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1 Originl Reserh TM DOI : Journl Home pge : «E-mil : eitor@je.o.in Journl of Environmentl Biology JEBTM p-issn: e-issn: CODEN: JEBIDP Antimiroil tivity of Cymopogon itrtus essentil oil ginst the myoflor of store rie fruits of Znthoxylum rmtum Authors Info D. Gupt, P. Bl n Y.P. Shrm* Deprtment of Botny, University of Jmmu, Jmmu8 6, Ini *Corresponing Author Emil : yshm3@yhoo.o.in Key wors Afltoxin B Antifungl Aspergillus flvus Cymopogon itrtus Essentil oil Astrt Aim : The stuy ime to investigte the fungl ontmintion in rie fruits of Znthoxylum rmtum n the effet of essentil oil of Cymopogon itrtus ginst the eteriorting fungi n fltoxin proution y Aspergillus flvus. Methoology : Myoflor nlysis of rie fruits of Z. rmtum ws one y ilution plte tehnique, while the ntifungl ssy of essentil oil of C. itrtus ginst seven isolte mouls ws rrie out y poisone foo tehnique. Afltoxigeni potentil of Aspergillus flvus n nti-fltoxigeni tivity of essentil oil of C. itrtus ws one y spetrophotometri metho. Results : A totl of 7 fungl speies were isolte with Aspergillus s the preominnt genus. The hrteriztion of C. itrtus essentil oil revele α- itrl n β- itrl s mjor omponents. Essentil oil signifintly exhiite ntifungl tivity ginst the growth of teste fungl speies. The miniml inhiitory onentrtion of essentil oil ws foun to e.5 µl ml for Fusrium grminerum, 2.5 µl ml for Alternri lternt, Aspergillus flvus, A. ohreus n Peniillium expnsum n 3. µl ml for A. prsitius n P. itrinum, respetively. The essentil oil ompletely heke the proution of fltoxin B y the toxigeni strins of A. flvus. Interprettion : Both myelil growth n fltoxin B proution followe elining tren with n inrese in essentil oil onentrtion, emonstrting its prtil effiy s plnt se ntimiroil for enhning the shelf-life of Z. rmtum. Isoltion of mouls from store rie fruits of Znthoxylum rmtum Extrtion n hrteriztion of Cymopogon itrtus essentil oil Anti fungl ssy of essentil oil ginst seven isolte mouls White Smoke Just write. Plgirism Detetor Pulition Info Pper reeive : Revise reeive :..28 Re-revise reeive :.3.28 Aepte : Assessment of fltoxin B prouing potentil of toxigeni isoltes of Aspergillus flvus Evlution of nti fltoxigeni effiy of essentil oil ginst three toxigeni strins of Aspergillus flvus Triveni Enterprises, Luknow (Ini) Journl of Environmentl Biology Novemer 28 Vol

2 952 D. Gupt et l. Introution Drie fruits re n importnt group of griulturl n onfetionry ommoities eing use sine the wn of humn iviliztion ll over the worl. Owing to their flvour, tste n inispensle nutrients, n rry of helth protetive iotive ingreients ielly inrese the iet qulity n help to erese the risk of unrelieve iseses (Chng et l., 26). Moisture ontent of rie fruits vries from 2 to 22 % n further reution in the wter ontent of these ommoities y rying inhiits miroil growth n slows own enzymti retions (Vinson et l., 25). In generl, most of the rie ommoities hve equtely low wter tivity to slow own teril growth. However, filmentous mouls, espeilly the xerophili mouls re the most importnt miro-orgnisms in rie fruits in terms of ioeteriortion, if their rying n storge is inequte. Some of the eteriorting mouls re lso potentil prouers of vrious myotoxins. Among these, fltoxins proue y the Aspergillus flvus n other relte speies re the most importnt myotoxins ourring in foo n fee (Uomkun et l., 27). Use of these ontminte ommoities uses fltoxiosis, isese hving symptoms of norml liver funtioning, epresse immune response, rinogenesis et. (Shukl et l., 29). To prolong the shelf-life of rie fruits n prevent the growth of filmentous fungi n proution of fltoxins, it is impertive to estlish n iel preservtive. Currently, to mnge storge mouls in rie ommoities, most of the syntheti hemils use uring posthrvest tretment re mn-me xenoiotis whose unte pplitions hve le to the evelopment of fungl resistne s well s resiul toxiity (Brent n Hollomon, 998). They re lso responsile for the stimultion of fltoxin iosynthesis (Jyshree n Surmnym, 2). The present isussion on the negtive effets of syntheti preservtives hs lso renewe interest of reserhers towrs nturl foo itives to improve the shelf-life of foo ommoities n to protet them from infesting miroes. One of suh lterntives is the pplition of eo-frienly, ost-effetive otnils to minimize fungl eteriortion of griulturl ommoities n to improve their qulity (Burt, 24). Sine, the essentil oils of higher plnts n their omponents re highly voltile n ioegrle, they re esily epte y onsumers. Consequently, there is n inresing ttention worlwie y vrious reserhers to stuy the ntimiroil tivities of vrious plnt extrts to ontrol mouls in store foo ommoities tht pose less risk to humn helth n environment (Wekesser et l., 27; Gnomi et l., 29; El-Ngeri et l., 23). Cymopogon itrtus (DC.) Stpf (Lemon grss) is known to possess vrious ntimiroil, ntiirrhel n ntioxint ttriutes owing to the presene of terpenois, phenoli ompouns n essentil oils (Tzortzkis n Eonomkis, 27; Mnvith n Biy, 24; Mosquer et l., 26; Kusr et l., 27; Oliveir et l., 28). Therefore, the present stuy ws unertken to sreen the fungi responsile for eteriorting store rie fruits of Znthoxylum rmtum (lolly known s timru) n etetion of toxigeni strins of Aspergillus flvus mong these spoilge mouls. Also, the essentil oil of C. itrtus ws evlute for its ntifungl n nti-fltoxigeni tivity in orer to ssess its effiy s prospetive foo itive. Mterils n Methos Prourement of rie fruits : A totl of 64 smples (2 g eh) of rie fruits of Znthoxylum rmtum were purhse from househols, wholeslers, mrkets n retil shops in ifferent lolities of Jmmu provine (Jmmu, Resi, Kthu, Rjouri, Poonh, Sm n Uhmpur). The smples were ollete in sterilize polythene gs, sele over flme to prevent further ontmintion n store t low temperture (5 C) for further myoflor n myotoxin nlysis. Myoflor nlysis : The filmentous fungi of rie fruits of Z. rmtum were isolte y ilution plte tehnique following the metho of Singh et l. (99). gm eh of powere smples ws homogenize in 9 ml sterile istille wter in Erlenmeyer flsk (25 ml). Dilutions of resiue were me with sterilize istille wter n one ml of liquot of eh smple ws then poure in Petri plte ontining ml freshly prepre Czpek'sox gr (CDA) meium. Three replites of eh smple were prepre n inute (27±2 C) for seven ys. Different fungl olonies were ounte n fungl speies were ientifie on the sis of their ulturl n miro-morphologil hrters using relevnt literture n reommene keys (Rper n Fennell, 965; Booth, 97; Ellis, 97; Pitt, 979). The ultures of fungl isoltes were mintine on PDA slnts t 4 C. The ourrene frequeny of isolte fungi ws etermine following Mneel's (25) formul. Extrtion of essentil oil : For extrtion of essentil oil, fresh leves (5 gm) of Cymopogon itrtus were ollete from the Botnil Gren, University of Jmmu, Jmmu. After wshing twie with istille wter, leves were sujete to Clevenger's hyro istilltion pprtus (3 hrs). The essentil oil ws seprte n wter tres were remove y nhyrous soium sulphte n store t 4 C in rk for further experiments. Chrteriztion of C. itrtus essentil oil : The essentil oil of C. itrtus ws nlyze through GC equippe with flme ioniztion etetor. The GC onitions were pillry olumn CP-Sil-8 (3m x.32mm x.25μm thik film); helium ws the rrier gs; injetion temperture 28 C, split rtio :5, olumn oven temperture 5 C for 5 min, then 25 C hel for 7 min. The ientifition of vrious ompouns ws se on their retention times reltive to those of stnrs n mthing spetrl peks ville. Antifungl ssy : The ntifungl tivity of essentil oil ws performe y using poisone foo tehnique using CDA meium (Perrui et l., 994) ginst seven spoilge mouls (Alternri lternt, Aspergillus flvus, A. ohreus, A. prsitius, F. Journl of Environmentl Biology, Novemer 28

3 Antimiroil effiy of Cymopogon itrtus ginst storge fungi 953 grminerum, Peniillium itrinum n P. expnsum) isolte from rie fruits of Z. rmtum. Different onentrtions of essentil oil (.5 3. μl ml ) were poure in Petri ishes ontining.5 ml etone n 9.5 ml molten CDA. Likewise, CDA pltes without essentil oil serve s ontrol. The pltes were then inoulte with iss of test fungi n inute t 27±2 C for 7 ys. Antifungl inex ws lulte s follows- Antifungl inex (%) = ( Dt/D) x Where, Dt : imeter of growth zone in the test plte; D : imeter of growth zone in the ontrol plte. Determintion of MIC n MFC : The miniml inhiitory onentrtion (MIC) of essentil oil of C. itrtus ws reore ginst the fungl speies y poisone foo tehnique following Perrui et l. (994) t ifferent onentrtions of C. itrtus oil viz.,.5,.,.5, 2., 2.5 n 3. μl ml. The lowest onentrtion tht i not permit ny visile growth ws tken s MIC. The inhiite iss of the fungi were su-ulture on tretment-free PDA pltes to fin out if the inhiition ws reversile. If the myelil growth ws inhiite, the plnt extrt ws fungiil t this onentrtion n llowe the etermintion of miniml fungiil onentrtion (MFC). Detetion of fltoxigeni isoltes : Fifty nine isoltes ientifie s A. flvus from ontminte rie fruit smples of Z. rmtum were exmine for their fltoxin B (AFB ) prouing potentil y thin lyer hromtogrphy (TLC) (Nney n Nesitt, 965). Eh isolte ws e to 25 ml Erlenmeyer flsk ontining 25 ml SMKY meium (Diener n Dvis, 966) n inute t 27±2 C for ys. The ontent of eh flsk ws then filtere n extrte with 2 ml hloroform. The extrt ws evporte to ryness on wter th n reissolve in one ml hloroform. Then 5 µl of hloroform extrts were pplie on the tivte TLC pltes. The fltoxin stnr (fltoxin B ) ws lso spotte on TLC pltes s referene spots n then evelope with solvent system (toluene:isomyl lohol:methnol; 9:32:2 v/v). After eveloping, the pltes were ir rie n ws oserve in UVtrnsillumintor (36 nm) n their presene were hemilly onfirme y sprying 25% H2SO 4. The intensity of the lue fluoresent spot in the UV trnsillumintor vries mong ifferent fltoxigeni strins (light lue to eep lue). The toxigeni isoltes of A. flvus (A6, A9 n A-2) proue mximum lue fluoresene uner UV light n were, therefore, selete for further investigtions. The lue spots were srthe from TLC pltes n then issolve in 5 ml methnol n entrifuge t 3 rpm for 5 min. Asorne of the superntnt ws reore t 36 nm n fltoxin B ws lulte y the following formul : Afltoxin B ontent (μg l ) = D M x E L D = sorne, M = moleulr weight (32), E = molr extintion oeffiient fltoxin B (28), L = pth length ( m) Effiy of C. itrtus essentil oil for nlyzing fltoxin B proution y A. flvus : Require mounts of C. itrtus essentil oil were issolve in.5 ml etone n e to 24.5 ml SMKY to hieve the vrious onentrtions from.5 to 3. μl ml. The meium inoulte seprtely with one ml spore 6 suspension ( spores ml ) of toxigeni isoltes of A. flvus (A- 6, A9 n A-2) ws inute for ten ys t 27±2 C. Likewise, ontrol sets were prepre using sme mount of istille wter without essentil oil. The meium ws filtere n myelium ws rie t 8 C for 2 hrs. Afltoxin B ws etete y thin lyer hromtogrphy following the metho of Nney n Nesitt (965). Sttistil nlysis : The sttistil signifine of the results ws nlyze y one wy nlysis of vrine (ANOVA) using SPSS softwre. Dt nlyze were expresse s men ± SE. Mens were seprte y the Tukey's multiple rnge tests when ANOVA ws signifint (P.5). Results n Disussion The result of the myoflor nlysis revele tht 64 smples of rie fruit of Z. rmtum were ontminte with ifferent fungl speies (Tle ). A totl of 279 isoltes elonging to 7 speies were reovere from these rie smples. They were represente y 7 gener viz., Aremonium (sp.), Alternri (sp.), Aspergillus (6 spp.), Closporium (2 spp.), Muor ( sp.), Fusrium (3 spp.) n Peniillium (3 spp.). These speies ontriute s mjor fungl omponents of rie fruits n represente group of tx of uiquitous nture tht oul exploit lmost ny sustrte n umulte toxi seonry metolites. Anlysis of myoflor inite genus Aspergillus s the most frequent olonizer tht omprise of six speies n thus, Tle : Myoflor nlysis of rie fruits of Z. rmtum Fungl speies Aremonium roseolum (G. Smith) W. Gms.7 Alternri lternt (Fr.) Keissl. 4.3 Aspergillus onjutus (Kwon n Fennell) 2.86 A. flvus Link 2.4 A. niger Vn Tieghem.47 A. ohreus Wilhelm 7.53 A. oryze (Ahlurg) Cohn 3.94 A. prsitius Spere.39 Closporium losporioies (Fresen.) e Vries 4.66 C. resine (Linu) e Vries.72 Muor mueo Fresen..36 Fusrium mptoers Wollenweer n Reinking 3.22 F. grminerum Shwe 6.8 F. oxysporum Shlehtenhl 4.66 Peniilllium psultum Rper n Fennell.7 P. itrinum Thom 6.8 P. expnsum Link ex Gry 8.96 Ourrene frequeny (%) Journl of Environmentl Biology, Novemer 28

4 954 D. Gupt et l. Tle 2 : Level of fltoxin B proution y Aspergillus flvus isoltes in SMKY meium A. flvus isoltes Conentrtion (µl l ) AF ±3.2* AF ±2.98 AF6 5.65±5.87 AF ±3.75 AF9 2.87±5.67 AF-2 2.±2.78 AF ±.9 AF ±.2 AF ±2.32 AF ±2.39 AF ±9.89 AF ±2.33 * men±se forme the lrgest iversity mong ll the reovere gener. The highest frequeny of ourrene ws reore for Aspergillus flvus (2.4%) followe y A. niger (.47%) n A. prsitius (.39%). Owing to their strong ompetitive ility to proue hyrolyti enzymes n unnt sporultion, the iniene of Aspergillus speies usully remins to e high (Bl et l., 24, 26; Gupt et l., 27). In view of the known fltoxin prouing potentil of A. flvus, 59 isoltes of A. flvus were evlute for their fltoxigeni poteny. Out of these, 2 isoltes were foun toxigeni in nture prouing lue fluoresene uner UV light. However, three of these toxi isoltes nmely AF6, AF9 n AF-2 proue mximum lue fluoresene uner UV light n further sujete to ntifltoxigeni iossy. The quntittive nlysis revele tht these 2 isoltes elorte fltoxin B in the rnge of µg l (Tle 2). Prior to evlution of ntifungl tivity, the Cymopogon itrtus oil ws hrterize y GC-MS nlysis. The essentil oil ws light yellow in olour n showe the presene of 8 ifferent omponents. Their retention time n perentge re tulte in Tle 3 n Fig.. Monoterpene lehyes viz., α Tle 3 : Chemil omposition of C. itrtus essentil oil Components Perentge (%) Retention time (Minutes) Methyl heptenone β pinene Limonene Cis Oimene Linlool (+)- Limonene oxie Cis Verenol β Citrl Gerniol α Citrl Methyl ette Methyl eugenol β Cryophyllene Germrene D Cinene Cryophyllene oxie α Selinene α Bisolol itrl (49.45 per ent) n β itrl (26.6 per ent) were the mjor omponents of C. itrtus essentil oil ounting for 75.6 per ent of the totl oil omposition. Perentge of eh of the remining onstituents of essentil oil ws less thn 4 perent. Citrl hs lso een foun in other meiinl plnts like Lippi l n Cymopogon khsns (Mishr et l., 22). The hemil profile of essentil oil of prtiulr plnt speies is influene y sesonl, limti n geogrphil onitions, plnt prts use, time of hrvesting n metho of isoltion, n these hemotypi vritions signifintly ffet the iologil effiy of oil (Shukl et l., 29; Prksh et l., 2). Sine hemil hrteriztion of essentil oil of C. itrtus in the present stuy showe itrl s its mjor omponent, it might hve prove effetive in retring the growth of filmentous fungi y inhiiting the myelil growth through mehnism of ell memrne mge, ompromising its integrity n permeility n inhiition of inter- n extrellulr enzymes (De Souz et l., 25). These mesures n our lone or in len n Tle 4 : Antifungl inex of C. itrtus essentil oil ginst ifferent fungl speies isolte from rie fruits of Z. rmtum Fungl speies Antifungl inex (% myelil growth inhiition) Essentil oil onentrtion (µl ml ) Alternri lternt 45.54± ± ± ±.88 Aspergillus flvus 46.± ± ± ±2.2 A. ohreus 47.72± ± ± ±.73 A. prsitius 39.5± ± ± ± ±.37 Fusrium grminerum 65.4± ±.67 Peniilllium itrinum 32.9± ± ± ± ±2.8 P. expnsum 48.± ± ± ±.78 e e e e ef Vlues represent men of three replites ±SE. The men followe y sme letters in the sme olumn re not signifintly ifferent oring to ANOVA n Tukeys multiple omprison tests Journl of Environmentl Biology, Novemer 28

5 Antimiroil effiy of Cymopogon itrtus ginst storge fungi 955 kcounts Ionizt... 4:5 Methyl Heptenone min + et pinene min Linlool 22.9 min et itrl min +.is-verenol 27.9 min Gerniol min lph itrl min Neryl ette min + Methyleugenol min + Germrene D min Cryophyllene oxie min + lph.-bisolol min TIC CYMBOPOGAN-C PM.SMS 4 Centroi minutes Fig. : GC-MS hromtogrm of essentil oil of Cymopogon itrtus. ulminte inhiition of myelium growth (Cown, 999). The ntifungl tivity of itrl ginst mouls n yests hs lrey een etete (Belletti et l., 28; Droy et l., 28; Aukwu et l., 26). Plhno et l. (24) reporte tht monoterpene (itrl) prove effetive in ontrolling myelil growth n oniil germintion of C. gloeosporioies. In the present stuy, esies itrl, other onstituent ompouns like linlool, limonene n gerniol lso might hve ugmente the ntifungl potentility of essentil oil of C. itrtus s onfirme erlier (Hylgr et l., 22). While linlool hs een ientifie to inhiit spore germintion y respirtory suppression of eril myeli (Mtsyoh et l., 2), gerniol enhnes the rte of potssium lekge out of the ells (Br et l., 988). The essentil oil of C. itrtus exhiite pronoune ntifungl tivity ginst ll the teste fungl speies isolte Tle 5 : Minimum inhiitory onentrtion (MIC) n minimum fungiil onentrtion (MFC) of C. itrtus essentil oil ginst fungl speies Fungl speies MIC (μl ml ) MFC (μl ml ) Alternri lternt Aspergillus flvus A. ohreus A. prsitius 3. >3. Fusrium grminerum Peniilllium itrinum 3. >3. P. expnsum from rie fruits of Znthoxylum. The MIC of essentil oil ws 2.5 µl ml for Alternri lternt, Aspergillus flvus, A. ohreus n Peniillium expnsum. F. grminerum ws most sensitive to essentil oil s it ws ontrolle even t.5 µl ml, wheres myelil growth of A. prsitius ws inhiite t 3. µl ml (Tle 4). The MIC of C. itrtus essentil oil ginst test fungi ws foun to e ifferent thn some erlier reporte essentil oils viz., Aenolymm llieum (Shukl et l., 28) n Anethum grveolens (Prksh et l., 22). The MFC of essentil oil for Alternri lternt, Aspergillus flvus, A. ohreus n P. expnsum ws reore t 3. µl ml while it ws 2.5 µl ml for F. grminerum. However, A. prsitius n P. itrinum showe MFC >3. µl ml, s the revivl growth of inhiite moul is ws oserve when they were su-ulture in fresh ulture meium suggesting fungistti nture of test smples (Tle 5). These results orroorte erlier reports on the effiy of essentil oil of C. itrtus n C. mrtini signifying strong inhiition of myelil growth of yest n other filmentous fungi (Hell et l., 26; Tzortzkis n Eonomkis, 27; Sonker et l., 24; Kusr et l., 27). In support of ntifungl tivity, Singtwi n Ktew (2) emonstrte the ility of C. itrtus essentil oil to inhiit the growth of mouls suh s Closporium sp., Aspergillus niger n Muor t lower onentrtions. Reently, Yooussef et l. (26) reporte tht C. itrtus essentil oils exhiite ro fungitoxi tivity ginst Aspergillus prsitius. The inhiition of fungl myelil my e ue to the ourrene of phenoli ompouns in the essentil oils. Besies, the lipophili nture of essentil oil llows it to ross Tle 6 : Effet of C. itrtus essentil oil on fltoxin B proution y A. flvus in SMKY meium ( µg l ) Conentrtion Afltoxin B (µg l ) (µl ml ) AF6 AF9 AF-2 CN 5.65± ± ± ± ± ± ± ± ± ± e 2.5 e 3. e Vlues represent men (n=3) ±SE. The men followe y sme letters in the sme olumn re not signifintly ifferent oring to ANOVA n Tukeys multiple omprison tests; CN- ontrol Journl of Environmentl Biology, Novemer 28

6 956 D. Gupt et l. the fungl ell memrne n intertion with the memrne enzymes n proteins therey, reting flux of protons towrs the ell s exterior tht lters the ell s n eventully uses eth (Bkkli et l., 28). The ntifungl effiy of C. itrtus oil ginst three toxigeni strins of A. flvus (AF6, AF9 n AF-2) is summrize in Tle 6. The essentil oil inhiite fltoxin B proution y AF6, AF9 n AF-2 t.5 µl ml,.5 µl ml n 2. µl ml, respetively. The essentil oil showe inhiition of fltoxin B proution t onentrtion lower thn their MIC for growth of A. flvus supporting the finings of erlier workers (Prksh et l., 22; Mishr et l., 25). Therefore, the essentil oil woul e ting y two iverse moe of tion (i) inhiitor of fungl growth, n (ii) fltoxin suppression (Rsooli n Ayneh, 24). The hemil onstituents of the essentil oils re responsile for the enzymti moultion n oul form soure for their potentil ntifltoxigeni roles (Hjre et l., 25). Citrl, the mjor omponent of C. itrtus essentil oil, is wiely use nturl ingreient n is e to foos, soft rinks n osmetis s flvouring n frgrne gent. Thus, there woul e no hne of off-flvour n hrmful effets on sensory qulity if the C. itrtus essentil oil is reommene s plnt se foo itive for omplete prevention ginst quntittive losses from foo-orne fungi, qulittive losses ue to fltoxins n s n lterntive to syntheti preservtive to improve the storge life of rie ommoities. Aknowlegments The uthors re thnkful to the He, Deprtment of Botny (SAP-DRS-II), University of Jmmu, Jmmu for proviing neessry lortory filities n UGC- BSR (F-25/234 (BSR)/-57/27) for proviing finnil ssistne. Referenes Aukwu, E.C., M. Bowles, V.E. Jones n H. Bone: Antimiroil tivity, ytotoxiity n hemil nlysis of lemongrss essentil oil (Cymopogon flexuosus) n pure itrl. Appl. Miroiol. Biotehnol.,, (26). Bkkli, F., S. Averek, D. Averek n M. Iomr : Biologil effets of essentil oils - A review. Foo Chem. Toxiol., 46, (28). Bl, P., D. 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