Aflatoxin M1 in UHT milk consumed

A flatoxin M 1in UHT milk consumed in Turkey and first assessment of its bioaccessibility using an in vitro digestion modelBulent Kabak *,Fatih OzbeyHitit University,Faculty of Engineering,Department of Food Engineering,TR-19030,Corum,Turkeya r t i c l e i n f oArticle history:Received 10February 2012Accepted 12May 2012Keywords:A flatoxin M 1UHT milk Occurrence Bioaccessibility HPLCDaily intakea b s t r a c tContamination of milk and dairy products with a flatoxin M 1(AFM 1)continues to receive increased attention because of its potential health hazard to humans.The first aim of this study was to know the occurrence and levels of AFM 1in whole UHT milk from main processors in Turkey in order to make a preliminary exposure assessment.A total of 40milk samples were analysed for AFM 1using high performance liquid chromatography with fluorescence detection (HPLC-FD)after immunoaf finity column clean-up.A flatoxin M 1was detected in 20%of samples at levels ranging from <0.004to 0.076m g l À1.Only two samples contained AFM 1above the EU limit of 0.05m g l À1.The second aim of this study was to determine the bioaccessibility of AFM 1from milk using an in vitro digestion model.The bioaccessibility of AFM 1in spiked and naturally contaminated milk samples ranged from 80.5to 83.8%and from 81.7to 86.3%,respectively.No difference (P >0.05)in AFM 1bioaccessibility was found between spiked and naturally contaminated milk samples.This study also assessed the binding of AFM 1by six probiotic bacteria under simulated gastrointestinal conditions.A 15.5e 31.6%reduction in AFM 1bioaccessibility was observed in the presence of probiotic bacteria.Based on the results obtained in the present study,the mean daily intake of AFM 1through milk consumption was estimated as 0.008ng kg À1b.w.day À1for Turkish adults.Ó2012Elsevier Ltd.All rights reserved.1.IntroductionA flatoxins (AFs)are major class of mycotoxins produced by some Aspergillus species (Aspergillus flavus ,Aspergillus parasiticus and the rare Aspergillus nomius )that occur in a wide variety of commodities including cottonseed,peanuts,tree nuts,spices,dried fruits and cereals (especially maize)during growth,harvest,post-harvest and storage (Pitt,2000).A flatoxinB 1(AFB 1)is the most potent hepa-totoxic AFs with a large variety of biological effects,such as carci-nogenicity,teratogenicity and mutagenicity in various animal species,including humans (Bognanno et al.,2006).A flatoxin M 1(AFM 1),the 4-hydroxy metabolite of AFB 1,is the predominant metabolite of AFB 1and can be found in milk and milk products obtained from lactating animals ingesting feed contami-nated with AFB 1.The conversion rate of ingested AFB 1to AFM 1varies from 0.5to 6%for lactating animals (Galvano,Galofaro,&Galvano,1996).The acute toxicity of AFM 1is similar or slightly less than that of AFB 1but its carcinogenic potential is about ten times less than that of AFB 1(JECFA,2001).Thus,AFB 1and AFM 1have been classi fied by the International Agency for Research on Cancer (IARC)of WHO as human carcinogens class I (carcinogenic)and 2B (possible carcinogenic),respectively (IARC,1993,pp.489e 521).As AFs are considered to be genotoxic carcinogens,the FAO/WHO Joint Expert Committee on Food Additives (JECFA)and the Scienti fic Committee on Food (SCF)of European Community did not establish a threshold for AFs,but recommended that its concen-trations in food should be As Low As Reasonable (ALARA).The frequency of occurrence of AFM 1in commercially available milk and dairy products,the high intake of these products by human population,especially by infants and young children and its prob-able carcinogenic effect,led to an increased concern about the establishment of measures to control AFM 1contamination.The European Commission Regulation 1881/2006sets a maximum permissible limit of 0.05m g kg À1for AFM 1in raw milk,heat-treated milk and milk for the manufacture of milk-based products (European Commission,2006a ).However,to show any detrimental effects on the speci fic tissue or organ,AFs must be first released from their matrix and then be absorbed from the gut via the intestinal cells.The termAbbreviations:AFs,a flatoxins;AFB 1,a flatoxin B 1;AFM 1,a flatoxin M 1;ALARA,As Low As Reasonably Achievable;HPLC,high performance liquid chromatography;IAC,immunoaf finity column;LOD,limit of detection;LOQ,limit of quanti fication;RSD,relative standard deviation;TDI,tolerable daily intake.*Corresponding author.Tel.:þ903642274533;fax:þ903642274535.E-mail address:bulentkabak@.tr (B.Kabak).Contents lists available at SciVerse ScienceDirectFood Controljournal homep age:/locate/foodcont0956-7135/$e see front matter Ó2012Elsevier Ltd.All rights reserved.doi:10.1016/j.foodcont.2012.05.029Food Control 28(2012)338e 344bioaccessibility is defined as the amount of ingested compound that is released from its matrix in the gastrointestinal tract and thus becomes available for intestinal absorption(Versantvoort,Oomen, Van de Kamp,Rompelberg,&Sips,2005).Several previous studies have evaluated the bioaccessibility and/or bioavailability of various mycotoxins in different food matrices.Avantaggiato, Havenaar,and Visconti(2003,2004)evaluated the intestinal absorption of zearalenone(ZEA)and deoxynivalenol(DON)and nivalenol(NIV)by a dynamic in vitro gastrointestinal model, respectively.Versantvoort et al.(2005)and Kabak,Brandon,Var, Blokland,and Sips(2009)determined the bioaccessibility of AFB1and OTA from peanuts,buckwheat and infant formula using in vitro digestion model.Similarly,Raiola,Meca,Mañes,and Ritieni(2012) and Meca,Mañes,Font,and Ruiz(2012)studied the bioaccessibility of DON and minor Fusarium mycotoxins,respectively.A recent study in our laboratory has shown that the bioaccessibility of AFs from various food matrices varied from80to98%.The bio-accessibility of AFs seems to be independent of the spiking level and food matrices.However,there is no data in the literature regarding the bio-accessibility of AFM1from milk and dairy products.Thus,this study aims to(i)assess AFM1bioaccessibility from spiked and naturally contaminated milk using an in vitro digestion model,(ii)test the effectiveness of probiotic bacteria in reducing AFM1bio-accessibility,(iii)detect AFM1contamination in commercially available milk in Turkey and(iv)estimate mean daily intake of AFM1through milk consumption for Turkish consumers for thefirst time.2.Materials and methods2.1.Reagents and chemicalsPotassium chloride(KCl),potassium thiocyanate(KSCN), sodium phosphate(NaH2PO4),sodium sulphate(NaSO4),sodium chloride(NaCl),sodium bicarbonate(NaHCO3),monopotassium phosphate(KH2PO4),calcium chloride(CaCl2),ammonium chloride (NH4Cl),urea,glucose,chloroform,n-pentane and HPLC-grade methanol were purchased from VWR(Leuven,Belgium).Magne-sium chloride(MgCl2),a-amylase,mucin,pepsin,pancreatin, lipase,bile salts,phosphoric acid and citric acid were from Sigma e Aldrich(St.Louis,MO,USA).Uric acid and D-glucosamine hydrochloride were purchased from AppliChem(Darmstadt, Germany).D-glucuronic acid was from Alfa Aesar(Ward Hill,MA, USA).Bovine serum albumin,hydrochloric acid(HCl),sodium hydroxide(NaOH),acetic acid and HPLC-grade acetonitrile were supplied by Merck(Darmstadt,Germany).The immunoaffinity columns(IAC,AflaM1ÔHPLC)were purchased from Vicam (Watertown,MA,USA).In all analytical steps,ultrapure water produced by Direct-Q3UV system,from Millipore(Molsheim, France)was used.2.2.Bacteria,culture conditions and enumerationThe bacteria used in this study and their sources are listed in Table1.These bacteria were selected based either on their use as dairy and probiotic cultures in food industry or on available infor-mation concerning their effects on AFs in aqueous solution.Bacterial cells were grown in De-Man-Rogosa-Sharpe Broth (MRS,Merck,Darmstadt,Germany)medium containing0.05%L-cysteine at37 C until the desired turbidity.The viable cell concentration was determined by plate counting on MRS Agar (Merck,Darmstadt,Germany)after a serial dilution.Petri plates were incubated at37 C for48h under anaerobic conditions (Anaerocoult A,Merck,Darmstadt,Germany).Then,colonies on the plates were counted and the population of bacteria were expressed as colony-forming units(CFU)per ml.Bacterial strains were used ata concentration of108CFU mlÀ1.2.3.Standard preparationAFM1standard(10m g mlÀ1,in1ml acetonitrile)was obtained from Supelco(Bellefonte,PA,USA).Stock solution of AFM1was diluted in25%acetonitrile to obtain an AFM1concentration of 50ng mlÀ1as an intermediate solution.From this intermediate solution,a series of working standards from0.05to1ng mlÀ1in mobile phase consisting of water-acetonitrile(75:25,v/v)was prepared.2.4.SamplesA total of40whole UHT milk(cow’s milk)samples from main milk processors of Turkey were analysed for AFM1.The milk samples were purchased randomly in different supermarkets in Corum,Turkey between February and March of2011.The samples were stored in refrigerator until analysis.The package size of the milk samples was1l.2.5.Immunoaffinity column clean-upMilk samples were extracted and cleaned-up by a method based on that described by Bognanno et al.(2006),with slight modifica-tions.An aliquot of40ml of milk was warmed at37 C and centrifuged(Rotafix32A,Hettich,Germany)at2750Âg for10min. After centrifugation,the upper cream layer was completely removed and the remaining milk wasfiltered through Whatman No.4filter paper.A25ml offiltered skimmed milk was passed through an IAC,placed in a vacuum manifold(Agilent Technologies, Santa Clara,CA,USA).The column was washed twice with10ml ultrapure water and AFM1was eluted from the column with4ml acetonitrile.The extract was then evaporated to dryness under rotary evaporator(Hidolph Hi-Vap Advantage,Germany),the residue was re-dissolved in1ml of mobile phase and collected in HPLC vials(Supelco,Bellefonte,PA,USA).2.6.HPLC analysisA liquid chromatographic system(Shimadzu,Tokyo,Japan) equipped with an LC-20AD pump,a DGU-20A3on-line degasser,an SIL-20AHT autosampler and afluorescence detector model RF-20AXL,controlled by a CBM-20Alite system controller was used. The analytical column was a reversed phase intersil ODS-3(5m m, 250mmÂ4.6mm,GL Sciences Inc,Tokyo,Japan),thermostated at 30 C.An isocratic mobile phase of water-acetonitrile(75:25,v/v) was used with aflow rate of1ml minÀ1.The detection wavelengths of excitation and emission were set at365and435nm,respectively. The injection volume was set to100m l using SIL-20AHT autosam-pler.The retention time for AFM1was about7min.Table1Bacteria and their sources used in this study.Bacteria SourceBifidobacterium longum Ezal-FranceBifidobacterium species420Danisco-Germany Lactobacillus acidophilus Danisco-Germany Lactobacillus acidophilus NCFM150B Rhodia Inc.,Wisconsin,USA Lactobacillus casei Shirota RIVM,a The Netherlands Lactobacillus rhamnosus Ezal-Francea Laboratory for Health Protection Research,The National Institute for Public Health and Environment,Bilthoven,The Netherlands.B.Kabak,F.Ozbey/Food Control28(2012)338e3443392.7.In vitro digestion procedureAn in vitro digestion model developed by the National Institute for Public Health and the Environment (RIVM,Bilthoven,The Netherlands)was used to assess AFM 1bioaccessibility and the performance of probiotic bacteria in reducing AFM 1bioaccessibility under simulated gastrointestinal conditions.The digestion model consists of initial saliva processing for 5min at 37 C to simulate the mouth compartment and the gastric conditions for 2h,followed by simulated small intestine compartment for 2h at 37 C.In the digestion model 4.5ml spiked milk was used.In order to prepare spiked samples at concentrations of 0.05,0.5and 1m g AFM 1l À1,the uncontaminated portion milk was spiked with an appropriate amount of AFM 1standard solution.The spiked samples were analysed for AFM 1according to the previously described to assess the homogeneity.The spiked materials were stored at 4 C until digestion experiment.A schematic diagram of the in vitro digestion model is repre-sented in Fig.1.The experiments were performed in five replicates for each spiked levels.Six probiotic bacteria were also tested for their ability to sequester AFM 1from spiked milk.The digestion was started with adding 6ml arti ficial saliva composed of KCl 0.9g l À1,KSCN 0.2g l À1,NaH 2PO 40.9g l À1,NaSO 40.57g l À1,NaCl 0.3g l À1,NaHCO 31.7g l À1,urea 0.2g l À1,290g of a -amylase,15mg of uric acid and 25mg of mucin to digestion tubes containing 4.5ml spiked milk and incubated for 5min at 37 ter,12ml of gastric juice consists of NaCl 2.75g l À1,NaH 2PO 40.27g l À1,KCl 0.82g l À1,CaCl 2.2H 2O 0.4g l À1,NH 4Cl 0.31g l À1,6.5ml of HCl (37%),glucose 0.65g l À1,glucuronic acid 0.02g l À1,urea 0.085g l À1,glucosamine hydrochloride 0.33g l À1,1g of BSA,2.5g of pepsin and 3g of mucin was added and the mixture was rotated for 2h (Multi RS-24rotator,Biosan,Latvia)at 37 C.Finally,12ml of simulated duodenal juice composed of NaCl 7.01g l À1,NaHCO 33.39g l À1,KH 2PO 40.08g l À1,KCl 0.56g l À1,MgCl 20.05g l À1,180m l of HCl,urea 0.1g l À1,CaCl 2.2H 2O 0.2g l À1,1g of BSA,9g of pancreatin and 1.5g of lipase and 6ml simulated bile consists of NaCl 5.26g l À1,NaHCO 35.79g l À1,KCl 0.38g l À1,150m l of HCl,urea 0.25g l À1,CaCl 2.H 2O 0.22g l À1,1.8g of BSA and 30g of bile,and 2ml of 1M NaHCO 3were added simultaneously,and the mixture was rotated for another 2h.The chyme (supernatant)and the digested matrix (pellet)were obtained by centrifugation (Rota fix 32A,Hettich,Germany)at 2750Âg for 5min.The AFM 1contents in the supernatant were measured by HPLC coupled with fluorescence detector after the clean-up of samples (20ml).AFM 1bioaccessibility is expressed as the ratio of AFM 1level in gastrointestinal phases to the total concentration of the compound before digestion.2.8.Validation of the analytical methodThe validation of the analytical method was based on the following criteria:selectivity,linearity,sensitivity,accuracy and precision.The selectivity of the method was evaluated by analysing blank and spiked samples of milk and biological fluid (obtained from in vitro digestion model)at levels of 0.05,0.5and 1m g AFM 1l À1.The linearity was assessed by constructing five-point calibration curve over the concentration range of 0.05e 1m g l À1,each concen-tration injected in triplicate.The linearity was evaluated by linear regression analysis using the least squares method and expressed as correlation coef ficient (R 2).The sensitivity of the method was expressed by the limits of detection (LOD)and quanti fication (LOQ).The LOD and LOQ were calculated as signal-to-noise ratio of 3:1and 10:1,respectively,from the chromatograms of spiked milk and biological fluids with lowest concentration level.To assess the accuracy (recovery),blank samples of milk and biological fluid spiked with appropriate amounts of AFM 1working standards to obtain final concentrations of 0.05,0.5and 1m g l À1.The recovery values were calculated by the analysis of three spiked samples with HPLC after extraction and IAC clean-up described previously.The precision of method was calculated in terms of intra-day and inter-day repeatability expressed as %RSD associated with the accuracy experiment on the same day (n ¼3)and on three consequent days (n ¼9)at the respective spiking levels.2.9.Statistical analysisStatistical analyses were performed by one-way analysis of variance (ANOVA),followed by Duncan multiple comparison test using the SPSS 10.0software package program.Probability (P )values of <0.05were considered signi ficant.A Student ’s t-test was also used in for pairwise comparison of data.3.Results and discussion 3.1.Method validationThe selectivity of the method was assured by the use of immunoaf finity column for clean-up and a very selective fluores-cence detector.To assess the selectivity,blank and spiked samples of milk and biological fluid were analysed according to previously described methods and the corresponding chromatograms were compared.No interfering peaks were observed at the retention time of AFM 1(7min).The calibration curve was linear over the concentration range of 0.05e 1m g l À1,with satisfactory coef ficient of determination (R 2¼0.99907).The regression line was y ¼0.073x À0.006.The LOD and LOQ values,accuracy and precision of analytical method for AFM 1in milk and supernatant samples are summarised in Table 2.The LODs (S/N ¼3),de fined as the lowest concentration of AFM 1that can be clearly detected above the baseline signal,were 0.004and 0.005m g l À1for milk and biological fluid,respectively.The LOQs (S/N ¼10),de fined as the lowest concentration of analyte that can be determined with acceptable precision andaccuracyFig.1.A schematic diagram representing the in vitro digestion experiment.The in vitro digestion model consists of three compartments:mouth,stomach and small intestine.B.Kabak,F.Ozbey /Food Control 28(2012)338e 344340were0.014and0.016m g lÀ1for milk and biologicalfluid,respec-tively.The intra-day and inter-day accuracy varied from84.3to 92.9%and from83.5to90.6%,respectively,for both matrices,and the intra-day and inter-day precision were satisfactory,with RSD values always lower than12%.There were no significant differences (P>0.05)in AFM1recoveries between the intra-day and inter-day studies.These recovery values(within the range60e120%for concentration of0.05m g lÀ1,and70e110%for concentration above 0.05m g lÀ1)meet the requirements of the Commission Regulation (EC).No.401/2006laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs.3.2.Occurrence of AFM1in UHT milkThe incidence and concentration of AFM1in whole UHT milk samples are summarised in Table3.Eight out of40samples(20%) contained AFM1.The contamination level of AFM1in milk samples ranged from<0.004to0.076m g lÀ1.A chromatogram of naturally contaminated UHT milk sample with AFM1(0.076m g lÀ1)is shown in Fig.2.Only2samples contained AFM1higher than permissible limit of0.05m g kgÀ1set by EU regulation(European Commission, 2006b).There are many studies on the occurrence of AFM1in milk and dairy products.In a survey on pasteurized and UHT milk in Turkey, AFM1was found59.3%of samples with levels ranging from<0.01to 0.05m g lÀ1(Gürbay,Aydın,Girgin,Engin,&S¸ahin,2006).Unusan (2006)found AFM1in75out of129UHT milk samples in Central Anatolia,Turkey,with a mean concentration of0.11m g lÀ1.In another survey,67of100of UHT milk samples were contaminated with AFM1at concentrations between0.01and0.63m g lÀ1 (Tekins¸en&Eken,2008).More recently,Atasever,Adıgüzel, Atasever,Özlü,andÖzturhan(2010)found AFM1in59%of UHT milk samples from Turkey ranging from0.005to0.185m g lÀ1.A comparison of the results of our study with those of other studies conducted in Turkey shows a rather low incidence of AFM1in UHT milk analysed in present work.The variations in AFM1levels among studies could be attributed to forage and feed quality,cows’diet, genetic variation in dairy cows,geographical and seasonal varia-tions,and analytical method procedures.The natural occurrence of AFM1in milk and dairy products is a global problem.In Portugal,Martins and Martins(2000)ana-lysed31raw milk and70UHT milk samples for AFM1,of which25 raw milk samples(range¼0.005e0.05m g lÀ1)and59UHT milk samples(range¼0.005e0.061m g lÀ1)contained AFM1.Galvano et al.(2001)found78%of UHT milk samples collected in Italy, with a maximum concentration of0.024m g lÀ1.In another survey conducted in Brazil,7of12pasteurized milk and10of12UHT milk samples were contaminated with AFM1,with mean values of 0.072and0.048m g lÀ1,respectively(Oliveira&Ferraz,2007). Cano-Sancho,Marin,Ramos,Peris-Vicente,and Sanchis(2010) detected AFM1in94.4%(68/72)of whole UHT milk samples from Spain at levels ranging from<0.005to0.030m g lÀ1.In Iran,AFM1 was detected in all samples analysed(49samples)and83.67%of them contained AFM1greater than EU limit of0.05m g lÀ1 (Movassagh,2011).3.3.Bioaccessibility of AFM1and efficacy of probiotic bacteria in reducing AFM1bioaccessibilityThe results of the AFM1bioaccessibility from spiked and natu-rally contaminated UHT milk are shown in Table4.AFM1concen-tration in the spiked milks ranged from0.047to0.939m g lÀ1.The bioaccessibility of AFM1from spiked milk varied from80.5to83.8%, depending on contamination level.In addition,the bioaccessibility of AFM1ranged from81.7to86.3%for naturally contaminated UHT milk samples containing0.011e0.076m g lÀ1AFM1.This is thefirst demonstration on the bioaccessibility of AFM1 from milk,while several authors have examined bioaccessibilities of AFB1,OTA and Fusarium toxins from different food matrices.The in vitro digestion model used in this study did not take the large intestine into account since mycotoxin absorptions mainly take place in the small intestine.Both in vivo and in vitro methods can be used to assessment of contaminant bioaccessibility and bioavail-ability.In vivo experiments are more convincing,but difficult to perform,time-consuming and for most of the known mycotoxins there are a lack of sensitive and specific bioassays(Avantaggiato et al.,2003).On the contrary,the in vitro methods are simple, rapid,low-cost and have high reproducibility(Cabanero,Madrid,& Camara,2004;He&Wang,2011).Table2The accuracy,precision,LOD and LOQ for AFM1in UHT milk and biologicalfluid samples.Matrix Spiking level(m g lÀ1)Intra-day a(n¼3)Inter-day b(n¼9)LOD c(m g lÀ1)LOQ d(m g lÀ1)Recovery(%)RSD(%)Recovery(%)RSD(%)Milk0.0588.0 5.690.67.30.0040.0140.592.1 4.288.79.4192.9 4.088.4 6.9Biologicalfluid0.0586.6 6.685.711.20.0050.0160.584.37.887.610.5185.9 4.983.59.7a Intra-day repeatability was estimated by analysis of three replicate samples on the same day.b Inter-day repeatability was estimated by analysis of three replicate samples on the consecutive days.c LOD,limit of detection of analytical method(S/N¼3).d LOQ,limit of quantification of analytical method(S/N¼10).Table3Occurrence and level of AFM1in whole UHT milk marketed in Turkey.SamplesAnalysed samples Positive samples(%)Frequency distribution,n(%)AFM1levels(m g lÀ1)<0.005m g lÀ10.005e0.015m g lÀ10.015e0.05m g lÀ1>0.05m g lÀ1Range a Average b408(20)321(12.5)5(62.5)2(25)<0.004e0.0760.029Æ0.02a Min e max.b Mean of positive samplesÆSD.B.Kabak,F.Ozbey/Food Control28(2012)338e344341Regarding the bioaccessibility of AFB 1,Versantvoort et al.(2005)described the applicability of an in vitro digestion model in assessing the bioaccessibility of myotoxins from food and found the bioaccessibility of AFB 1in the duodenal compartment to be 94%.In another study,the total intestinal absorption of AFB 1was 44%in the 6h of digestion of the contaminated feeds (Avantaggiato,Havenaar,&Visconti,2007).More recently,Kabak et al.(2009)showed that the bioaccessibilities of AFB 1from pistachio nuts,low spiked infant food and high spiked infant food were 86%,94%and 88%,respectively.For OTA,a few studies have shown that OTA in buckwheat (Versantvoort et al.,2005)and feeds (Avantaggiato et al.,2007)is highly bioaccessible (83e 100%)in the intestinal compartment.However,Kabak et al.(2009)showed that its bioaccessibility in buckwheat (22%)and spiked infant food (30%)was much lower than that of AFB 1.Concerning Fusarium toxins,it was shown that 32%of ZEA intake through arti ficially contaminated wheat (4.1mg kg À1)was released from the food matrix to the bioaccessible fraction during 6h of digestion (Avantaggiato et al.,2003).In another work by the same group,the intestinal absorptions recorded by Avantaggiato,Havenaar,and Visconti (2004)using the same model were 51%and 21%,respectively,as referred to 170m g DON and 230m g NIV ingested through spiked wheat.Raiola et al.(2012)showed that the mean DON bioaccessibility value after duodenal process was of 12.1%,with values ranging from 1.1%to 24.1%.In a recent work,Meca et al.(2012)evaluated the enniatins (ENs)bioaccessibility,spiked in commercial wheat crispy bread at 1.5and 3m mol g À1concentrations,their transepithelial transport and bioavailability using Caco-2cells as a model of the human intestinal epithelium.The overall ENs bioaccessibility was 80%.It has been also demon-strated that all the studied mycotoxins were similarly transported from the apical to the basolateral side of the cell monolayers,reporting a main bioavailability of 47.4%.It has been reported that mycotoxin bioaccessibility depends on several factors,such as food matrices,contamination level,compound and type of contamination (spiked versus naturally contaminated food materials)(Kabak et al.,2009).According to present study,there was no signi ficant variation in AFM 1bio-accessibility (P >0.05)among the spiked and naturally contami-nated milk samples.Similarly,the amount of AFM 1in the digestion model did not in fluence (P >0.05)the percentage of its bio-accessibility.These results are consistent with that reported by Versantvoort et al.(2005)who showed that different amounts of OTA (4e 51ng)in the digestion model had no effect on the bio-accessibility of this compound.Moreover,He and Wang (2011)observed no signi ficant difference in bioaccessibility of mercury (61e 62%)and methylmercury (68%)between the two concentra-tion treatments.Fig.3shows the reductions in the AFM 1bioaccessibility in milk following addition of probiotic bacteria to the digestion model.The use of probiotic bacteria resulted in reductions in AFM 1bio-accessibility ranging from 15.5to 31.6%,in comparison to the control.These results showed that when AFM 1are released from its matrix into the biological fluid,they are partially bound by pro-biotic bacteria.Among test bacteria,Lactobacillus acidophilus NCFM 150B was the most ef ficient binder for AFM 1,while Lactobacillus casei Shirota (Yakult bacteria)was observed to posses the poorest capacity.Although the precise mechanism of action is not yet well understood,it is thought that the primary cellular components involved are peptidoglycan,as well as,cell wall polysaccharides and proteins (Lahtinen,Haskard,Ouwehand,Salminen,&Ahokas,2004).However,it is possible that multiple components are involved in mycotoxin binding (Peltonen,El-Nezami,Haskard,Ahokas,&Salminen,2001).In vitro binding experiments demonstrate that viable probiotic bacteria can bind AFM 1in aqueous solution and reconstituted milk with ranging from 10.22to 26.65%and from 7.85to 25.94%,respectively (Kabak &Var,2008).In another work,Pierides,El-Nezami,Peltonen,Salminen,and Ahokas (2000)found that Lacto-bacillus strains decreased AFM 1level in the range from 18.1to 53.8%in aqueous solution.In agreement with the in vitro screening test,the intestinal absorption of AFB 1was decreased by 74%in chickens by the addition of Lactobacillus rhamnosus GG (El-Nezami,Mykkänen,Kankaanpää,Salminen,&Ahokas,2000).According to our results,the different amount of AFM 1in spiked milk in the digestion model had no effect (P >0.05)in the reduc-tions of AFM 1bioaccessibility by probiotic bacteria.These results are in agreement with that reported by El-Nezami,Kankaanpää,Salminen,and Ahokas (1998)who observed that the removal of AFB 1by bacteria was independent of toxin concentration.3.4.Estimated daily intake of AFM 1The mean daily intake of AFM 1was estimated based on the results in real samples,its bioaccessibility reported in this paper and the consumption of milk by the average Turkish consumers.Since 80%of results were less than LOD,a reasonable estimate ofmi n0.000.250.500.75mVDetector A:Ex:365nm,Em:435nmA F M 1/7.043Fig.2.HPLC chromatogram of a naturally contaminated UHT milk sample with AFM 1(0.076m g l À1).Table 4Bioaccessibility of AFM 1from spiked and naturally contaminated UHT milk samples.MatrixAFM 1level (m g l À1)Amount of AFM 1in digestion model (ng)Bioaccessibility (%,mean ÆSD)Spiked milk 0.0470.2183.75Æ4.99Spiked milk 0.464 2.0981.35Æ3.49Spiked milk 0.939 4.2280.53Æ5.06Sample 60.0290.1385.45Æ4.72Sample 80.0440.2083.13Æ4.28Sample 110.0380.1785.29Æ5.53Sample 190.0760.3483.34Æ4.30Sample 270.0460.2181.67Æ6.43Sample 320.0580.2683.12Æ4.77Sample 350.0110.0586.26Æ6.53Sample 380.0270.1284.64Æ3.82B.Kabak,F.Ozbey /Food Control 28(2012)338e 344342。