Research Article Potential of Aqueous Ozone to Control Aflatoxigenic Fungi in Brazil Nuts

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1 ISRN Biotechnology Volume 2013, Article ID , 6 pges Reserch Article Potentil of Aqueous Ozone to Control Afltoxigenic Fungi in Brzil Nuts Otniel Freits-Silv, 1,2 Héctor Morles-Vlle, 1 nd Armndo Venâncio 1 1 Institute for Biotechnology nd Bioengineering IBB, Centre of Biologicl Engineering, Universidde do Minho, Cmpus de Gultr, Brg, Portugl 2 EMBRAPA Food Technology, Avenid ds Américs 29501, Rio de Jneiro, RJ, Brzil Correspondence should e ddressed to Otniel Freits-Silv; otniel.freits@emrp.r Received 31 My 2013; Accepted 26 June 2013 Acdemic Editors: S. Pn nd M. Shod Copyright 2013 Otniel Freits-Silv et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. This study imed to verify the use of queous ozone s lterntive technology for fungl control. Brzil nuts sterilized were inocultedwitheither or conidi ml 1 of Aspergillus flvus (MUM 9201) to determine optiml tretment prmeters nd different queous ozone contct times. These ssys showed tht the effect of ozone is lmost immedite ginst A. flvus, nd the optimum ozone concentrtion depended on the numer of initil vile spores on the shell. The remining vile spores in the ozone solution were recorded, nd the rte of inctivtion for ech tretment ws determined y ssessing the rtio etween the cfu of ech tretment nd the control. The ozonized nuts were lso cultured to recover the fungl popultion. Aqueous ozone ws effective in reducing theconidi of A. flvus nd the nturl fungl popultion ssocited with Brzil nuts. Aqueous ozone presented gret potentil to reduce microorgnisms counts in Brzil nuts with gret potentil use in pcking houses for decontmintion step. 1. Introduction The Brzil nut (Bertholieti excels) is n importnt nontimer forest product (NTFP) from the Amzon forest. Brzil produces pproximtely 24% of the totl world supply of this nut. Brzil nuts exploittion is n importnt socil nd economic ctivity for people living in forest res. Besides this, it is n importnt fctor in forestry conservtion nd environmentl sustinility [1]. Despite the positive nutrition nd helth-relted spects of Brzil nuts, they re susceptile to coloniztion y mycotoxin-producing fungi nd consequently to contmintion with mycotoxins. There re reports of the presence of Aspergillus flvus, nd A. nomius, which re the min producers of fltoxins (AF) [2], nd more recently two other fltoxigenic species A. omycis nd A. rchidicol [3]nd new nonfltoxigenic species A. ertholletius [4], ll of them from this sme section, were isolted nd identified in Brzil nut smples. EU countries nd the United Sttes hve een the mjor importers of in-shell nd shelled Brzil nuts, respectively. AF contmintion constitutes not only n economic prolem for Brzil nut producing countries ut lso serious helth risk forconsumersllovertheworld[5]. Both industries nd producers hve een mking considerle efforts in the pst 15 yers to minimize fungl growth nd AF contmintion of tree nuts. Prticulrly in the cse of Brzil nuts, the climte conditions in the Amzon environment nd the chrcteristics of the exploittion ctivity (collecting nd primry hndling) cnnot e controlled, exerting direct or indirect effects on the toxigenic fungi nd on AF or multitoxin production [6]. Processes nd/or tretments tht hve een proven to reduce AF levels in Brzil nuts include shelling nd sorting y size, specific grvity, color, or dmge [1]. Furthermore, severl strtegies hve een reported for the mngement of fungi nd mycotoxins lredy present in rw mteril nd food [7]. Of the ville methods to ensure food sfety, ozone ppliction is one of the most promising ones. Ozontion is chemicl mens of food processing tht involves exposing food or n intermedite product to O 3 [7, 8]. Ozone cn e pplied s gs or dissolved in n queous

2 2 ISRN Biotechnology solution.thefirstmethodismoreuseful,utothhve een used successfully on mny food products to reduce () posthrvest diseses [9], () viility of potentil mycotoxinproducing fungi, nd (c) mycotoxin ccumultion [7]. The fct tht ozontion is nonresidul tretment, n dvntgeinlightoftheincresingconcernreltedtotheuse of chemicls on food chins, mkes it promising method to tret Brzil nuts. The ssys performed in the present work re focused on the use of queous ozone since it is known to e strong ntimicroil gent [7, 10] nd its use for mycotoxin destruction hs een studied erlier [11]. Also, ozone is generlly recognized s sfe (GRAS) product, nd it hs een lredy used in mny griculturl products, including orgniclly leled ones [12], like Brzil nuts. In view of this, the present work imed to investigte the potentil of queous ozone to control the fungi ssocited with Brzil nuts, focusing on Aspergillus flvus. 2. Mterils nd Methods The smples used were collected from the 2009 Brzil nuts hrvest, otined from n groforestry production re in Prá stte, Brzil. The initil smple weighed out 40 kg nd ws homogenized to otin working smple. The working smples of Brzil nuts were divided into two groups: (i) nturlly contminted nuts (without steriliztion) nd (ii) sterilized nuts intended for studies with rtificilly contminted nuts. For the ltter group, the steriliztion ws crried out y utoclving t 121 Cduring20mint1tm, nd the contmintion ws with the MUM 9201 strin of A. flvus. This strin is well-known producer of oth AF nd cyclopizonic cid (CPA) [13, 14]. To otin the stock solution, wter sturted with ozone ws prepred y uling gs, generted y the pssge of extr dry oxygen through rector (Anseros, Model CD- COM-HF-4) for 15 minutes (power genertor t 100%; gs flow of 25 Lh 1 ) in ottle with 1000 ml of wter, t 3 C. The finl queous ozone concentrtion ws determined y the colorimetric method with JASCO V560 spectrophotometer (λ mx = 258 nm nd ε = 2900 M 1 cm 1 ) [15]. The ozone concentrtion in the stock solution rnged from 25 to 40 mg/l. The different ozone concentrtions required for the ssys were otined from the stock solution diluted with ozone demnd-free wter s necessry, until otining the ozone working dilution. Nonozonted wter ws used for control tretments. Only purified wter otined y successive reverse osmosis y Milli-Q plus system (Millipore, Molsheim, Frnce) ws used in the tests. A MUM 9201 suspension of conidi ml 1 ws prepred in peptone wter (0.1%). From tht, 10x diluted conidil suspension ws prepred nd the numer of vile conidi of oth suspensions ws ssessed y inocultion on mlt extrct gr (MEA: mlt extrct, 20 g/l; glucose, 20 g/l; peptone, 1 g/l, gr, 20 g/l). The high-concentrte conidil solution (HC) yielded out 400 vile conidi, wheres the low-concentrte suspension (LC) yielded out 40. From now on ll the ssys resedonthenumerofvileconidi Effect of Ozontion Time on Vile Conidi. Suspensions of A. flvus (MUM9201)with0.1mLofeitherHCorLC conidi were wshed in 20 ml of ozone solution for 2.5 min, 5 min, 10 min, 20 min, 30 min, or 60 min. Susequently, the ozone solution ws filtered in Millipore filtrtion system. Filters (0.2 μm) with wshed conidi were plted directly on MEA nd incuted t 25 C for 3 dys for ssessment of colony-forming units (cfu). Control tests were crried out y filtrtion of ech conidil suspension in solution of Tween 80 in Milli-Q wter (0.005% v/v). The durtion of the ozone tretment ws controlled y the ddition of 1 ml of 1 M regent-grde sodium formte solution to neutrlize the remining dissolved ozone fter the desired contct time [16]. Three replictions of ech tretment were crried out, nd the results underwent full fctoril testing t α < In order to ssess whether the fctor time ws ffected y the ozone concentrtion (interction etween effects), the whole ssy ws crried out t four different ozone concentrtions (0,1,10,nd20mgL 1 ). In summry, the ssy consisted of two different conidil concentrtions (HC or LC), six sumersion times (2.5, 5, 10, 20, 30, nd 60 min), four ozone concentrtions(0,1,10,nd20mgl 1 ), nd three replictions ( = 144 experiments) Effect of Ozone Concentrtion on Recovery of Vile Conidi from Brzil Nuts. Six sterilized nuts were inoculted with 0.1 ml of either the LC or HC conidil solution of A. flvus MUM The nuts were stored overnight t room temperture.thenthenutsweresumergedin20mlof ozone solution t different concentrtions (1, 5, 10, 15, 20, 25, nd 30 mg L 1 ). Control tests were crried out y sumerging nuts in solution of Tween 80 in Milli-Q wter (0.005% v/v). After 15 min, 20 ml of the ozone or Tween 80 solution ws filtered (0.2 μm filter) in Millipore filtrtion system, nd the filtrte ws plted directly on MEA nd incuted t 25 C for 3 dys for determintion of cfu. The whole experiment ws crried out twice, with three replictions per ssy. The effect of oth the ozone concentrtion nd initil conidil concentrtion on the vile spores fter ozontion ws studied y full fctoril tests t α < Effect of Ozontion on Shell Color. The effect of the ozontion on the color of the nutshell ws tested. For tht purpose, 20 nuts were sumerged in queous ozone during 30 min. Five different ozone concentrtions were tested (0, 0.1, 1.0, 10, nd 20 mg L 1 ). The control smple consisted on 20 untreted nuts chosen rndomly. Chromticity vlues of the shells in the L spce coordintes were recorded [17]. The vlues of L (lightness or rightness) vried from lck (0) to white (100), nd the chrom vlues of rnged from green ( 60) to red (+60) nd rnged from lue ( 60) to yellow (+60). The hue [defined s rctn( / )] ws clculted. An Avntes spectrometer (AvMouse 2.0, Eereek, Netherlnd) ws used. The chnge in shell color ws evluted y compring the prmeters hue nd L of the control smples with those of the treted ones. The significnce of the results ws ssessed y t-tests for independent smples t α < 0.05.

3 ISRN Biotechnology 3 Tle 1:Vile conidi ofa. flvus (MUM 9201) fter different tretment durtion nd queous ozone concentrtion. Ozone (mg L 1 ) Time (min) Vile conidi (%) fter different time exposure (min) to ozone 2.5 (±sd) 5 (±sd) 10 (±sd) 20 (±sd) 30 (±sd) 60 (±sd) 0 96 ± 7 94 ± 2 92 ± 0 93 ± 5 97 ± 5 94 ± ± 1 69 ± 2 64 ± ± 4 58 ± 7 61 ± ±4 5±2 6±6 5±3 3±2 2±2 20 2±3 1±0 2±1 2±1 1±1 3± Assys with Inoculted nd Nturl Contminted Nuts. In order to pply the correct ozone concentrtion during the tretment, it ws necessry to know the degree of contmintion of the nutshell. For tht purpose, previous ssy with rtificilly contminted nuts ws crried out to lern the percentge of vile conidi tht migrte from the nutshell to nqueoussolution.forthispurpose,100μlofeitherthelc or HC conidil suspension ws inoculted onto the sterilized nut surfce. After one hour t room temperture, the nut ws sumerged in 20 ml of solution of Tween 80 (0.005% v/v) in wter. After 1 hour of shking, this solution ws filtered (0.2 μm filter) in Millipore filtrtion system, nd the filtrte ws plted directly on MEA nd incuted t 25 Cfor3dys for cfu determintion. The ssy ws crried out three times with three replictions (three nuts) ech time. In prllel, to know the degree of contmintion of the nturlly contminted nuts, they were sumerged in 20 ml of solution of Tween 80 (0.005% v/v) in wter. Just s in the previous ssy, fter 1 hour shking, 20 ml of the queous solution ws filtered (0.2 μm filter), nd the filtrte ws plted directly on MEA nd incuted t 25 C for 3 dys for cfu determintion. Three tests were performed with three nuts ech. The percentge of migrtion otined in the previous ssyisusedtossessthetotlinitilvileconidionthe nutshell. Next, three more sterilized nuts were inoculted with 100 μl of either the LC or HC conidil solution nd left 1 hour t room temperture. In prllel, three nturlly contminted nuts were rndomly picked. Ech smple ws sumergedin20mlofozonesolution(20mgl 1 ). Ech ozone solution ws filtered (0.2 μm filter)in Millipore filtrtion system nd the filtrte ws plted directly on MEA. The determintion of cfu ws crried out fter 3 dys t 25 C. The whole ssy ws crried out three times. The fungi isolted from the nturlly contminted smples were identified y genus ccording to the identifiction key of Pitt nd Hocking [18]. The significnce of the efficiency of ozontion ws ssessed y comprison of vile conidi in queous solution to vile conidi in ozone solution y mens of t-test for seprte smples t α < Results Neither the durtion of queous ozone tretment nor the interction with the ozone concentrtion ffected the efficiency of the tretment. Tle 1 shows the percentge of vile spores fter different tretment durtions. However, the ozone concentrtion fctor s single effect ws highly significnt (P < 0.001) nd so ws further tested (see elow). Therefore, the effect of the ozone ginst the spores ws instntneous t ll ozone concentrtions. This ws corroorted y compring the results of n ssy in which sodium formtesolutionwsusedwiththeresultsofnotherssy in which the tretment durtion ws determined only y the time of sumersion of the nuts in the ozone solution (dt not shown). Therefter, the sodium formte solution ws no longer used, nd the durtion of the tretment (which influences the shell color) ws studied The effect of the ozone concentrtion on the tretment efficiency ws highly significnt (P < 0.001). In generl, whtever the initil chrge of spores, the higher the ozone concentrtion, the lower the finl concentrtion of vile conidi (Figure 1). However, the interction of ozone concentrtion with initil vile spores ws lso highly significnt (P < 0.001). This interction showed tht for lower initil vile spore chrge it ws enough to use n ozone concentrtion of 20 mg L 1, s no significntly etter results were oserved with higher concentrtions. The sumersion of LC or HC inoculted nuts in solution oftween80showedtht,whtevertheinitilnumerof vile spores (HC or LC), out 60% of them migrted to the wter solution. The sumersion of nturlly contminted nuts in the Tween 80 solution yielded 32 ± 5 conidi per nut. This mens the initil contmintion ws 53 ± 9 conidi per nut. According to the results otined with different ozone concentrtions (Figure 1), concentrtion of 20 mg L 1 of ozone ws chosen to ssy with HC nd LC inoculted nuts nd nturlly contminted nuts. The most frequently isolted filmentous fungi from the nturlly contminted nuts were, in descending order, Aspergillus section Nigri, Cunninghmell sp, Penicillium spp., nd Aspergillus section Flvi, with incidences of 26%, 21%, 16%, nd 5%, respectively. The rest of the fungl isoltes (33%) were yests. The results of sumersion of () nuts rtificilly inoculted with LC solution of A. flvus MUM 9201, () nturlly contminted nuts, nd (c) nuts rtificilly contminted with HC suspension of A. flvus MUM 9201 in 20 mg L 1 ozone solution showed lmost 0 vile spores fter tretment incses()nd()ndout3vilesporespernutincse (c) (Figure 2).Inllcses,thereductionofvilesporesws highly significnt (P < 0.001). The influence of the queous ozone on the shell color ws evident t higher ozone concentrtions. The vlues of the prmeter L incresed with rising ozone concentrtion

4 4 ISRN Biotechnology Vile spores (%) c d e f Ozone (mg L 1 ) Vile spores (%) c d e e e Ozone (mg L 1 ) () 400 spores per nut () 40 spores per nut Figure 1: Percentiles of A. flvus vile conidi recovered fter queous ozone exposure up to 30 mg L 1 in high () nd low () concentrtion of conidi per nut. Totl vile spores LC Nturl HC Milli-Q tween 80 Ozone solution Figure 2: Reduction of vile spores on Brzil nuts from nturl popultion nd inoculted A. flvus (MUM 9201) fter queous ozone 20 mg ml 1. The low-concentrte suspension (LC) nd the high-concentrte conidil solution (HC). nd significntly differed from the control ones t concentrtions higher thn 10 mg L 1. On the other hnd, the hue vlues decresed with incresing ozone concentrtion nd significntly differed from control ones t concentrtions of 10 mg L 1 nd higher (Figure 3). 4. Discussion The genus Aspergillus is minly responsile for fungl contmintionndsusequentproductionofafinthefield, t hrvest, nd during posthrvest opertions nd storge of nuts [13]. Due to high potentil risk of nut contmintion, decontmintion methods re of gret interest from economic, pulic helth, nd environmentl spects. Improving posthrvest processing followed y further prevention of funglgrowthwithozonetretmentisoneofthemosteffective wys to restrict AF contmintion nd would go long wy to reduce helth-relted risks from eting contminted nuts nd economic losses during production [19]. This work focused on fungl decontmintion of Brzil nuts using different ozone concentrtions. The use of ozone in dried fruits nd nuts hs een studied efore. The contmintion of dried figs using 0.01 to 0.02 g m 3 of gseous O 3 for 3 nd 4 h resulted in the reduction of totl mycoiot counts [20]. When the efficiency of gseous ozone ws evluted for the degrdtion of AF on pistchio kernels nd ground pistchios [21], pistchios were contminted with known concentrtions of AFB 1,AFB 2,AFG 1,ndAFG 2,ndthen smples were exposed to gseous ozone t 5.0, 7.0, nd 9.0 g m 3 concentrtionsfor140nd420mint20 Cnd70% RH. This works lthough presented significnt reduction in totl AF nd in AFB 1 for pistchio kernels, no significnt chnge in the ftty cid composition ws oserved. Likewise, no significnt chnges were found in sweetness, rncidity, flvor, ppernce nd overll pltility etween ozonted nd nonozonted pistchio kernels. In Brzil nuts, other uthors hve studied the potentil of gseous ozone. In reserch conducted y Giordno nd couthors [22] theyhdshownthto 3 tretment for 5 h t 31 mg L 1 inhiited the viility of fungi of the nut-contminting mycoiot, including the fltoxigenic Aspergillus. Innotherwork[23] lsowsreportedthepotentilof gseous ozone to tret Brzil nuts nd considered the est method, especilly when ssocited with vcuum. According to these uthors, ozone tretment ws le to () degrde AF (15 μgkg 1 ), () reduce fungi nd yest spores from cfu g 1 to no growth, (c) keep the ftty cid oxidtion indictor (i.e., mlondildehyde) stle, nd (d) even improve the sensory ttriutes for consumer cceptnce. Such fetures re of gret importnce for the gseous ozone tretment to e considered good snitizer. Unfortuntely, the use of gseous ozoneinsomecsescouldcusetoxicitysincetheconcentrtions of gseous O 3 necessry to inctivte the conidi of Penicillium digittum, P. itlicum, ndbotrytis cinere from tle grpes were reltively high. The need for such high O 3 concentrtion mkes it hrd to chieve complete continment of the gs, nd thus the worker sfety my

5 ISRN Biotechnology 5 L Hue Ozone concentrtion (mg L 1 ) Ozone concentrtion (mg L 1 ) () () Figure 3: Effect of queous ozone concentrtion on color prmeters () L nd () hue. e jeoprdized [24]. Gseous ozone tretment needs some djustments in its ppliction to comply with regultions since O 3 concentrtions cnnot exceed μll 1 during n 8hworkdy[25]. According to our results, the rection to the ozone is lmost instntneous. The ppliction of queous ozone tretments in industries does not seem to e very complicted. It is necessry to lern the degree of contmintion of the nuts in dvnce, ecuse if it is not very high, lower ozone concentrtions cn e used. For the sme reson it is necessry to lern which degree of contmintion is cceptle fter tretment, ecuse higher ozone concentrtions will e required if lower levels of spores per nut re to e chieved. Although the effect of gseous ozone on red pepper treted with concentrtions up to 66 g m 3 for up to 1 hour did not significntly chnge the product s color [26], it seems tht some chnges could hppen in the cse of Brzil nuts. Such effect could depend on the ozone concentrtion s well s the sumersiontime.tkingintoccountthttheeffectofozone ginst conidi seems to e immedite, it seems fesile to reduce color chnges y minimizing s much s possile the sumersion time nd djusting the ozone concentrtion to the minimum needed for the initil degree of contmintion. When the effectiveness of gseous nd queous ozone on mycoiot nd AFB 1 content of dried figs [27] wsstudied, in oth tretments, degrdtion of AFB 1 incresed with incresing ozontion time. Long gseous ozone tretment, s mentioned previously, is hrd to control nd cn jeoprdize worker sfety. These uthors [27] sttedthtgseousozone ws more effective thn ozonted wter for reduction of AFB 1, wheres ozonted wter ws more effective for decresing microil counts. Our results show tht the use of queous ozone is vile lterntive technique to control fungi on Brzil nuts, showingoptimumcontroloflrgespectrumoffungind good pplicility to complex mtrices such s nuts. The queous O 3 solution cn lso e recommended to control other mycotoxin-producing Aspergilli. However, dditionl experiments re needed to djust the method for use in Brzil nut pcking houses. Food-relted ppliction of ozone is currently restricted in pcking houses, especilly for lef nd fruit steriliztion [7]. The food processing industry constntly serches for new technologies to improve commercil steriliztion of food commodities, especilly those ssocited with mycotoxins nd other hzrds. As shown in this pper, queous ozone ppliction with ccurte O 3 dosifiction cn e n efficient method for disinfection of dry food surfces. O 3 disinfection of food surfces opens up n lterntive for the elimintion of fungl contmintion since ozone ppliction on food is esy nd, ccording to the results shown here, effective t low doses nd short durtions, yielding no residues on the product or in the environment, for which reson it hs GRAS certifiction [12]. More reserch must e undertken to evlute this techniquetoecomecommoninthefoodindustry,todetermine the idel concentrtion nd projected cost of lrge-scle tretment of Brzil nuts. Conflict of Interests The uthors declre no conflict of interests. Acknowledgments H. Morles-Vlle is grteful to the Portuguese Government (Ministério d Ciênci, Tecnologi e Ensino Superior; FundçãoprCiêncieTecnologiFCT)Grntref. SFRH/BPD/38011/2007). References [1] O. Freits-Silv nd A. Venâncio, Brzil nuts: enefits nd risks ssocited with contmintion y fungi nd mycotoxins, Food Reserch Interntionl,vol.44,no.5,pp ,2011. [2] M. Olsen, P. Johnsson, T. Möller, R. Pldino, nd M. Lindld, Aspergillus nomius, n importnt fltoxin producer in Brzil nuts? World Mycotoxin Journl, vol. 1, pp , [3] J. S. Gonçlves, L. M. Ferrcin, M. L. C. Vieir, B. T. Imnk, M. H. Tniwki, nd M. H. P. Fungro, Moleculr nlysis

6 6 ISRN Biotechnology of Aspergillus section Flvi isolted from Brzil nuts, World Journl of Microiology nd Biotechnology, vol.28,no.4,pp , [4] M.H.Tniwki,J.I.Pitt,B.T.Imnketl., Aspergillus ertholletius sp. nov. from Brzil Nuts, PLoS ONE, vol. 7, no. 8, Article ID e42480, [5] O. Freits-Silv, A. Teixeir, F. Q. d Cunh, R. L. O. de Godoy, nd A. Venâncio, Predominnt mycoiot nd fltoxin content in Brzil nuts, Journl for Consumer Protection nd Food Sfety,vol.6,no.4,pp ,2011. [6] O. Freits-Silv, M. D. L. M. de Souz, nd A. Venâncio, Trcing fungi secondry metolites in Brzil nuts using LC- MS/MS, Drug Metolism Letters,vol.5,no.3,pp ,2011. [7] O. Freits-Silv nd A. Venâncio, Ozone pplictions to prevent nd degrde mycotoxins: review, Drug Metolism Reviews,vol.42,no.4,pp ,2010. [8] Z. B. Guzel-Seydim, A. K. Greene, nd A. C. Seydim, Use of ozone in the food industry, Leensmittel-Wissenschft und- Technologi,vol.37,no.4,pp ,2004. [9] J.-G. Kim, A. E. Yousef, nd S. Dve, Appliction of ozone for enhncing the microiologicl sfety nd qulity of foods: review, Journl of Food Protection,vol.62,no.9,pp , [10] M. A. Khdre, A. E. Yousef, nd J.-G. Kim, Microiologicl spects of ozone pplictions in food: review, JournlofFood Science,vol.66,no.9,pp ,2001. [11] K. S. McKenzie, A. B. Srr, K. Myur et l., Oxidtive degrdtion nd detoxifiction of mycotoxins using novel source of ozone, Food nd Chemicl Toxicology, vol. 35, no. 8, pp , [12] F. M. Gler, J. L. Smilnick, M. F. Mnsour, nd H. Krc, Influence of fumigtion with high concentrtions of ozone gs on posthrvest gry mold nd fungicide residues on tle grpes, Posthrvest Biology nd Technology, vol.55,no.2,pp , [13] P. Rodrigues, A. Venâncio, Z. Kozkiewicz, nd N. Lim, A polyphsic pproch to the identifiction of fltoxigenic nd non-fltoxigenic strins of Aspergillus section Flvi isolted from Portuguese lmonds, Interntionl Journl of Food Microiology,vol.129,no.2,pp ,2009. [14] C.Sores,P.Rodrigues,O.Freits-Silv,L.Arunhos,ndA. Venâncio, HPLC method for simultneous detection of fltoxins nd cyclopizonic cid from Aspergillus section Flvi, World Mycotoxin Journl,vol.3,no.3,pp ,2010. [15] H. Bder nd J. Hoigne, Determintion of ozone in wter y the indigo method, Wter Reserch,vol.15,no.4,pp , [16] K.Bisws,S.Crik,D.W.Smith,ndM.Belosevic, Synergistic inctivtion of Cryptosporidium prvum using ozone followed y free chlorine in nturl wter, Wter Reserch,vol.37, no.19, pp , [17] R. G. McGuire, Reporting of ojective colour mesurements, HortScience,vol.27,pp ,1992. [18] J. I. Pitt nd A. D. Hocking, Fungi nd Food Spoilge, Blckie Acdemic nd Professionl, London, UK, 3rd edition, [19] P. Bsrn, N. Bsrn-Akgul, nd L. Oksuz, Elimintion of Aspergillus prsiticus from nut surfce with low pressure cold plsm (LPCP) tretment, Food Microiology, vol. 25, no. 4, pp , [20] S. Öztekin, B. Zorlugenç,nd F. K. Zorlugenç, Effects of ozone tretment on microflor of dried figs, Journl of Food Engineering,vol.75,pp ,2006. [21] M. Y. Aks nd M. Ozdemir, Effect of different ozone tretments on fltoxin degrdtion nd physicochemicl properties of pistchios, Journl of the Science of Food nd Agriculture,vol. 86, no. 13, pp , [22] B. N. E. Giordno, V. Simo, D. Mnfio et l., Reduction of inshellbrzilnut(bertholleti excels H.B.K.) fltoxin contmintion y ozone gs ppliction during storge, Julius-Kühn- Archiv,vol.425,pp ,2010. [23] V. M. Scussel, B. N. Giordno, V. Simo et l., Effect of oxygenreducing tmospheres on the sfety of pckged shelled Brzil nuts during storge, Interntionl Journl of Anlyticl Chemistry, vol. 2011, Article ID , 9 pges, [24] R. Ozkn, J. L. Smilnick, nd O. A. Krulut, Toxicity of ozone gs to conidi of Penicillium digittum, Penicillium itlicum, ndbotrytis cinere nd control of gry mold on tle grpes, Posthrvest Biology nd Technology, vol.60,no.1,pp , [25] USEPA, Ntionl mient ir qulity stndrds for ozone, finl rule, Federl Register,vol.73,pp ,2008. [26] F. Inn, M. Pl, nd I. Doymz, Use of ozone in detoxifiction of fltoxin B 1 in red pepper, Journl of Stored Products Reserch,vol.43,no.4,pp ,2007. [27] B. Zorlugenç, F. Kiroǧlu Zorlugenç, S. Öztekin, ndi. B. Evliy, The influence of gseous ozone nd ozonted wter on microil flor nd degrdtion of fltoxin B 1 in dried figs, Food nd Chemicl Toxicology, vol. 46, no. 12, pp , 2008.

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