Susceptibility of Date Fruits (Phoenix dactylifera) to Aflatoxin Production
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J Sci Food Agric 1997, 74, 64È68 Susceptibilit y of Date Fruits dactylifera) ( Phoenix to Aflatoxin Production Imad A Abdul Wahab K Ahmed Ahmed, ¤ Central Food Control and Consultancy Laboratory, Sharjah, UAE and Richard K Robinson * Department of Food Science and Technology, University of Reading, PO Box 226, Whiteknights, Reading, RG6 2AP, UK (Received 15 March 1996 ; revised version received 23 September 1996 ; accepted 28 October 1996) Abstract : Segments of fruits from 12 varieties of date (Phoenix dactylifera) and three stages of maturation were inoculated with a toxigenic strain of Aspergillus parasiticus. During growth at 28¡C for 10 days, 8 varieties supported appreciable aÑatoxin production at the Khalal stageÈthe most popular stage for direct human consumption, with a maximum value in excess of 300 kgg~1 of fruit. Marked di†erences in susceptibility to infection and/or aÑatoxin production were observed between varieties and/or stage of maturation. It was concluded that toxigenic aspergilli could proliferate on any date fruits that su†ered mechanical damage in the Ðeld or during harvesting, and hence that such fruits should be considered as likely to be unÐt for human or animal consumption. Key words : date fruit, aÑatoxin, Aspergillus parasiticus. INTRODUCTION AÑatoxins, as secreted by toxigenic strains of Aspergillus Ñavus and A parasiticus, are amongst the most carcino- genic, mutagenic and teratogenic substances found naturally in foods and feeds (Coulombe 1991). Although it was at Ðrst believed that the groundnuts were the only agricultural product that needed screening for aÑa- toxin, the number of products now recorded as support- ing toxin synthesis has increased dramatically (Ellis et al 1991). Moreover, it is believed that the reported increase in exposure of human beings to aÑatoxin in some areas might be due to the consumption of products whose susceptibility to aÑatoxin production remains unre- corded (Ellis et al 1991). One such product might be the fruit of the date (Phoenix dactylifera L), considered as the major fruit of the Near East and North Africa and consumed in large quantities, both directly or incorporated into other food * To whom correspondence should be addressed. ¤ Present address : Central Food Control Laboratory, PO Box 3717, Ajman, UAE. preparations. It is eaten at all stages of fruit develop- ment, namely KimriÈthe green immature stage, KhalalÈwhen the fruit is mature and full-coloured, RutabÈthe soft, brown stage and the hard, raisin-like Tamr stage (Downson and Aten 1962) ; all but the last stage are susceptible to fungal invasion. In particular, Aspergillus has been found to be abundant on dates at all stages of ripening (Al-Shaikly et al 1986 ; Mahjoub et al 1989) and the natural presence of toxigenic strains on date fruits has also been conÐrmed (Abu Zinada and Ali 1982 ; Saxena et al 1988). Furthermore, a random sample of date fruits of Buchibal variety at the Khalal stage examined in our laboratory contained aÑatoxins at levels of 113 and 133 kg kg~1 for aÑatoxins and B 1 respectively, and Kulkarni et al (1986) also reported G 1 , the natural occurrence of aÑatoxin in one insect- damaged sample of dried dates. Obviously each variety of date shows distinct charac- teristics with respect to morphology and physical struc- ture, as well as chemical composition (Ahmed et al 1995) and, for any given variety, there are di†erences between stages of ripening. Nevertheless, an overall assessment of the available analyses suggests that dates 64 J Sci Food Agric 0022-5142/97/$17.50 1997 SCI. Printed in Great Britain (
Transcript of Susceptibility of Date Fruits (Phoenix dactylifera) to Aflatoxin Production
J Sci Food Agric 1997, 74, 64È68
Susceptibility of Date Fruits dactylifera)(Phoenixto Aflatoxin ProductionImad A Abdul Wahab K AhmedAhmed,¤
Central Food Control and Consultancy Laboratory, Sharjah, UAE
and Richard K Robinson*
Department of Food Science and Technology, University of Reading, PO Box 226, Whiteknights, Reading,RG6 2AP, UK
(Received 15 March 1996 ; revised version received 23 September 1996 ; accepted 28 October 1996)
Abstract : Segments of fruits from 12 varieties of date (Phoenix dactylifera) andthree stages of maturation were inoculated with a toxigenic strain of Aspergillusparasiticus. During growth at 28¡C for 10 days, 8 varieties supported appreciableaÑatoxin production at the Khalal stageÈthe most popular stage for directhuman consumption, with a maximum value in excess of 300 kg g~1 of fruit.Marked di†erences in susceptibility to infection and/or aÑatoxin production wereobserved between varieties and/or stage of maturation. It was concluded thattoxigenic aspergilli could proliferate on any date fruits that su†ered mechanicaldamage in the Ðeld or during harvesting, and hence that such fruits should beconsidered as likely to be unÐt for human or animal consumption.
Key words : date fruit, aÑatoxin, Aspergillus parasiticus.
INTRODUCTION
AÑatoxins, as secreted by toxigenic strains of AspergillusÑavus and A parasiticus, are amongst the most carcino-genic, mutagenic and teratogenic substances foundnaturally in foods and feeds (Coulombe 1991). Althoughit was at Ðrst believed that the groundnuts were theonly agricultural product that needed screening for aÑa-toxin, the number of products now recorded as support-ing toxin synthesis has increased dramatically (Ellis et al1991). Moreover, it is believed that the reported increasein exposure of human beings to aÑatoxin in some areasmight be due to the consumption of products whosesusceptibility to aÑatoxin production remains unre-corded (Ellis et al 1991).
One such product might be the fruit of the date(Phoenix dactylifera L), considered as the major fruit ofthe Near East and North Africa and consumed in largequantities, both directly or incorporated into other food
* To whom correspondence should be addressed.¤ Present address : Central Food Control Laboratory, POBox 3717, Ajman, UAE.
preparations. It is eaten at all stages of fruit develop-ment, namely KimriÈthe green immature stage,KhalalÈwhen the fruit is mature and full-coloured,RutabÈthe soft, brown stage and the hard, raisin-likeTamr stage (Downson and Aten 1962) ; all but the laststage are susceptible to fungal invasion. In particular,Aspergillus has been found to be abundant on dates atall stages of ripening (Al-Shaikly et al 1986 ; Mahjoub etal 1989) and the natural presence of toxigenic strains ondate fruits has also been conÐrmed (Abu Zinada and Ali1982 ; Saxena et al 1988). Furthermore, a randomsample of date fruits of Buchibal variety at the Khalalstage examined in our laboratory contained aÑatoxinsat levels of 113 and 133 kg kg~1 for aÑatoxins andB1
respectively, and Kulkarni et al (1986) also reportedG1,the natural occurrence of aÑatoxin in one insect-damaged sample of dried dates.
Obviously each variety of date shows distinct charac-teristics with respect to morphology and physical struc-ture, as well as chemical composition (Ahmed et al1995) and, for any given variety, there are di†erencesbetween stages of ripening. Nevertheless, an overallassessment of the available analyses suggests that dates
64J Sci Food Agric 0022-5142/97/$17.50 1997 SCI. Printed in Great Britain(
AÑatoxin production on date fruits 65
should, at least at some stages of development, be ableto support fungal growth and, perhaps, mycotoxin pro-duction. Consequently, we decided to establish whethertissues from the fruits of dates of di†erent varieties andstages of ripening would support the growth of Asper-gillus parasiticus and the formation of aÑatoxins.
MATERIALS AND METHODS
Materials
Twelve varieties of date of commercial importance inthe United Arab Emirates (UAE) were selected to rep-resent both early and late-maturing cultivars. Typicalfruits (100) of each variety were collected at each stageof ripening (except for the hard, raisin-like fruits atTamr which do not support mycelial growth) and, onthe day of harvesting, were frozen at [20¡C for sub-sequent use. At the end of the growing season, batchesof fruit from each variety and stage of ripening wereremoved from the freezer and thawed at room tem-perature (20¡C) overnight. The fruits were surface steril-ized by immersion in 10 g litre~1 silver nitrate solutionfor 1 min. After neutralising the sterilant with sodiumchloride (10 g litre~1 solution) and rinsing with steriledistilled water, the calyxes and stones (pits) wereremoved aseptically. The fruits (including the skins)were then cut into small pieces with a sterilised dispos-able blade and sub-samples (20 g) samples were weighedinto 250 ml sterile, Erlenmeyer Ñasks Ðtted with screwcaps.
Aspergillus parasiticus IMI 9109b was grown onslopes of potato dextrose agar (100 ml amounts ofmedium in screw-cap, medical ÑatsÈ150 ml capacity) at28¡C for 10 days. A dense spore suspension was thenprepared by adding 25 ml of sterile distilled water to theculture and agitating the surface of the colony with asterile glass rod. After decanting the suspension into asterile conical Ñask (50 ml), the spore counts ml~1 oftwo serial dilutions (1] 9 and 1] 99) were made usinga haemocytometer. The original suspension was thendiluted so that 1 ml of spore suspension of A parasiticuscontained 1] 106 spores. All manipulations werecarried out under a laminar-Ñow hood (SterilGardHood, Bader Company Inc, Maine, USA) designed forhandling pathogenic organisms.
One millilitre of the prepared suspension was intro-duced into each Ñask and the Ñasks shaken in order todistribute the inoculum over the fruit segments. ThreeÑasks were inoculated for each maturation stage of eachvariety, plus a control Ñask which was inoculated with1 ml of sterile distilled water. All Ñasks were incubatedat 28¡C for 10 days. At the end of the incubation period,the Ñasks were taken for extraction of aÑatoxins asdescribed below.
Extraction and determination of aÑatoxins
The BF (best food) procedure of the AOAC (1990) wasused for the extraction and clean-up of aÑatoxin fromthe inoculated fruits. The resultant dry extracts were re-dissolved in 1 ml acetonitrile-190 (UniChrom-AjaxChemicals, Australia)/water (Hi PerSolv-BDHChemicals), 40 : 60, v/v. After shaking vigorously with avortex mixer, the dissolved extract was Ðltered througha 0É45 km disposable PTFE membrane microÐlter unit(Supelco Inc, USA) and injected into a high per-formance liquid chromatograph (HPLC).
The HPLC used in this experiment was a HewlettPackard (HP) 1090, equipped with a PV5 Ternary SDSthree-channel pump. Each channel was connected to aproportioning valve, which controlled the compositionof the solvent. A Ðlter frit (0É45 km) was attached toeach channel and the solvents were degassed internallywith helium. A manual injector, with RheodyneTMModel 7010 valve, was built into the HP 1090 main-frame together with thermo-controlled column com-partment. A stainless-steel (250 ] 4É6 mm) RP columnpacked with 5 km Spherisorb ODS1 was used and themobile phase was water/acetonitrile/methanol(60 : 30 : 10, v/v/v, all HPLC Grade) with a Ñow rate of0É75 ml min~1. The HPLC was connected to an HP-1046A Programmable Fluorescence Detector (PFD)and both were controlled via an HP 79994A AnalyticalWork Station consisting of an HP 9000/300 computer,HP 9153 integrator and HP 3574A colour monitor.
In order to enhance the Ñuorescent characteristics ofaÑatoxins and a post-column derivatisationB1 G1,(PCD) reaction with a saturated solution of iodine, asdescribed by Shepherd et al (1986), was employed. Theinstrument used for PCD was the PC] 3100 Reactor(Pickering Laboratory, USA) and the method of Beaverand Wilson (1990) was followed for the preparation ofthe saturated solution of iodine. Authentic standardswere employed to identify the peaks and a conÐrmatoryTLC procedure using 25% methanolic sulphuric acid(AOAC 1990) was used as well.
Decontamination of the laboratory glassware andwastes was carried out according to the methoddescribed by Castegnaro et al (1980) using sodiumhypochlorite and acetone.
RESULTS AND DISCUSSION
With the exception of a few varieties at certain stages ofdevelopment, the fruits were found to support the syn-thesis of substantial amounts of aÑatoxin, and theresults are summarised in Tables 1, 2 and 3.
In general, the Khalal stage appeared to supportmaximum aÑatoxin production compared with theother two stages, Kimri and Rutab. This pattern couldbe attributed to the high moisture content (545È
66 I A Ahmed, A W K Ahmed, R K Robinson
TABLE 1AÑatoxin production (kg g~1) by A parasiticus (IMI 9109b) grown on date segments of di†erent
varieties at Kimri stagea
V ariety AFB1 AFB2 AFG1 AFG2 T otal
Naghal 19É91 ^ 1É03 0É56 ^ 0É06 11É54 ^ 0É98 NDb 31.87^ 1.98Buchibal 27É84 ^ 2É38 0É86 ^ 0É18 16É10 ^ 2É92 0É30 44É16 ^ 6É66Khunaizy 9É22 ^ 1É94 0É19 ^ 0É03 2É92 ^ 0É43 0É00 12É26 ^ 2É33Khulas 59É61 ^ 4É72 2É40 ^ 0É32 32É26 ^ 3É23 0É78 94É53 ^ 8É11Gush Rabei 19É93 ^ 6É43 0É74 ^ 0É36 11É46 ^ 5É99 0É44 32É27 ^ 12É90Hilali Ahmr 7É14 ^ 1É02 0É33 ^ 0É09 5É12 ^ 1É32 0É10 12É66 ^ 2É48Barhi 11É02 ^ 4É23 0É50 5É07 ^ 1É67 ND 16É26 ^ 6É11Lulu 60É68 ^ 4É60 2É63 ^ 0É24 34É29 ^ 1É92 0É74 97É84 ^ 5É79Fard 4É86 ^ 0É93 0É11 ^ 0É01 2É04 ^ 0É42 ND 6É97 ^ 1É30Naghal Hilali ND ND ND ND NDKhasab 12É85 ^ 2É54 0É39 ^ 0É11 6É05 ^ 1É16 ND 19É29 ^ 3É80Hilali Pakistan ND ND ND ND ND
a Mean of six replicates^ standard deviation.b ND, not detected.
765 g kg~1) and optimum sugar level (188È319 g kg~1FW) of the fruits at Khalal stage (Ahmed et al 1995)compared with Kimri 801È855 g kg~1 moisture and34È77 g kg~1 FW total sugars) and Rutab (359È505 g kg~1 moisture and 408È502 g kg~1 FW totalsugars). Whether the low sugar concentrations at Kimrior reduced available water at Rutab a†ected mycelialgrowth and/or aÑatoxin production was not deter-mined, but these proposed associations would not beunreasonable (Mateles and Adye 1965 ; Davis et al1966 ; Llewellyn et al 1980).
However, the correlation is not a simple one, forwhile Lulu and Barhi have almost identical composi-tions with respect to moisture and sugar content atboth Kimri and Khalal, the patterns of aÑatoxin pro-
duction were quite di†erent (see Tables 1 and 2). Thelower moisture level of the Rutab stage of Barhi(397 g kg~1) compared with Lulu (452 g kg~1) couldexplain the contrasted Ðgures for aÑatoxin in Table 3,for it was noticeable that Khunaizy also had low mois-ture (379 g kg~1) and aÑatoxin contents at Rutab.However, it remains likely that other factors areinvolved as well for, while the moisture level of Buchi-bal at Rutab was only 359 g kg~1, aÑatoxin productionwas abundant.
It is notable also that some of the varieties, with theexception of the early-maturing varieties Naghal andBuchibal, supported the production of aÑatoxin atKimri but showed reduced levels at the more matureRutab stage, eg Khulas and Fard. This pattern is the
TABLE 2AÑatoxin production (kg g~1) by A parasiticus (IMI 9109b) grown on date segments of di†erent varieties at
Khalal stagea
V ariety AFB1 AFB2 AFG1 AFG2 T otal
Naghal 72É26 ^ 11É78 4É10 ^ 0É10 163É93 ^ 12É16 8É60 ^ 2É69 248É88 ^ 12É16Buchibal 120É3 ^ 25É63 11É37 ^ 2É90 234É70 ^ 2É71 11É94 ^ 2É24 378É35 ^ 73É48Khunaizy 23É17 ^ 2É29 1É92 ^ 0É06 64É04 ^ 3É90 3É39 ^ 0É15 92É52 ^ 1É69Khulas 5É16 ^ 0É65 0É36 ^ 0É09 17É29 ^ 5É10 0É73 ^ 0É23 23É53 ^ 6É08Gush Rabei 43É30 ^ 3É78 1É98 ^ 0É01 59É97 ^ 4É13 1É80 ^ 0É07 107É05 ^ 0É4Hilali Ahmr 24É71 ^ 2É60 1É03 ^ 0É15 24É29 ^ 0É11 0É74 ^ 0É02 50É76 ^ 2É61Barhi 48É38 ^ 3É81 2É44 ^ 0É53 53É05 ^ 4É81 1É91 ^ 0É76 105É78 ^ 7É64Lulu 26É30 ^ 2É64 1É08 ^ 0É11 37É44 ^ 5É23 1É04 ^ 0É14 65É86 ^ 8É05Fard 0É51 ^ 0É10 NDb ND 0É06 ^ 0É03 0É58 ^ 0É06Naghal Hilali 4É97 ^ 1É84 0É19 ^ 0É03 4É57 ^ 0É67 0É15 ^ 0É02 9É83 ^ 1É84Khasab ND ND ND ND NDHilali Pakistan 4É84 ^ 1É49 ND 2É42 ^ 0É28 ND 7É26 ^ 1É78
a Mean of six replicates^ standard deviation.b ND, not detected.
AÑatoxin production on date fruits 67
TABLE 3AÑatoxin production (kg g~1) by A parasiticus (IMI 9109b) grown on date segments of di†erent
varieties at Rutab stagea
V ariety AFB1 AFB2 AFG1 AFG2 T otal
Naghal 9É69 ^ 2É72 1É34 ^ 0É28 63É31 ^ 7É82 3É71 ^ 0É73 76É53 ^ 12É22Buchibal 5É23 ^ 0É47 0É68 ^ 0É06 48É49 ^ 4É90 2É02 ^ 0É26 56É41 ^ 5É63Khunaizy 0É17 NDb 0É38 ND 0É55Khulas ND ND ND ND NDGush Rabei 1É45 ^ 0É29 0É22 13É16 ^ 2É59 0É65 ^ 0É19 15É33 ^ 3É14Hilali Ahmr 0É87 ^ 0É12 0É09 6É73 ^ 0É84 ND 7É64 ^ 1É01Barhi 0É26 ND 6É10 ^ 0É92 0É30 6É66 ^ 0É92Lulu 8É62 ^ 2É03 1É55 86É71 ^ 18É65 3É52 ^ 0É75 99É36 ^ 21É16Fard ND ND ND ND NDNaghal Hilali 8É65 ^ 2É53 0É80 ^ 0É25 31É74 ^ 1É03 0É82 ^ 0É08 42É00 ^ 3É39Khasab ND ND ND ND NDHilali Pakistan 4É62 ^ 0É36 0É03 ^ 0É10 4É26 ^ 0É37 0É14 ^ 0É03 9É31 ^ 0É06
a Mean of six replicates^ standard deviation.b ND, not detected.
opposite of most agricultural crops which show moresusceptibility to fungal growth/aÑatoxin production asthe fruits advance in maturity (Buchanan et al 1975).
In general, it was noted that the late-maturing vari-eties (Fard, Naghal Hilali, Khasab and Hilali Pakistan)supported the least development of aÑatoxin. No causeÈe†ect relationship could be established on the basis ofthe chemical components, but the slow development ofthe fruit in the late-maturing varieties might indicatethat certain biological substances which facilitate thedevelopment of the fruit from Kimri to Khalal mightstill be functioning in late varieties and, perhaps, beexerting an inhibitory e†ect on toxin production.
Moreover, although the observations were not quan-tiÐed, it appeared that early-maturing fruits like Naghal,Buchibal and Khunaizy were characterised by having,at Khalal stage for example, soft tissues which facili-tated the utilisation of nutrients by the fungus, whereasthe late-maturing varieties had, at the same stage ofdevelopment, hard and compact tissues which conÐnedmycelial growth to the outer layers of the fruit seg-ments. This pattern is broadly similar to that observedby Priyadarshini and Tulpule (1978), who correlatedvariations in fungal growth between varieties of maizewith the hardness of the endosperm.
Despite the fact that only one strain of A parasiticuswas used, there were clear di†erences between the threestages of ripening with respect to the proportions of thedi†erent types of aÑatoxin. Fruits at Kimri stage con-tained more aÑatoxin (about 2É5 times) than aÑatox-B1in along with small amounts of aÑatoxin andG1, B2traces of aÑatoxin However, at Khalal stage theG2 .situation was completely reversed. AÑatoxin wasG1produced at almost twice the level of with aÑatoxinB1,
either equal to or double the quantity of aÑatoxinG2As the fruits reached the Rutab stage, largeB2 .
amounts of aÑatoxin were producedÈabout sixG1times the level of aÑatoxin likewise, aÑatoxinB1Èand
increased comparative to (see Table 3). TheseG2 B2variations in the proportions of the B-group aÑatoxinsrelative to the G-group could be due to a number offactors, such as changes in the pH value of the fruitsfrom very acidic at Kimri to neutral at Rutab(Downson and Aten 1962), or alterations in the chemi-cal constituents of the fruits with ripening (Ahmed et al1995). Thus, Davis et al (1966) found a balance infavour of in a medium rich in sucrose, while LaceyG1(1986) concluded that more aÑatoxin was producedB1on poor media ; in the present work, the low sugarcontent at Kimri might have encouraged the dominanceof aÑatoxin B1.
However, irrespective of the type of toxin formed, ourinvestigation with segments of date fruits has providedevidence that toxigenic aspergilli can proliferate andproduce aÑatoxins at all stages of fruit development.Obviously the experimental conditions were conduciveto fungal growth but, given that the inner tissues offruits are often exposed on farms or in stores followingbird or other damage, it is reasonable to speculate thatcontamination of dates with aÑatoxins could easily arisein commercial situations. At present, it is likely thatmost fruits damaged in this way are removed duringharvesting, but the need for vigilance is self-evident.
ACKNOWLEDGEMENTS
The authors are extremely grateful to the SharjahMunicipality for encouragement and support duringthis work and to Dr H R Shabana for his help with theidentiÐcation of the varieties and di†erent stages ofripening.
68 I A Ahmed, A W K Ahmed, R K Robinson
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