogenic fungus termites andnematodesrdquo International Biodeterioration and Biodegradationvol 65 no 5 pp 703ndash707 2011

[15] A Alizadeh E Zamani R Sharaifi M Javan-Nikkhah andS Nazari ldquoAntifungal activity of some essential oils againsttoxigenic Aspergillus speciesrdquo Communications in agriculturaland applied biological sciences vol 75 no 4 pp 761ndash767 2010

[16] I Ipsilantis C Samourelis and D G Karpouzas ldquoThe impactof biological pesticides on arbuscular mycorrhizal fungirdquo SoilBiology and Biochemistry vol 45 pp 147ndash155 2012

[17] M Razzaghi-Abyaneh M Shams-Ghahfarokhi M-B Rezaeeet al ldquoChemical composition and antiaflatoxigenic activity ofCarum carvi L Thymus vulgaris and Citrus aurantifolia essen-tial oilsrdquo Food Control vol 20 no 11 pp 1018ndash1024 2009

[18] H Tadeg Phytopharmaceutical Studies of Some SelectedMedici-nal Plants Locally Used in the Treatment of Skin Disorders AddisAbaba University Addis Ababa Ethiopia 2004

[19] H Wagner and S Bladt Plant Drug Analysis a Thin LayerChromatography Atlas Springer New York NY USA 2ndedition 1996

[20] K Das R K S Tiwari and D K Shrivastava ldquoTechniques forevaluation of medicinal plant products as antimicrobial agentcurrent methods and future trendsrdquo Journal of Medicinal PlantsResearch vol 4 no 2 pp 104ndash111 2010

[21] S A Uldahl and G Knutsen ldquoSpore swelling and germinationas a bioassay for the rapid screening of crude biological extractsfor antifungal activityrdquo Journal of Microbiological Methods vol79 no 1 pp 82ndash88 2009

[22] A BalowsManual of Clinical Microbiology ASM InternationalMaterials Park Ohio USA 6th edition 1991

[23] A Kumar R Shukla P Singh C S Prasad and N K DubeyldquoAssessment of Thymus vulgaris L essential oil as a safe botan-ical preservative against post harvest fungal infestation of foodcommoditiesrdquo Innovative Food Science and Emerging Technolo-gies vol 9 no 4 pp 575ndash580 2008

[24] J I Pitt and A D Hocking Fungi and Food Spoilage SpringerNew York NY USA 3rd edition 2009

[25] E Maier K Kurz M Jenny H Schennach F Ueberall and DFuchs ldquoFood preservatives sodiumbenzoate and propionic acidand colorant curcumin suppress Th1-type immune response invitrordquo Food and Chemical Toxicology vol 48 no 7 pp 1950ndash1956 2010

[26] F Bakkali S Averbeck D Averbeck and M Idaomar ldquoBio-logical effects of essential oilsmdasha reviewrdquo Food and ChemicalToxicology vol 46 no 2 pp 446ndash475 2008

[27] P Sivamani and A S S Hameed ldquoMycosporicidal activityof essential oils from selected herbals against isolates fromHIVAIDS patientsrdquo Journal of Pharmacy Research vol 3 no3 pp 679ndash683 2010

[28] S Paul R C Dubey D K Maheswari and S C Kang ldquoTrach-yspermum ammi (L) fruit essential oil influencing on mem-brane permeability and surface characteristics in inhibitingfood-borne pathogensrdquo Food Control vol 22 no 5 pp 725ndash7312011

[29] VUniyal R P Bhatt S Saxena andA Talwar ldquoTifungal activityof essential oils and their volatile constituents against respira-tory tract pathogens causing Aspergilloma and Aspergillosis bygaseous contactrdquo Journal of Applied and Natural Science no 1pp 65ndash70 2012

[30] G J E Nychas ldquoNatural antimicrobials from plantsrdquo in NewMethods of Food Preservation G W Gould Ed pp 58ndash89Blackie Academic London UK 1995

[31] J Tian X Ban H Zeng J He Y Chen and Y Wang ldquoThemechanism of antifungal action of essential oil from dill(Anethum graveolens l) on Aspergillus flavusrdquo PLoS ONE vol7 no 1 Article ID e30147 2012

[32] S Bansod andM Rai ldquoAntifungal activity of essential Oils fromIndian medicinal plants against human pathogenic Aspergillusfumigatus and A nigerrdquo World Journal of Medical Sciences vol3 no 2 pp 81ndash88 2008

[33] G I K Marei M A Abdel Rasoul and S A M AbdelgaleilldquoComparative antifungal activities and biochemical effects ofmonoterpenes on plant pathogenic fungirdquo Pesticide Biochem-istry and Physiology vol 103 no 1 pp 56ndash61 2012

[34] Javed A A Shahid M S Haider et al ldquoNutritional phyto-chemical potential and pharmacological evaluation of NigellaSativa (Kalonji) andTrachyspermumammi (Ajwain)rdquoMedicinalPlants Research vol 6 no 5 pp 768ndash775 2012

[35] R Kazan A Levine and H Abeliovich ldquoBenzoic acid aweak organic acid food preservative exerts specific effects onintracellular membrane trafficking pathways in SaccharomycescerevisiaerdquoApplied and EnvironmentalMicrobiology vol 70 no8 pp 4449ndash4457 2004

[36] A Ahmad A Khan S Yousuf L A Khan and N ManzoorldquoProton translocating ATPase mediated fungicidal activity ofeugenol and thymolrdquo Fitoterapia vol 81 no 8 pp 1157ndash11622010

[37] S S Hajare S N Hajare and A Sharma ldquoAflatoxin inactivationusing aqueous extract of ajowan (Trachyspermum ammi) seedsrdquoJournal of Food Science vol 70 no 1 pp C29ndashC34 2005

[38] J Tian X Ban H Zeng J He B Huang and Y WangldquoChemical composition and antifungal activity of essential oilfrom Cicuta virosa L var latisecta Celakrdquo International Journalof Food Microbiology vol 145 no 2-3 pp 464ndash470 2011

[39] WHO The WHO Recommended Classification of Pesticidesby Hazard and Guidelines to Classification WHO GenevaSwitzerland 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


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BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


biodegradability nature [14 15] European Union allowedthe use of essential oils in food and aromatherapy [16] SoEOs with antimicrobial activity are possible candidates forthe preservativation of food commodities against Aspergillusspecies [17]

Cymbopogon martinii L Foeniculum vulgare Miller andTrachyspermum ammi L Sprague ex Turrillare medicinalaromatic plants of Ethiopia They are used traditionally asfood additives and also are used for the treatment of variousdiseases [18] However as far as our knowledge is concernedthere is no reliable evidence that indicated that these plantsessential oils have fungitoxic and antiaflatoxigenic potentialsagainst aflatoxigenic Aspergillus species in Ethiopia The aimof this study was to evaluate the effect of essential oilson growth spore and mycotoxin production of Aspergillusspecies that could alternate synthetic chemical preservatives

2 Material and Method

21 Chemicals and Media Our media chemicals and sol-vents used in the study were obtained from differentcompanies in different countries Potato Dextrose Agarwas obtained from HiMedia Laboratories Pvt Ltd IndiaSabouraud dextrose agar from Oxoid Ltd EnglandSabouraud dextrose broth from Difco Ltd England sucroseand yeast extract from Labort fine Chem Pvt Ltd IndiaAflatoxin Mix Kit-M from Supelco USA anhydrous Sodiumsulphate MgSO

4sdot7H2O Potassium hydroxide Vanillin and

Silicagel60 thin layer chromatography plate 02mm fromMerck-Schuchardt Germany potasium nitrate from RhonePoulenc US sodium benzoate from Codex FarmacopiaItaly thymol Chloroform Ethyl acetate sulphuric acidTween 20 and Tween 80 from Sigma-Aldrich ChemieGermany acetone from Labort Fine Chem Pvt Ltd Indiaethanol absolute and Methanol from Finken India andtoluene from AnalaR England

22 Plant Materials Cymbopogon martinii (aerial part) wascollected from the botanical garden of TMMRD EHNRIEthiopia Foeniculum vulgare (leaf lamina and leaf sheath)from Shashamane Ethiopia and Trachyspermum ammi(fruits) from Tepi Ethiopia Following collection identifi-cation of plants was carried out in Traditional and ModernDrug Research Department of the Ethiopian Health andNutrition Research Institute (EHNRI) Fresh areal partsof C martinii F vulgare and dried fruits of T ammi(250 g) were placed in a 5 L round-bottom distillation flaskand the plant material was wetted with 3 L distilled waterThe essential oils were obtained by hydrodistillation usingClevenger-type apparatus for continuous 3 h The volatile oilwas taken from the upper layer The excess aqueous layerwas further portioned using dichloromethane to extract andenrich the essential oil from the water layer The organiclayer (dichloromethane extract) was filtered and dried withanhydrous sodium sulfate and concentrated using rotaryevaporator to give the crude essential oil

23 Phytochemical Analysis of the Essential Oils

231 TLC Analysis Following the extraction of essentialoils that are intended for biological assay a portion ofessential oils were subjected to thin-layer chromatography(pre coated Silica gel G60F254) finger print analyses Wagnerand Bladtsrsquo procedure for plant drug analysis was used forthe developing of chromatogram and for the identificationof major secondary metabolites responsible for biologicalactivity [19]

232 GC-Analysis Gas chromatographic analysis of the oilof T ammi was performed on a Shimadzu GC-2010 systemwith split mode The column used was a ZB-1MS equivalentto OV-1 fused silica capillary column 30m times 025mm idfilm thickness 025120583m coated with 5 diphenyl-95 poly-dimethylsiloxane and operated with the following oven tem-perature programs 50∘C held for 2min rising at 3∘Cminto 210∘C Injection temperature and volume were 250∘C and10 120583L respectively injection mode was split split ratio was10 1 carrier gas was nitrogen at 652 cms linear velocityand inlet pressure was 100KPa detector temperature was270∘C nitrogen flow rate was 521mLmin air flow rate was400mLmin make-up 32 (H2air) flow rate was 40mLminand sampling rate was 40ms

24 Fungal Strains Toxigenic strains of Aspergillus flavus(AF001 AF0061015840 AF009 AF019 AF027 and AF037) andAspergillus niger (AN002) were selected for this study Thestrains were isolated from food commodities that werecollected from different local markets in Addis Ababa priorto the study In addition standard strains of Aspergillusflavus (ATCC 13697) and Aspergillus niger (ATCC 10535)were used in the study as a control The standard strainswere obtained fromMicrobiology laboratory Traditional andModern Medicine Research Directorate Ethiopian Healthand Nutrition Research Institute

25 Antifungal Activity

251 Determination of Sporicidal Activity Sporicidal activi-ties of C martinii F vulgare and T ammi were conductedusing spore germination assay according to standard ref-erence method [20] The test organisms were grown onPDA medium for sporulation and spores were harvestedafter 10 days of incubation Spores were collected by adding5mL of sterile water containing 01 (vv) Tween 80 toeach Petri dish and rubbing the surface with a sterile L-shaped spreader (3 times) The suspension was collectedand then centrifuged at room temperature at 2000 rpm for5minThe supernatant was discarded and recentrifuged until1mL of highly concentrated spore solution remained Ahaemocytometer slide was used to count spore production tohave approximately 108 sporemL [21]

Concentrations of essential oils were prepared in 5mLof sabouraud dextrose broth in 100mL flask and then 1mLof the spore suspension was added to each flask The flaskswere then incubated for 24 h at 25∘C on a rotary shaker








mycelial dry weight



(2)


4sdot7H2O 05 g KNO









(i) (ii) (iii) (iv)

(a) (b)



3 Results



















































385380375370365360

99

95908070605040302010

5

1

()


Table of statistics



Inverse log






4 Discussion











5 Conclusions





Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








mycelial dry weight



(2)


4sdot7H2O 05 g KNO









(i) (ii) (iii) (iv)

(a) (b)



3 Results



















































385380375370365360

99

95908070605040302010

5

1

()


Table of statistics



Inverse log






4 Discussion











5 Conclusions





Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





(i) (ii) (iii) (iv)

(a) (b)



3 Results



















































385380375370365360

99

95908070605040302010

5

1

()


Table of statistics



Inverse log






4 Discussion











5 Conclusions





Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












































385380375370365360

99

95908070605040302010

5

1

()


Table of statistics



Inverse log






4 Discussion











5 Conclusions





Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Discussion











5 Conclusions





Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





5 Conclusions





Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Effect of Cymbopogon martinii, Foeniculum vulgare, and ...

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