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1 ISSN: Journl of Glol Phrm Tehnology Aville Online t Reserh Pper Afltoxins nd Afltoxigeni Fungi Contmintion of Dried Fruits in Irqi Mrket Tkw S. Al-Memr 1, Mohmmd J. Al-Jssni 2 *, Nid Shih Hmd 1 1 Deprtment of Biology, Collge of Siene for Women/ University of Bylon, Irq. 2 DNA Reserh Center/ University of Bylon, Irq. *Corresponding Author s E-mil: pr2000@yhoo.om Astrt Ojetives: Studying the inidene of Aspergillus sp. nd Afltoxins (AFs) ontmintion in dried fruits olleted from lol mrket of Hill ity nd other origins. Methods: Intergeni fungi were isolted y diret plte method nd were morphologilly identified on speified medi. AFs prodution y Aspergillus spp. were sreened nd determined y thin lyer hromtogrphy (TLC) nd high performne liquid hromtogrphy (HPLC). Results: Aspergillus spp. represented 93.33% of the totl isolted mold speies. A. niger ws dominnt (79.61%) nd A. flvus ws only 12.5%. Of the totl Aspergillus spp.,54.4% found tive fltoxin B1 (AFB1) nd fltoxin B2 (AFB2) produers mong 85.24% elong to A. niger nd 14.75% elong to A. flvus. HPLC shows tht 100% of dried fruit smples tested positive for AFs. Conlusion: Through our study the mold isoltes showed different AFs prodution ehvior tht they re le to produe AFB1 nd AFB2 t ph rnge (3.1 to 4.5), optimum temperture t rnge (28-32ºC) nd low w for est AFs prodution nd this needs muh onern espeilly with dried fruit storge nd tretment onditions. Keywords: Dried fruit, Afltoxin, Afltoxigeni fungi, TLC, HPLC. Introdution Dried fruits re vulnerle to fungl nd my o toxin ontmintion for their fvorle humidity, high sugr ontent nd other nutrients. Mold infetion in dried fruits my our on the tree through ripening stges, fter flling from the tree, through drying proess [1] nd during storge or refrigertion where Aspergillus niger, found the most undnt (14.92%) followed y Aspergillus flvus (10.14%) in refrigertors [2]. The Mediterrnen regions re very fvorle for dried fruits prodution. Now dys, dried fruits onsumption is wide spred. Almost hlf of the dried fruits sold ll over the world re risins, Dte, nd Prune, Fig, Apriot, Peh, Apple nd Pers. Dried fruits n support fungl growth, some of whih produe myotoxins. Ourrene of toxigeni molds nd myotoxins on these dried fruits n use serious helth nd eonomi prolem in the other prts of the world. The most importnt myotoxins ourring in the Mediterrnen rops re AFs nd OTA where the toxin type, inidene rte of myotoxins nd toxigeni molds vry y rop, ountry nd geogrphi lotion [3]. All strins tested from A. flvus nd A. prsitius showed fltoxigeni potentil. A. flvus isoltes otined from priot nd Fig showed the highest ilities (334 nd 333pp), respetively ompred with two isoltes otined from dried vine fruits10.2 nd 79.4pp, respetively nd n isolte from plum (37.4pp). Two isoltes of A. prsitius from Fig showed mrked vrition in their AF potentil (81 nd 344 pp) [4]. All smples from the rops or dried fruit smples from Irqi mrkets were ontminted with mold speies. A. niger, A. flvus, A. prsitius nd F. oxysporium dominnt molds when ompred with other speies , JGPT. All Rights Reserved 299

2 Mohmmd J. Al-Jssni et. l.: Journl of Glol Phrm Tehnology. 2017; 10(9): AFB1 only in high onentrtion rnged from µg/kg in Fig smples while, the rnges µg/kg nd µg/kg in penut nd dried priot smples, respetively however, other AFs types (AFB2, AFG1 nd AFG2) were not deteted in ll smples [5]. In previous study onerning Dte fruit, Aspergillus spp. represented 84% of the totl isolted mold speies. A. niger ws dominnt (65%) nd A. flvus ws only 19%. Of the totl A. spp., 16.6% found tive in AFB1 nd AFB2 prodution mong of whih 45.45% elong to A. niger nd 54.54% elong to A. flvus. HPLC shows tht 88.8 % of Dte fruit smples tested positive for fltoxins AFs. AFB1 nd AFB2 were deteted produed y A. niger isoltes y TLC. This ws the first report tht AFG is produed y oth A. niger nd A. flvus s deteted y HPLC. The results showed tht Dte fruits re ontminted with totl AFs rnged from 5.7 to 274µg/kg [6]. The AFs onentrtion rnges exeeded t lest with some smples the llowed levels in food mterils in Irq or other ountries stndrd tht is 20µg/kg in food smples. Totl of 30ipriotind 15 prune fruit smples were nlyzed to evlute the onentrtion of AFs y HPLC AFB1, AFB2, nd AFG1 were deteted in 7 priot smples. AFB1 nd AFG1were deteted in 2 prune smples. AFG2 ws not deteted in ny of the priot nd prunes smple. All ontminted smples hd level of totl AFs nd AFB1 elow the Irnin Ntionl StndrdiNo (5ng/g for AFB1 nd 15ng/g for totl AFs) Therefore, they hve no serious prolem for the puli helth. However, it is neessry to monitor the food qulity routinely. The results proved the suitility nd the effetiveness the immune ffinity olumn len-up tehniques for my o toxins level determintion [7, 8]. Mterils nd Methods Smple Colletion Fruit smples whih inlude (6) dried fruits (Apriot (Prunusrmeni), Fig (Fiusri), plum (Prunusdomesti) nd Jujue (Ziziphusjuju)) were olleted from lol mrket of Hill ity of different soures nd origins. Wter tivity (w) nd ph of the smples were mesured nd s the following: w ws lulted y mesuring the Reltive Humidity using Dul Moisture Meter (Exteh Instruments, USA), nd divided y 100 s the following formul: w = RH / 100 The ph ws mesured y mixing 10g of dried fruit smple with 90ml distilled wter (dh2o) (1/10 smple/wter) then homogenized in lender. The ph vlue mesurements were rried out using ph meter (Exteh Instruments, USA) [9]. Smples were olleted in sterilized seled ontiners then trnsferred to the lortory nd refrigerted until use. Fungl Isoltion nd Identifition Dried fruits tken rndomly in triplites nd they were surfe-sterilized with 1% sodium hypohlorite solution for 3min [10] then wshed y sterile dh2o nd dried on filter pper under septi onditions. Smples were ut in smll piees then plted on PDA. All pltes were inuted in drk t º C for 7 dys [11]. After the inution period, totl fungl olonies were ounted nd those elonging to Aspergillus genus were trnsferred to new PDA pltes nd inuted t º C for 5 dys in order to otin pure isoltes. For the identifition of Aspergillus spp., pure olonies were grown on two medi in triplites. The medi used were s follows: CYA nd MEA eh medium ws supplemented with 250mg/L hlormpheniol. Therefter, pltes were inoulted t three points ording to the stndrdized proedure nd inuted in drk t 37 º C nd 25 º C for CYA nd t 25 º C for MEA, for 7 dys [12]. Aspergillus spp. were identified y olony morphology on CYA nd mirosopilly using lto phenol otton lue stin through its onidiophores hrteristi fetures, s desried y the keys provided y [13-19]. Detetion of Aspergillus Toxigeni Strins The isoltes of Aspergillus sp. were sreened for the prodution of the AFs using SMKY liquid medium (surose 200g; potssium nitrte KNO3, 0.3g; mgnesium sulfte MgSO4, 7H2O, 0.5g nd yest extrt, 7g in 1L of dh2o) [20]. Aout 1ml of the mold spore , JGPT. All Rights Reserved 300

3 Mohmmd J. Al-Jssni et. l.: Journl of Glol Phrm Tehnology. 2017; 10(9): suspension ws inoulted in 25ml of SMKY medium in sterile ups nd inuted t 28 ± 2ºC for 10 dys in triplites, for AFs estimtion [21]. Toxins Extrtion After inution, the ontents of eh up were filtered through whtmn filter pper No.1 under vuum. The filtrte ws extrted with 25ml of hloroform in seprting funnel with gentle shking for 1min then let until two lyers seprted. The lower lyer (orgni) ws tken nd evported to dryness in oven t º C. The residue ws re-dissolved in 1ml of hloroform nd the ontiners were stored t -20ºC until use. TLC Test TLC ws rried out on sili gel 60 plte 20 20m (Merk, Germny) nd with modifition using toluene: hloroform: etone (15:75:10 v/v) s moile phses [22]. AFs mix stndrd (20μl) (Sigm, USA) nd 50μl of test smples were spotted on TLC pltes nd ws run for 60min t room temperture. Pltes were ir-dried nd heked over UV Trnsillumintor (360nm). The AF spots were mrked nd the retention ftor (Rf) vlue ws lulted for eh spot in omprison with the stndrds [23]. To mke sure tht there might e other types of AFs like AFG1 nd AFG2 nd did not pper on TLC plte. We seleted est fltoxigeni isoltes tht produe AFB1 nd AFB2 nd they were nlyzed y HPLC tehnique using the sme onditions s mentioned in Tle 2. Afltoxins nlysis in Dried Fruit Smples A modified method of AFs extrtion nd purifition ws onduted ording to the mnul instrutions [24]. Totl of 10g of dried fruit smple were homogenized in lender for 3min with 100ml of methnol: wter (80:20 v/v) plus 1g NCl ws dded in order to filitte seprtion then it ws filtrted under vuum through Whtmn filter pper NO.1. The filtrte ws trnsferred to seprting funnel then sme volume of hloroform ws dded nd mixed gently. The orgni lyer ws evported t º C in oven. The resultnt dried rude extrt ws dissolved in 1ml hloroform nd the ontiners were stored t -20 º C for further nlysis. Afltoxins Determintion TLC Test TLC tests for the fruit AFs extrts were performed s mentioned ove. Spetrophotometer For totl AFs quntittive estimtion, the dried rude extrt ws dried then dissolved in 1ml enzene: etonitrile (98:2 v/v), vortexed then entrifuged t 5000xg for 5min. The optil density (OD) of AFs present in the superntnt ws determined t 360nm using the informtion in Tle 1 nd lulted ording to the following formul [25]. Purified spots were stored in drk t -20 C until use. D: sorne, M: moleulr weight, E: molr extintion oeffiient of AFs, nd L: pth length (1m). Tle 1: Afltoxins Dt in enzene : etonitrile (98:2) Afltoxin Formul Mol. Wt. g/mol Molr Asorptivity AFB1 C17H12O ,800 AFB2 C17H14O ,900 AFG1 C17H12O ,100 AFG2 C17H14O ,200 HPLC Anlysis Aout 5g of Grnulr Ativted Chrol (GAC) were dded with 10 ml of HPLC grde methnol : wter (1:9 v/v). Smple ws shked t 300 rpm for 15min. Therefter, the liquid phse ws disrded y syringe nd 5ml of solute ethnol ws dded to the (GAC) nd sonited for 10min t room temperture. Therefter, solvent ws trnsferred y syringe to new up nd evported t 40 º C in vuum drying oven (Guomiing, Kore). The resultnt dried extrt ws dissolved in 1ml of HPLC grde methnol nd entrifuged t 10000xg for 1min then Millipore filtered. Aout 20µl were , JGPT. All Rights Reserved 301

4 Mohmmd J. Al-Jssni et. l.: Journl of Glol Phrm Tehnology. 2017; 10(9): injeted to the HPLC instrument (Shimdzu, Jpn) [26]. The HPLC onditions with some modifition re summrized in Tle 2. Tle 2: HPLC onditions [27] Column Wht mn C18, 25 x 4.6mm ID, 5μm prtil size? Moile Phse Deionized wter: etonitrile: methnol (60:20:20). Flow Rte 1ml/min. Temp ºC. Detetion DAD 365nm. Injetion Afltoxin Mix 20μl. The smples were nlyzed with AFs stnders mix using diode-rry detetion (DAD) t 365nm. AFs in smples were heked depending on retention time (RT) nd spetrum of eh stndrd AF tht ws ompred with those of eh smple nd AFs onentrtion ws lulted. Sttistil Anlysis Sttistil nlysis of the dt ws done y using IBM SPSS softwre version 20, onewy ANOVA (Dunn) nlysis. All p vlues <0.05 were onsidered s sttilly signifint. Results nd Disussion Isoltion of Molds from Dried Fruit Smples Totl of 112 isoltes were otined from dried fruit smples elonging to different fungl gener where the numer of A. niger 95 isoltes (79%), A. flvus 15 isoltes (12%) nd A. terreus 2 isoltes (2%). The other fungl speies were 8 isoltes (7%) (Figure 1). A. niger nd A. flvus showed high pperne rte in dried fruit smples A. niger rehed 100% in S12 nd S14 while the lowest rte rehed 57.8% in S11. A. flvus reorded highest rte of pperne in S11 rehed 31.5% nd the lowest rte of pperne rehed 8.4% in S15. As generl result, A. niger ppered in high inidene in omprison with A. flvus in ll fruit smples under study (Figure 2) tht disgree with wht ws reported efore [28]. Figure1: Rtio of s per gill us sp. inidene in dried fruit smples Figure 2: Perentge of A. flvus nd A. niger isoltes from dried fruit smples , JGPT. All Rights Reserved 302

5 Mohmmd J. Al-Jssni et. l.: Journl of Glol Phrm Tehnology. 2017; 10(9): The high inidene of Aspergillus genus in most food nd feedstuff, reflets its ility to serete vrious enzymes nd some speies n tolerte poor environmentl onditions nd grow in low moister ontent in ddition to high reltive density of spores [29]. The mold isoltes were identified morphologilly s in Figure 3. Figure 3: Morphologil identifition of Aspergillus isoltes where A: A. flvus, B: A. niger A1, B1 on CYA t 25ºC, A2, B2 on CYA t 37ºC, A3, B3 on MEA t 25ºC fter 7 dys [14-16, 19, 36] It is lerly visile tht S11 Fig smple (Figure 2) imported from Irn ontin vriility of mold speies (A. niger, A. flvus nd A. terreus) in omprison with S12 Irqi Fig smple tht showed 100% A. niger inidene. This result is highly vlule in fruit origin determintion, qulity ssessment nd possile new fungl infetion outrek espeilly tht S11 smple showed higher AFs ontent tht must e tken in onsidertion (Figure 7). Tle 3 shows tht, despite signifint differene in ph nd w within rnge nd , respetively, A. niger nd A. flvus n grow suessfully in ll smples. There ws no signifint differene in A. flvus nd A. niger ount etween smples. However, S10 smple showed signifint differene with ll smples (ut S13) in A. niger ount. There ws no signifint differene etween smples S10 nd S14 in ph nd w ut showed signifint differene A. niger inidene. While there ws signifint differenes etween S10 nd S15 in ph, w, mold nd totl Aspergillums isoltes. Tle 3: Dried Fruit smples ph, w nd mold popultion sttistis nlysis Code S10 S11 S12 S13 S14 S15 Desription Plum Fig Fig Apriot Apriot Jujue ph 3.2±0.2 4±0.1 4± ± ± ±0.2 C 0.08± ± ± ± ±0.002 w d e A Molds 11.3±2.08 7±1.7 4±0 8± ± ±0.57 d A A. niger 8.6± ±0.57 4±0 6± ±1.5 4±0 A A. flvus 2±1.73 2±2 0.6± ±0.57 A Totl Aspergillus 10.66± ±1.52 4±0 6.66± ±1.52 4±0 A High sugr onentrtion nd low wter tivity in dried fruits ssist the development of Aspergillus speies, espeilly A. niger, A. ronr nd A. ohreus sine they re xerophili nd A. niger is the most predominnt mold in most dried fruit smples [28, 30-32]. A. niger ws the most deteted fungus followed y A. flvus in dried Fig, Apriot nd plum smples [4, 32-35] , JGPT. All Rights Reserved 303

6 Mohmmd J. Al-Jssni et. l.: Journl of Glol Phrm Tehnology. 2017; 10(9): The w of dried fruit smples, filitted the growth of xerophili fungi nd ws in rnge of 0 to 0.13 in dried fruit smples under study. This rnge disgree with [28, 32], ut S11. This might e euse of hot nd dry onditions nd or long storge time of the olleted smples. Detetion of Toxigeni Aspergillus sp. y TLC Totl fltoxigeni Aspergillus sp. isoltes from dried fruit smples were 61 isoltes t rte of 54.4 % ( 85.24% A. niger nd 14.75% A. flvus), AFB1 nd AFB2 were the only AFs deteted (Tle 4). Tle 4: Detetion fltoxins prodution in Aspergillus isoltes using TLC tehnique Code Toxigeni sp. No. % Afltoxins B1 B2 S10 A. flvus A. niger S11 A. niger A. flvus S13 A. niger S14 A. niger S15 A. niger A. flvus Totl Aspergillus AFs Produer % A. niger % A. flvus % The results in Tle 4 showed tht 100% of A.niger isoltes from S11 nd S14 smples nd A. flvus from ll smples, produe AFB1 nd AFB2. Figure 4 showed tht fltoxigeni isoltes, A. niger nd A. flvus found produing AFB1 nd AFB2 only nd ll other dried fruit smples showed no AFB1 or AFB2 on TLC plte. The Rf vlue for the AFB1, AFB2, AFG1 nd AFG2 stndrds on TLC plte were 0.29, 0.23, 0.20 nd 0.16 respetively. Figure 4: AFs Sreening of Aspergillus isoltes nd dried fruit smples using TLC. 1: AFs mix stnders, 3-4: Negtive dried fruit smples, 2, 5 7: A flt oxygeni isoltes produing AFB1 nd AFB2 Flse negtive results were otined y TLC sine it is not s sensitive s HPLC for low onentrtion of AFs (low detetion limit) less thn 5pp [37] (Figure 4). All seleted isoltes produed AFB1 nd AFB2. Furthermore, B4N isolte produed AFG1 nd AFG2 while DS1F isolte produed AFG1 (Tle 4; Figure 5). This is the first report through this study tht AFG is produed y oth A. niger nd A. flvus isolted from dried fruit. Tle 4: HPLC results of the seleted mold isoltes Molds smples A flt oxins AFB1 AFB2 AFG1 AFG2 DS1F D2F D2F B2F C1F C2F F14F B4N , JGPT. All Rights Reserved 304

7 Mohmmd J. Al-Jssni et. l.: Journl of Glol Phrm Tehnology. 2017; 10(9): Figure 5: Detetion of AFs prodution in mold using HPLC method with AFs stnder (STD) Afltoxins Anlysis in Dried Fruit Smples Results hve reveled tht ll smples of dried fruits were found positive with AFs. Most of the smples were ove the suggested limit for totl AFs 4µg/kg set y EU regultions ut S12 [38]. RT for AFG2, AFG1 AFB2 nd AFB1 stndrds were 6.8, 7.8, 9.2 nd 10.6 respetively s shown in the Figures elow. Signifint differene in AFs prodution ws deteted etween S10 nd S15. While etween S10 nd S12 ppered in AFB2 prodution. High onentrtion of AFB1 ws deteted in S15, S13, S11, S14 nd S3 smples (132.3, 7.33, 2.87 nd 1.59µg/kg,respetively). While, AFB2 ws found only in S10 nd S15 (12.86 nd 9.66µg/kg, respetively), AFG1 ws found in S11, S14, S10 nd S12 (3.5, 3, 2.69 nd 1.93µg/kg, respetively), nd AFG2 were found only in S13, S12 nd S15 smples t 8.35, 2 nd 1.4µg/kg, respetively (Tle 5). Tle 5: Afltoxins onentrtion (µg/kg) in Dried fruit smples y HPLC Code S10 S11 S12 S13 S14 S15 AFB1 2.87± ±0.20 d 1.59± ±0.60 e AFB ± ±0.90 AFG1 2.69± ± ±1.04 3±0.55 AFG ±1.75 2±1.73 In omprison with previous study [33] tht mentioned Plum nd Apriot dried fruit AFs ontmintion, our results showed vrile results s AFB1 ontmintion ws deteted in S13 nd S14 only wheres S10 nd S14 smples found ontminted with AFG1 in higher onentrtion. Both S10 nd S14 were not ontminted with AFG2. S10 found ontminted with AFB2 while S13 found ontminted with AFG2. Figure7: Detetion of AFs in dried fruits using HPLC method with AFs stnder (STD) , JGPT. All Rights Reserved 305

8 Mohmmd J. Al-Jssni et. l.: Journl of Glol Phrm Tehnology. 2017; 10(9): Dried Fig nd priot smples hd shown no ontmintion with AFB2. AFG2 ws not deteted in Fig smples (S11) tht gree with [4], ut these smples re ontminted with AFB1 t lower onentrtions thn the study mentioned s shown in Tle (4). However, it ws reported tht no AFs ontmintion ws deteted in the Fig smples [39]. This is the first report through this study tht the dried Fig nd priot smples were found ontminted with AFG1 nd AFG2. S11 ws found ontminted with AFB1 t 2.87µg/kg tht is lower thn wht ws previously reported [30]. S10, S11 nd S12 smples found ontminted with AFG1 t lower onentrtion s ompred with previous study [40]. Apriot smples ontminted with AFs t onentrtion rnge 4.59 to15.5µg/kg whih is lower thn wht ws reported y [32, 41], ut higher rnge s ompred with [12, 42]. Dried Plum smple ws found ontminted with totl AFs t 15.55µg/kg, whih is higher thn wht ws previously reported [24, 32]. Despite the lk of signifint differenes etween S10 nd S12 smples in ph nd w, they were AFB1 negtive. The result shows tht dried fruit smples filitte the prodution of AFG1 nd AFG2 t ph < 6. This disgrees with previous study [43].The results of the present study show tht the presene or sene of AFB1 nd/or AFB2 is not relted with w nd ph. It is most proly relted with the dried fruits omposition. Conlusion The mold isoltes showed different AFs prodution ehvior tht they re le to produe AFB1 nd AFB2 t ph rnge (3.1 to 4.5) nd optimum temperture t rnge (28-32ºC) tht disgree with wht ws reported previously [44]. This indites tht our isoltes prefer idi onditions, mient temperture nd low w for est AFs prodution nd this needs muh onern espeilly with dried fruit storge nd tretment onditions s reported efore [5]. From our knowledge, this is the first study reporting tht Jujue smple (S15) ppered ontminted with AFB1, AFB2 nd AFG2 t high onentrtion. Referenes 1. Ozy G, Arn M, (1995) Pl M Influene of hrvesting nd drying tehnique on myo flor nd my o toxins of figs. Die Nhrung, 39(2): Al-kemwee, AAA nd Mohmed, DY (2017) Detetion of Molds whih Present in Domesti s Refrigertors in Bghdd City, Journl of Glol Phrm Tehnology, 6(9): Ozer H, Okty Bsseymez HI, Ozy G (2012) My o toxin risks nd t oxigeni fungi in dte prune nd dried priot mong Mediterrnen rops. Phytopth. Medit., 51(1): Sdullh AA, Adullh, SK (2014) Detetion of Aspergillus Speies in Dried Fruits Colleted from Duhok Mrket nd Study their Afltoxigeni Properties. Irq. Rf. J. Si., 25 (1): Thlij, KM, Hjeej JM, Mohmmed MJ (2015) Study the Ourrene of Afltoxins in some Crops nd Dry Fruits in Irqi Mrkets. J. of Nturl Sienes Reserh, 5 (11). 6. Al-Memr TS, Al-Jssni MJ, Hmed NS (2017) Contmintion of Dte fruit y fltoxigeni fungi nd fltoxins in Hill ity, Irq. Journl of Glol Phrm Tehnology, 11(9) (Aepted rtile). 7. Jnti SSF, Beheshti HR, Asdi M, Mihnprst S et.l. (2011) Preliminry Survey of Afltoxins nd Ohrtoxin A in Dried Fruits from Irn.Bulletin of environmentl ontmintion nd (3): toxiology, 8. Jnti SSF, Beheshti HR, Asdi M, Mihnprst, S. et.l (2012) Preliminry survey of fltoxins nd ohrtoxin A in dried fruits from Irn. Bulletin of environmentl ontmintion nd toxiology, 88(3): Mndeel QA (2005) Fungl ontmintion of some imported spies. Myopthologi, 159(2): Prksh B, Singh P, Mishr PK, Duey NK (2012) Sfety ssessment of Zntho xylumltum Rox. Essentil oil, its ntifungl, nti-fltoxin, ntioxidnt , JGPT. All Rights Reserved 306

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