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Aflatoxin M1 in UHT milk consumed in Turkey and first assessment of its bioaccessibility using an in vitro digestion model
Food Control 28 (2012) 338e344
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Aflatoxin M1 in UHT milk consumed in Turkey and first assessment of its bioaccessibility using an in vitro digestion model Bulent Kabak*, Fatih Ozbey Hitit University, Faculty of Engineering, Department of Food Engineering, TR-19030, Corum, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 February 2012 Accepted 12 May 2012
Contamination of milk and dairy products with aflatoxin M1 (AFM1) 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 AFM1 in whole UHT milk from main processors in Turkey in order to make a preliminary exposure assessment. A total of 40 milk samples were analysed for AFM1 using high performance liquid chromatography with fluorescence detection (HPLC-FD) after immunoaffinity column clean-up. Aflatoxin M1 was detected in 20% of samples at levels ranging from <0.004 to 0.076 mg l1. Only two samples contained AFM1 above the EU limit of 0.05 mg l1. The second aim of this study was to determine the bioaccessibility of AFM1 from milk using an in vitro digestion model. The bioaccessibility of AFM1 in spiked and naturally contaminated milk samples ranged from 80.5 to 83.8% and from 81.7 to 86.3%, respectively. No difference (P > 0.05) in AFM1 bioaccessibility was found between spiked and naturally contaminated milk samples. This study also assessed the binding of AFM1 by six probiotic bacteria under simulated gastrointestinal conditions. A 15.5e31.6% reduction in AFM1 bioaccessibility was observed in the presence of probiotic bacteria. Based on the results obtained in the present study, the mean daily intake of AFM1 through milk consumption was estimated as 0.008 ng kg1 b.w. day1 for Turkish adults. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Aflatoxin M1 UHT milk Occurrence Bioaccessibility HPLC Daily intake
1. Introduction Aflatoxins (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). Aflatoxin B1 (AFB1) is the most potent hepatotoxic AFs with a large variety of biological effects, such as carcinogenicity, teratogenicity and mutagenicity in various animal species, including humans (Bognanno et al., 2006). Aflatoxin M1 (AFM1), the 4-hydroxy metabolite of AFB1, is the predominant metabolite of AFB1 and can be found in milk and milk products obtained from lactating animals ingesting feed contaminated with AFB1. The conversion rate of ingested AFB1 to AFM1 varies from 0.5 to 6% for lactating animals (Galvano, Galofaro, & Galvano,
Abbreviations: AFs, aflatoxins; AFB1, aflatoxin B1; AFM1, aflatoxin M1; ALARA, As Low As Reasonably Achievable; HPLC, high performance liquid chromatography; IAC, immunoaffinity column; LOD, limit of detection; LOQ, limit of quantification; RSD, relative standard deviation; TDI, tolerable daily intake. * Corresponding author. Tel.: þ90 364 2274533; fax: þ90 364 2274535. E-mail address: [email protected] (B. Kabak). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.05.029
1996). The acute toxicity of AFM1 is similar or slightly less than that of AFB1 but its carcinogenic potential is about ten times less than that of AFB1 (JECFA, 2001). Thus, AFB1 and AFM1 have been classified 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. 489e521). As AFs are considered to be genotoxic carcinogens, the FAO/ WHO Joint Expert Committee on Food Additives (JECFA) and the Scientific Committee on Food (SCF) of European Community did not establish a threshold for AFs, but recommended that its concentrations in food should be As Low As Reasonable (ALARA). The frequency of occurrence of AFM1 in commercially available milk and dairy products, the high intake of these products by human population, especially by infants and young children and its probable carcinogenic effect, led to an increased concern about the establishment of measures to control AFM1 contamination. The European Commission Regulation 1881/2006 sets a maximum permissible limit of 0.05 mg kg1 for AFM1 in 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 specific tissue or organ, AFs must be first released from their matrix and then be absorbed from the gut via the intestinal cells. The term
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bioaccessibility 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 AFB1 and 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 from 80 to 98%. The bioaccessibility of AFs seems to be independent of the spiking level and food matrices. However, there is no data in the literature regarding the bioaccessibility of AFM1 from milk and dairy products. Thus, this study aims to (i) assess AFM1 bioaccessibility from spiked and naturally contaminated milk using an in vitro digestion model, (ii) test the effectiveness of probiotic bacteria in reducing AFM1 bioaccessibility, (iii) detect AFM1 contamination in commercially available milk in Turkey and (iv) estimate mean daily intake of AFM1 through milk consumption for Turkish consumers for the first time. 2. Materials and methods 2.1. Reagents and chemicals
339
Table 1 Bacteria and their sources used in this study. Bacteria
Source
Bifidobacterium longum Bifidobacterium species 420 Lactobacillus acidophilus Lactobacillus acidophilus NCFM 150B Lactobacillus casei Shirota Lactobacillus rhamnosus
Ezal-France Danisco-Germany Danisco-Germany Rhodia Inc., Wisconsin, USA RIVM,a The Netherlands Ezal-France
a Laboratory for Health Protection Research, The National Institute for Public Health and Environment, Bilthoven, The Netherlands.
plates were counted and the population of bacteria were expressed as colony-forming units (CFU) per ml. Bacterial strains were used at a concentration of 108 CFU ml1. 2.3. Standard preparation AFM1 standard (10 mg ml1, in 1 ml acetonitrile) was obtained from Supelco (Bellefonte, PA, USA). Stock solution of AFM1 was diluted in 25% acetonitrile to obtain an AFM1 concentration of 50 ng ml1 as an intermediate solution. From this intermediate solution, a series of working standards from 0.05 to 1 ng ml1 in mobile phase consisting of water-acetonitrile (75:25, v/v) was prepared. 2.4. Samples A total of 40 whole 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 of 2011. The samples were stored in refrigerator until analysis. The package size of the milk samples was 1 l.
Potassium 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). Magnesium chloride (MgCl2), a-amylase, mucin, pepsin, pancreatin, lipase, bile salts, phosphoric acid and citric acid were from SigmaeAldrich (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-Q 3 UV system, from Millipore (Molsheim, France) was used.
Milk samples were extracted and cleaned-up by a method based on that described by Bognanno et al. (2006), with slight modifications. An aliquot of 40 ml of milk was warmed at 37 C and centrifuged (Rotafix 32A, Hettich, Germany) at 2750 g for 10 min. After centrifugation, the upper cream layer was completely removed and the remaining milk was filtered through Whatman No. 4 filter paper. A 25 ml of filtered skimmed milk was passed through an IAC, placed in a vacuum manifold (Agilent Technologies, Santa Clara, CA, USA). The column was washed twice with 10 ml ultrapure water and AFM1 was eluted from the column with 4 ml acetonitrile. The extract was then evaporated to dryness under rotary evaporator (Hidolph Hi-Vap Advantage, Germany), the residue was re-dissolved in 1 ml of mobile phase and collected in HPLC vials (Supelco, Bellefonte, PA, USA).
2.2. Bacteria, culture conditions and enumeration
2.6. HPLC analysis
The bacteria used in this study and their sources are listed in Table 1. These bacteria were selected based either on their use as dairy and probiotic cultures in food industry or on available information concerning their effects on AFs in aqueous solution. Bacterial cells were grown in De-Man-Rogosa-Sharpe Broth (MRS, Merck, Darmstadt, Germany) medium containing 0.05% Lcysteine at 37 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 at 37 C for 48 h under anaerobic conditions (Anaerocoult A, Merck, Darmstadt, Germany). Then, colonies on the
A liquid chromatographic system (Shimadzu, Tokyo, Japan) equipped with an LC-20AD pump, a DGU-20A3 on-line degasser, an SIL-20AHT autosampler and a fluorescence detector model RF20AXL, controlled by a CBM-20Alite system controller was used. The analytical column was a reversed phase intersil ODS-3 (5 mm, 250 mm 4.6 mm, GL Sciences Inc, Tokyo, Japan), thermostated at 30 C. An isocratic mobile phase of water-acetonitrile (75:25, v/v) was used with a flow rate of 1 ml min1. The detection wavelengths of excitation and emission were set at 365 and 435 nm, respectively. The injection volume was set to 100 ml using SIL-20AHT autosampler. The retention time for AFM1 was about 7 min.
2.5. Immunoaffinity column clean-up
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2.7. In vitro digestion procedure An in vitro digestion model developed by the National Institute for Public Health and the Environment (RIVM, Bilthoven, The Netherlands) was used to assess AFM1 bioaccessibility and the performance of probiotic bacteria in reducing AFM1 bioaccessibility under simulated gastrointestinal conditions. The digestion model consists of initial saliva processing for 5 min at 37 C to simulate the mouth compartment and the gastric conditions for 2 h, followed by simulated small intestine compartment for 2 h at 37 C. In the digestion model 4.5 ml spiked milk was used. In order to prepare spiked samples at concentrations of 0.05, 0.5 and 1 mg AFM1 l1, the uncontaminated portion milk was spiked with an appropriate amount of AFM1 standard solution. The spiked samples were analysed for AFM1 according 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 represented 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 AFM1 from spiked milk. The digestion was started with adding 6 ml artificial saliva composed of KCl 0.9 g l1, KSCN 0.2 g l1, NaH2PO4 0.9 g l1, NaSO4 0.57 g l1, NaCl 0.3 g l1, NaHCO3 1.7 g l1, urea 0.2 g l1, 290 g of a-amylase, 15 mg of uric acid and 25 mg of mucin to digestion tubes containing 4.5 ml spiked milk and incubated for 5 min at 37 C. Later, 12 ml of gastric juice consists of NaCl 2.75 g l1, NaH2PO4 0.27 g l1, KCl 0.82 g l1, CaCl2.2H2O 0.4 g l1, NH4Cl 0.31 g l1, 6.5 ml of HCl (37%), glucose 0.65 g l1, glucuronic acid 0.02 g l1, urea 0.085 g l1, glucosamine hydrochloride 0.33 g l1, 1 g of BSA, 2.5 g of pepsin and 3 g of mucin was added and the mixture was rotated for 2 h (Multi RS-24 rotator, Biosan, Latvia) at 37 C. Finally, 12 ml of simulated duodenal juice composed of NaCl 7.01 g l1, NaHCO3 3.39 g l1, KH2PO4 0.08 g l1, KCl 0.56 g l1, MgCl2 0.05 g l1, 180 ml of HCl, urea 0.1 g l1, CaCl2.2H2O 0.2 g l1, 1 g of BSA, 9 g of pancreatin and 1.5 g of lipase and 6 ml simulated bile consists of NaCl 5.26 g l1, NaHCO3 5.79 g l1, KCl 0.38 g l1, 150 ml of HCl, urea 0.25 g l1, CaCl2.H2O 0.22 g l1, 1.8 g of BSA and 30 g of bile, and 2 ml of 1 M NaHCO3 were added simultaneously, and the mixture was rotated for another 2 h. The chyme (supernatant) and the digested matrix (pellet) were obtained by centrifugation (Rotafix 32A, Hettich, Germany) at 2750 g for 5 min. The AFM1 contents in the supernatant were measured by HPLC coupled with fluorescence detector after the clean-up of samples (20 ml). AFM1 bioaccessibility is expressed as the ratio of AFM1 level in gastrointestinal phases to the total concentration of the compound before digestion.
2.8. Validation of the analytical method The 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, 0.5 and 1 mg AFM1 l1. The linearity was assessed by constructing five-point calibration curve over the concentration range of 0.05e1 mg l1, each concentration injected in triplicate. The linearity was evaluated by linear regression analysis using the least squares method and expressed as correlation coefficient (R2). The sensitivity of the method was expressed by the limits of detection (LOD) and quantification (LOQ). The LOD and LOQ were calculated as signal-to-noise ratio of 3:1 and 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 AFM1 working standards to obtain final concentrations of 0.05, 0.5 and 1 mg l1. 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 ¼ 3) and on three consequent days (n ¼ 9) at the respective spiking levels. 2.9. Statistical analysis Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by Duncan multiple comparison test using the SPSS 10.0 software package program. Probability (P) values of <0.05 were considered significant. A Student’s t-test was also used in for pairwise comparison of data. 3. Results and discussion 3.1. Method validation The selectivity of the method was assured by the use of immunoaffinity column for clean-up and a very selective fluorescence 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 AFM1 (7 min). The calibration curve was linear over the concentration range of 0.05e1 mg l1, with satisfactory coefficient of determination (R2 ¼ 0.99907). The regression line was y ¼ 0.073x 0.006. The LOD and LOQ values, accuracy and precision of analytical method for AFM1 in milk and supernatant samples are summarised in Table 2. The LODs (S/N ¼ 3), defined as the lowest concentration of AFM1 that can be clearly detected above the baseline signal, were 0.004 and 0.005 mg l1 for milk and biological fluid, respectively. The LOQs (S/N ¼ 10), defined as the lowest concentration of analyte that can be determined with acceptable precision and accuracy
Fig. 1. A schematic diagram representing the in vitro digestion experiment. The in vitro digestion model consists of three compartments: mouth, stomach and small intestine.
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Table 2 The accuracy, precision, LOD and LOQ for AFM1 in UHT milk and biological fluid samples. Matrix
Spiking level (mg l1)
Milk
0.05 0.5 1 0.05 0.5 1
Biological fluid
a b c d
Intra-daya (n ¼ 3)
Inter-dayb (n ¼ 9)
Recovery (%)
RSD (%)
Recovery (%)
RSD (%)
88.0 92.1 92.9 86.6 84.3 85.9
5.6 4.2 4.0 6.6 7.8 4.9
90.6 88.7 88.4 85.7 87.6 83.5
7.3 9.4 6.9 11.2 10.5 9.7
LODc (mg l1)
LOQd (mg l1)
0.004
0.014
0.005
0.016
Intra-day repeatability was estimated by analysis of three replicate samples on the same day. Inter-day repeatability was estimated by analysis of three replicate samples on the consecutive days. LOD, limit of detection of analytical method (S/N ¼ 3). LOQ, limit of quantification of analytical method (S/N ¼ 10).
were 0.014 and 0.016 mg l1 for milk and biological fluid, respectively. The intra-day and inter-day accuracy varied from 84.3 to 92.9% and from 83.5 to 90.6%, respectively, for both matrices, and the intra-day and inter-day precision were satisfactory, with RSD values always lower than 12%. There were no significant differences (P > 0.05) in AFM1 recoveries between the intra-day and inter-day studies. These recovery values (within the range 60e120% for concentration of 0.05 mg l1, and 70e110% for concentration above 0.05 mg l1) meet the requirements of the Commission Regulation (EC). No. 401/2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs.
The natural occurrence of AFM1 in milk and dairy products is a global problem. In Portugal, Martins and Martins (2000) analysed 31 raw milk and 70 UHT milk samples for AFM1, of which 25 raw milk samples (range ¼ 0.005e0.05 mg l1) and 59 UHT milk samples (range ¼ 0.005e0.061 mg l1) contained AFM1. Galvano et al. (2001) found 78% of UHT milk samples collected in Italy, with a maximum concentration of 0.024 mg l1. In another survey conducted in Brazil, 7 of 12 pasteurized milk and 10 of 12 UHT milk samples were contaminated with AFM1, with mean values of 0.072 and 0.048 mg l1, respectively (Oliveira & Ferraz, 2007). Cano-Sancho, Marin, Ramos, Peris-Vicente, and Sanchis (2010) detected AFM1 in 94.4% (68/72) of whole UHT milk samples from Spain at levels ranging from <0.005 to 0.030 mg l1. In Iran, AFM1 was detected in all samples analysed (49 samples) and 83.67% of them contained AFM1 greater than EU limit of 0.05 mg l1 (Movassagh, 2011).
3.2. Occurrence of AFM1 in UHT milk The incidence and concentration of AFM1 in whole UHT milk samples are summarised in Table 3. Eight out of 40 samples (20%) contained AFM1. The contamination level of AFM1 in milk samples ranged from <0.004 to 0.076 mg l1. A chromatogram of naturally contaminated UHT milk sample with AFM1 (0.076 mg l1) is shown in Fig. 2. Only 2 samples contained AFM1 higher than permissible limit of 0.05 mg kg1 set by EU regulation (European Commission, 2006b). There are many studies on the occurrence of AFM1 in milk and dairy products. In a survey on pasteurized and UHT milk in Turkey, AFM1 was found 59.3% of samples with levels ranging from <0.01 to 0.05 mg l1 (Gürbay, Aydın, Girgin, Engin, & S¸ahin, 2006). Unusan (2006) found AFM1 in 75 out of 129 UHT milk samples in Central Anatolia, Turkey, with a mean concentration of 0.11 mg l1. In another survey, 67 of 100 of UHT milk samples were contaminated with AFM1 at concentrations between 0.01 and 0.63 mg l1 (Tekins¸en & Eken, 2008). More recently, Atasever, Adıgüzel, Atasever, Özlü, and Özturhan (2010) found AFM1 in 59% of UHT milk samples from Turkey ranging from 0.005 to 0.185 mg l1. A comparison of the results of our study with those of other studies conducted in Turkey shows a rather low incidence of AFM1 in UHT milk analysed in present work. The variations in AFM1 levels among studies could be attributed to forage and feed quality, cows’ diet, genetic variation in dairy cows, geographical and seasonal variations, and analytical method procedures.
3.3. Bioaccessibility of AFM1 and efficacy of probiotic bacteria in reducing AFM1 bioaccessibility The results of the AFM1 bioaccessibility from spiked and naturally contaminated UHT milk are shown in Table 4. AFM1 concentration in the spiked milks ranged from 0.047 to 0.939 mg l1. The bioaccessibility of AFM1 from spiked milk varied from 80.5 to 83.8%, depending on contamination level. In addition, the bioaccessibility of AFM1 ranged from 81.7 to 86.3% for naturally contaminated UHT milk samples containing 0.011e0.076 mg l1 AFM1. This is the first 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 bioavailability. 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).
Table 3 Occurrence and level of AFM1 in whole UHT milk marketed in Turkey. Samples Analysed samples
Positive samples (%)
1
40 a b
8 (20)
Minemax. Mean of positive samples SD.
AFM1 levels (mg l1)
Frequency distribution, n (%) 1
1
1
<0.005 mg l
0.005e0.015 mg l
0.015e0.05 mg l
>0.05 mg l
Rangea
Averageb
32
1 (12.5)
5 (62.5)
2 (25)
<0.004e0.076
0.029 0.02
342
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AFM1 /7 .0 4 3
0.75
mV Detector A:Ex:365nm,Em:435nm
0.50 0.25 0.00 0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
min
Fig. 2. HPLC chromatogram of a naturally contaminated UHT milk sample with AFM1 (0.076 mg l1).
Regarding the bioaccessibility of AFB1, 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 AFB1 in the duodenal compartment to be 94%. In another study, the total intestinal absorption of AFB1 was 44% in the 6 h of digestion of the contaminated feeds (Avantaggiato, Havenaar, & Visconti, 2007). More recently, Kabak et al. (2009) showed that the bioaccessibilities of AFB1 from 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 (83e100%) 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 AFB1. Concerning Fusarium toxins, it was shown that 32% of ZEA intake through artificially contaminated wheat (4.1 mg kg1) was released from the food matrix to the bioaccessible fraction during 6 h 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 170 mg DON and 230 mg 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 1.5 and 3 mmol g1 concentrations, their transepithelial transport and bioavailability using Caco-2 cells as a model of the human intestinal epithelium. The overall ENs bioaccessibility was 80%. It has been also demonstrated 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, Table 4 Bioaccessibility of AFM1 from spiked and naturally contaminated UHT milk samples. Matrix
AFM1 level (mg l1)
Amount of AFM1 in digestion model (ng)
Bioaccessibility (%, mean SD)
Spiked milk Spiked milk Spiked milk Sample 6 Sample 8 Sample 11 Sample 19 Sample 27 Sample 32 Sample 35 Sample 38
0.047 0.464 0.939 0.029 0.044 0.038 0.076 0.046 0.058 0.011 0.027
0.21 2.09 4.22 0.13 0.20 0.17 0.34 0.21 0.26 0.05 0.12
83.75 81.35 80.53 85.45 83.13 85.29 83.34 81.67 83.12 86.26 84.64
4.99 3.49 5.06 4.72 4.28 5.53 4.30 6.43 4.77 6.53 3.82
compound and type of contamination (spiked versus naturally contaminated food materials) (Kabak et al., 2009). According to present study, there was no significant variation in AFM1 bioaccessibility (P > 0.05) among the spiked and naturally contaminated milk samples. Similarly, the amount of AFM1 in the digestion model did not influence (P > 0.05) the percentage of its bioaccessibility. These results are consistent with that reported by Versantvoort et al. (2005) who showed that different amounts of OTA (4e51 ng) in the digestion model had no effect on the bioaccessibility of this compound. Moreover, He and Wang (2011) observed no significant difference in bioaccessibility of mercury (61e62%) and methylmercury (68%) between the two concentration treatments. Fig. 3 shows the reductions in the AFM1 bioaccessibility in milk following addition of probiotic bacteria to the digestion model. The use of probiotic bacteria resulted in reductions in AFM1 bioaccessibility ranging from 15.5 to 31.6%, in comparison to the control. These results showed that when AFM1 are released from its matrix into the biological fluid, they are partially bound by probiotic bacteria. Among test bacteria, Lactobacillus acidophilus NCFM 150B was the most efficient binder for AFM1, 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 AFM1 in aqueous solution and reconstituted milk with ranging from 10.22 to 26.65% and from 7.85 to 25.94%, respectively (Kabak & Var, 2008). In another work, Pierides, ElNezami, Peltonen, Salminen, and Ahokas (2000) found that Lactobacillus strains decreased AFM1 level in the range from 18.1 to 53.8% in aqueous solution. In agreement with the in vitro screening test, the intestinal absorption of AFB1 was 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 AFM1 in spiked milk in the digestion model had no effect (P > 0.05) in the reductions of AFM1 bioaccessibility 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 AFB1 by bacteria was independent of toxin concentration. 3.4. Estimated daily intake of AFM1 The mean daily intake of AFM1 was 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 of
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Fig. 3. Bioaccessibility of AFM1 from spiked milk at 0.042, 0.439 and 0.882 mg l1 in the presence of probiotic bacteria.
mean was obtained by setting all
and dairy products should be conducted by the government agencies. This is also the first time that bioaccessibility of AFM1 from UHT milk has been examined using an in vitro digestion model. The results show that AFM1 is highly bioaccessible (80.5e86.3%) in milk. The bacteria used in this study showed only partially reduction (15.5e31.6%) in the bioaccessibility of AFM1 under simulated gastrointestinal conditions. However, more non-pathogenic bacteria must be tested for their ability in reducing AFM1 bioaccessibility either with in vivo experiments or with the digestion models. Acknowledgements This work was funded by the Scientific and Technological Research Council of Turkey (TUBITAK, project no: TOVAG108O502). References Atasever, M. A., Adıgüzel, G., Atasever, M., Özlü, H., & Özturhan, K. (2010). Occurrence of aflatoxin M1 in UHT milk in Erzurum-Turkey. Kafkas Universitesi Veteriner Fakultesi Dergisi, 16, 119e122. Avantaggiato, G., Havenaar, R., & Visconti, A. (2003). Assessing the zearalenonebinding activity of adsorbent materials during passage through a dynamic in vitro gastrointestinal model. Food and Chemical Toxicology, 41, 1283e1290. Avantaggiato, G., Havenaar, R., & Visconti, A. (2004). Evaluation of the intestinal absorption of deoxynivalenol and nivalenol by an in vitro gastrointestinal model, and the binding efficacy of activated carbon and other adsorbent materials. Food and Chemical Toxicology, 42, 817e824. Avantaggiato, G., Havenaar, R., & Visconti, A. (2007). Assessment of the multimycotoxin-binding efficacy of a carbon/aluminosilicate-based product in an in vitro gastrointestinal model. Journal of Agricultural and Food Chemistry, 55, 4810e4819. Bognanno, M., La Fauci, L., Ritieni, A., Tafuri, A., De Lorenzo, A., Micari, P., et al. (2006). Survey of the occurrence of aflatoxin M1 in ovine milk by HPLC and its confirmation by MS. Molecular Nutrition and Food Research, 50, 300e305. Cabanero, A. I., Madrid, Y., & Camara, C. (2004). Selenium and mercury bioaccessibility in fish samples: an in vitro digestion method. Analytica Chimica Acta, 526, 51e61. Cano-Sancho, G., Marin, S., Ramos, A. J., Peris-Vicente, J., & Sanchis, V. (2010). Occurrence of aflatoxin M1 and exposure assessment in Catalonia (Spain). Revista Iberoamericana de Micología, 27, 130e135. Coffey, R., Cummins, E., & Ward, S. (2009). Exposure assessment of mycotoxins in dairy milk. Food Control, 20, 239e249. El-Nezami, H., Kankaanpää, P., Salminen, S., & Ahokas, J. (1998). Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1. Food and Chemical Toxicology, 36, 321e326. El-Nezami, H., Mykkänen, K., Kankaanpää, P., Salminen, S., & Ahokas, J. (2000). Ability of Lactobacillus and Propionibacterium strains to remove aflatoxin B1 from the chicken duodenum. Journal of Food Protection, 63, 549e552. European Commission. (2006a). Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union, L364, 5e24.
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European Commission. (2006b). Commission Regulation (EC) No 401/2006 of 23 February 2006 laying down the methods of sampling analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Union, L70, 12e34. Galvano, F., Galofaro, V., & Galvano, G. (1996). Occurrence and stability of aflatoxin M1 in milk and milk products: a worldwide review. Journal of Food Protection, 59, 1079e1090. Galvano, F., Galofaro, V., Ritieni, A., Bognanno, M., De Angalis, A., & Galvano, G. (2001). Survey of the occurrence of aflatoxin M1 in dairy products marketed in Italy: second year of observation. Food Additives and Contaminants, 18, 644e646. Gürbay, A., Aydın, S., Girgin, G., Engin, A. B., & S¸ahin, G. (2006). Assessment of aflatoxin M1 levels in milk in Ankara, Turkey. Food Control, 17, 1e4. He, M., & Wang, W.-X. (2011). Factors affecting the bioaccessibility of methylmercury in several marine fish spices. Journal of Agricultural and Food Chemistry, 59, 7155e7162. International Agency for Research on Cancer (IARC). (1993). Some naturally occurring substances, food items and constituents, heterocyclic aromatic amines and mycotoxins, Vol. 56. Lyon France: World Health Organization. International Dairy Federation (IDF). (2010). Bulletin of the International Dairy Federation, 446/2010. Brussels, Belgium: The World Dairy Situation 2010. Joint FAO/WHO Expert Committee on Food Additives (JECFA). (6e15 February 2001). Aflatoxin M1. In Safety evaluations of specific mycotoxins, Prepared by the fifty-sixth meeting of the Joint FAO/WHO Expert Committee on Food Additives, Geneva. Kabak, B., Brandon, E. F. A., Var, I., Blokland, M., & Sips, A. J. A. M. (2009). Effects of probiotic bacteria on the bioaccessibility of aflatoxin B1 and ochratoxin A using an in vitro digestion model under fed conditions. Journal of Environmental Science and Health, Part B, 44, 472e480. Kabak, B., & Var, I. (2008). Factors affecting the removal of aflatoxin M1 from food model by Lactobacillus and Bifidobacterium strains. Journal of Environmental Science and Health, Part B, 43, 617e624. Kuiper-Goodman, T. (1990). Uncertainties in the risk assessment of three mycotoxins: aflatoxin, ochratoxin and zearalenone. Canadian Journal of Physiology and Pharmacology, 68, 1017e1024. Lahtinen, S. J., Haskard, C. A., Ouwehand, A. C., Salminen, S. J., & Ahokas, J. T. (2004). Binding of aflatoxin B1 to cell wall components of Lactobacillus rhamnosus strain GG. Food Additives and Contaminants, 21, 158e164.
Martins, M. L., & Martins, H. M. (2000). Aflatoxin M1 in raw and ultra high temperature-treated milk commercialized in Portugal. Food Additives and Contaminants, 17, 871e874. Meca, G., Mañes, J., Font, G., & Ruiz, M.-J. (2012). Study of the potential toxicity of commercial crispy breads by evaluation of bioaccessibility and bioavailability of minor Fusarium mycotoxins. Food and Chemical Toxicology, 50, 288e294. Movassagh, M. H. (2011). Presence of aflatoxin M1 in UHT milk in Tabriz (Northwest of Iran). Journal of Food Safety, 31, 238e241. Oliveira, C. A. F., & Ferraz, J. C. O. (2007). Occurrence of aflatoxin M1 in pasteurised, UHT milk and milk powder from goat origin. Food Control, 18, 375e378. Peltonen, K., El-Nezami, H., Haskard, C., Ahokas, J., & Salminen, S. (2001). Aflatoxin B1 binding by dairy strains of lactic acid bacteria and bifidobacteria. Journal of Dairy Science, 84, 2152e2156. Pierides, M., El-Nezami, H., Peltonen, K., Salminen, S., & Ahokas, J. (2000). Ability of dairy strains of lactic acid bacteria to bind aflatoxin M1 in a food model. Journal of Food Protection, 63, 645e650. Pitt, J. I. (2000). Toxigenic fungi: which are important. Medical Mycology, 38, 17e22. Raiola, A., Meca, G., Mañes, J., & Ritieni, A. (2012). Bioaccessibility of deoxynivalenol and its natural co-occurrence with ochratoxin A and aflatoxin B1 in Italian commercial pasta. Food and Chemical Toxicology, 50, 280e287. Ruangwises, N., Saipan, P., & Ruangwises, S. (2011). Estimated daily intake of aflatoxin M1 in Thailand. In R. Guevara-González (Ed.), AflatoxinseBiochemistry and molecular biology (pp. 439e446). Intech. Signorini, M. L., Gaggiotti, M., Molineri, A., Chiericatti, C. A., Zapata de Basílico, M. L., Basílico, J. C., et al. (2012). Exposure assessment of mycotoxins in cow’s milk in Argentina. Food and Chemical Toxicology, 50, 250e257. Tekins¸en, K. K., & Eken, H. S. (2008). Aflatoxin M1 levels in UHT milk and Kashar cheese consumed in Turkey. Food and Chemical Toxicology, 46, 3287e3289. Unusan, N. (2006). Occurrence of aflatoxin M1 in UHT milk in Turkey. Food and Chemical Toxicology, 44, 1897e1900. Versantvoort, C. H. M., Oomen, A. G., Van de Kamp, E., Rompelberg, C. J. M., & Sips, A. A. J. (2005). Applicability of an in vitro digestion model in assessing the bioaccesibility of mycotoxins from food. Food and Chemical Toxicology, 43, 31e40. Zinedine, A., González-Osnaya, L., Soriano, J. M., Moltó, J. C., Idrissi, L., & Mañes, J. (2007). Presence of aflatoxin M1 in pasteurized milk from Morocco. International Journal of Food Microbiology, 114, 25e29.
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Aflatoxin M1 in UHT milk consumed in Turkey and first assessment of its bioaccessibility using an in vitro digestion model Bulent Kabak*, Fatih Ozbey Hitit University, Faculty of Engineering, Department of Food Engineering, TR-19030, Corum, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 February 2012 Accepted 12 May 2012
Contamination of milk and dairy products with aflatoxin M1 (AFM1) 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 AFM1 in whole UHT milk from main processors in Turkey in order to make a preliminary exposure assessment. A total of 40 milk samples were analysed for AFM1 using high performance liquid chromatography with fluorescence detection (HPLC-FD) after immunoaffinity column clean-up. Aflatoxin M1 was detected in 20% of samples at levels ranging from <0.004 to 0.076 mg l1. Only two samples contained AFM1 above the EU limit of 0.05 mg l1. The second aim of this study was to determine the bioaccessibility of AFM1 from milk using an in vitro digestion model. The bioaccessibility of AFM1 in spiked and naturally contaminated milk samples ranged from 80.5 to 83.8% and from 81.7 to 86.3%, respectively. No difference (P > 0.05) in AFM1 bioaccessibility was found between spiked and naturally contaminated milk samples. This study also assessed the binding of AFM1 by six probiotic bacteria under simulated gastrointestinal conditions. A 15.5e31.6% reduction in AFM1 bioaccessibility was observed in the presence of probiotic bacteria. Based on the results obtained in the present study, the mean daily intake of AFM1 through milk consumption was estimated as 0.008 ng kg1 b.w. day1 for Turkish adults. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Aflatoxin M1 UHT milk Occurrence Bioaccessibility HPLC Daily intake
1. Introduction Aflatoxins (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). Aflatoxin B1 (AFB1) is the most potent hepatotoxic AFs with a large variety of biological effects, such as carcinogenicity, teratogenicity and mutagenicity in various animal species, including humans (Bognanno et al., 2006). Aflatoxin M1 (AFM1), the 4-hydroxy metabolite of AFB1, is the predominant metabolite of AFB1 and can be found in milk and milk products obtained from lactating animals ingesting feed contaminated with AFB1. The conversion rate of ingested AFB1 to AFM1 varies from 0.5 to 6% for lactating animals (Galvano, Galofaro, & Galvano,
Abbreviations: AFs, aflatoxins; AFB1, aflatoxin B1; AFM1, aflatoxin M1; ALARA, As Low As Reasonably Achievable; HPLC, high performance liquid chromatography; IAC, immunoaffinity column; LOD, limit of detection; LOQ, limit of quantification; RSD, relative standard deviation; TDI, tolerable daily intake. * Corresponding author. Tel.: þ90 364 2274533; fax: þ90 364 2274535. E-mail address: [email protected] (B. Kabak). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.05.029
1996). The acute toxicity of AFM1 is similar or slightly less than that of AFB1 but its carcinogenic potential is about ten times less than that of AFB1 (JECFA, 2001). Thus, AFB1 and AFM1 have been classified 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. 489e521). As AFs are considered to be genotoxic carcinogens, the FAO/ WHO Joint Expert Committee on Food Additives (JECFA) and the Scientific Committee on Food (SCF) of European Community did not establish a threshold for AFs, but recommended that its concentrations in food should be As Low As Reasonable (ALARA). The frequency of occurrence of AFM1 in commercially available milk and dairy products, the high intake of these products by human population, especially by infants and young children and its probable carcinogenic effect, led to an increased concern about the establishment of measures to control AFM1 contamination. The European Commission Regulation 1881/2006 sets a maximum permissible limit of 0.05 mg kg1 for AFM1 in 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 specific tissue or organ, AFs must be first released from their matrix and then be absorbed from the gut via the intestinal cells. The term
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bioaccessibility 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 AFB1 and 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 from 80 to 98%. The bioaccessibility of AFs seems to be independent of the spiking level and food matrices. However, there is no data in the literature regarding the bioaccessibility of AFM1 from milk and dairy products. Thus, this study aims to (i) assess AFM1 bioaccessibility from spiked and naturally contaminated milk using an in vitro digestion model, (ii) test the effectiveness of probiotic bacteria in reducing AFM1 bioaccessibility, (iii) detect AFM1 contamination in commercially available milk in Turkey and (iv) estimate mean daily intake of AFM1 through milk consumption for Turkish consumers for the first time. 2. Materials and methods 2.1. Reagents and chemicals
339
Table 1 Bacteria and their sources used in this study. Bacteria
Source
Bifidobacterium longum Bifidobacterium species 420 Lactobacillus acidophilus Lactobacillus acidophilus NCFM 150B Lactobacillus casei Shirota Lactobacillus rhamnosus
Ezal-France Danisco-Germany Danisco-Germany Rhodia Inc., Wisconsin, USA RIVM,a The Netherlands Ezal-France
a Laboratory for Health Protection Research, The National Institute for Public Health and Environment, Bilthoven, The Netherlands.
plates were counted and the population of bacteria were expressed as colony-forming units (CFU) per ml. Bacterial strains were used at a concentration of 108 CFU ml1. 2.3. Standard preparation AFM1 standard (10 mg ml1, in 1 ml acetonitrile) was obtained from Supelco (Bellefonte, PA, USA). Stock solution of AFM1 was diluted in 25% acetonitrile to obtain an AFM1 concentration of 50 ng ml1 as an intermediate solution. From this intermediate solution, a series of working standards from 0.05 to 1 ng ml1 in mobile phase consisting of water-acetonitrile (75:25, v/v) was prepared. 2.4. Samples A total of 40 whole 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 of 2011. The samples were stored in refrigerator until analysis. The package size of the milk samples was 1 l.
Potassium 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). Magnesium chloride (MgCl2), a-amylase, mucin, pepsin, pancreatin, lipase, bile salts, phosphoric acid and citric acid were from SigmaeAldrich (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-Q 3 UV system, from Millipore (Molsheim, France) was used.
Milk samples were extracted and cleaned-up by a method based on that described by Bognanno et al. (2006), with slight modifications. An aliquot of 40 ml of milk was warmed at 37 C and centrifuged (Rotafix 32A, Hettich, Germany) at 2750 g for 10 min. After centrifugation, the upper cream layer was completely removed and the remaining milk was filtered through Whatman No. 4 filter paper. A 25 ml of filtered skimmed milk was passed through an IAC, placed in a vacuum manifold (Agilent Technologies, Santa Clara, CA, USA). The column was washed twice with 10 ml ultrapure water and AFM1 was eluted from the column with 4 ml acetonitrile. The extract was then evaporated to dryness under rotary evaporator (Hidolph Hi-Vap Advantage, Germany), the residue was re-dissolved in 1 ml of mobile phase and collected in HPLC vials (Supelco, Bellefonte, PA, USA).
2.2. Bacteria, culture conditions and enumeration
2.6. HPLC analysis
The bacteria used in this study and their sources are listed in Table 1. These bacteria were selected based either on their use as dairy and probiotic cultures in food industry or on available information concerning their effects on AFs in aqueous solution. Bacterial cells were grown in De-Man-Rogosa-Sharpe Broth (MRS, Merck, Darmstadt, Germany) medium containing 0.05% Lcysteine at 37 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 at 37 C for 48 h under anaerobic conditions (Anaerocoult A, Merck, Darmstadt, Germany). Then, colonies on the
A liquid chromatographic system (Shimadzu, Tokyo, Japan) equipped with an LC-20AD pump, a DGU-20A3 on-line degasser, an SIL-20AHT autosampler and a fluorescence detector model RF20AXL, controlled by a CBM-20Alite system controller was used. The analytical column was a reversed phase intersil ODS-3 (5 mm, 250 mm 4.6 mm, GL Sciences Inc, Tokyo, Japan), thermostated at 30 C. An isocratic mobile phase of water-acetonitrile (75:25, v/v) was used with a flow rate of 1 ml min1. The detection wavelengths of excitation and emission were set at 365 and 435 nm, respectively. The injection volume was set to 100 ml using SIL-20AHT autosampler. The retention time for AFM1 was about 7 min.
2.5. Immunoaffinity column clean-up
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2.7. In vitro digestion procedure An in vitro digestion model developed by the National Institute for Public Health and the Environment (RIVM, Bilthoven, The Netherlands) was used to assess AFM1 bioaccessibility and the performance of probiotic bacteria in reducing AFM1 bioaccessibility under simulated gastrointestinal conditions. The digestion model consists of initial saliva processing for 5 min at 37 C to simulate the mouth compartment and the gastric conditions for 2 h, followed by simulated small intestine compartment for 2 h at 37 C. In the digestion model 4.5 ml spiked milk was used. In order to prepare spiked samples at concentrations of 0.05, 0.5 and 1 mg AFM1 l1, the uncontaminated portion milk was spiked with an appropriate amount of AFM1 standard solution. The spiked samples were analysed for AFM1 according 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 represented 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 AFM1 from spiked milk. The digestion was started with adding 6 ml artificial saliva composed of KCl 0.9 g l1, KSCN 0.2 g l1, NaH2PO4 0.9 g l1, NaSO4 0.57 g l1, NaCl 0.3 g l1, NaHCO3 1.7 g l1, urea 0.2 g l1, 290 g of a-amylase, 15 mg of uric acid and 25 mg of mucin to digestion tubes containing 4.5 ml spiked milk and incubated for 5 min at 37 C. Later, 12 ml of gastric juice consists of NaCl 2.75 g l1, NaH2PO4 0.27 g l1, KCl 0.82 g l1, CaCl2.2H2O 0.4 g l1, NH4Cl 0.31 g l1, 6.5 ml of HCl (37%), glucose 0.65 g l1, glucuronic acid 0.02 g l1, urea 0.085 g l1, glucosamine hydrochloride 0.33 g l1, 1 g of BSA, 2.5 g of pepsin and 3 g of mucin was added and the mixture was rotated for 2 h (Multi RS-24 rotator, Biosan, Latvia) at 37 C. Finally, 12 ml of simulated duodenal juice composed of NaCl 7.01 g l1, NaHCO3 3.39 g l1, KH2PO4 0.08 g l1, KCl 0.56 g l1, MgCl2 0.05 g l1, 180 ml of HCl, urea 0.1 g l1, CaCl2.2H2O 0.2 g l1, 1 g of BSA, 9 g of pancreatin and 1.5 g of lipase and 6 ml simulated bile consists of NaCl 5.26 g l1, NaHCO3 5.79 g l1, KCl 0.38 g l1, 150 ml of HCl, urea 0.25 g l1, CaCl2.H2O 0.22 g l1, 1.8 g of BSA and 30 g of bile, and 2 ml of 1 M NaHCO3 were added simultaneously, and the mixture was rotated for another 2 h. The chyme (supernatant) and the digested matrix (pellet) were obtained by centrifugation (Rotafix 32A, Hettich, Germany) at 2750 g for 5 min. The AFM1 contents in the supernatant were measured by HPLC coupled with fluorescence detector after the clean-up of samples (20 ml). AFM1 bioaccessibility is expressed as the ratio of AFM1 level in gastrointestinal phases to the total concentration of the compound before digestion.
2.8. Validation of the analytical method The 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, 0.5 and 1 mg AFM1 l1. The linearity was assessed by constructing five-point calibration curve over the concentration range of 0.05e1 mg l1, each concentration injected in triplicate. The linearity was evaluated by linear regression analysis using the least squares method and expressed as correlation coefficient (R2). The sensitivity of the method was expressed by the limits of detection (LOD) and quantification (LOQ). The LOD and LOQ were calculated as signal-to-noise ratio of 3:1 and 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 AFM1 working standards to obtain final concentrations of 0.05, 0.5 and 1 mg l1. 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 ¼ 3) and on three consequent days (n ¼ 9) at the respective spiking levels. 2.9. Statistical analysis Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by Duncan multiple comparison test using the SPSS 10.0 software package program. Probability (P) values of <0.05 were considered significant. A Student’s t-test was also used in for pairwise comparison of data. 3. Results and discussion 3.1. Method validation The selectivity of the method was assured by the use of immunoaffinity column for clean-up and a very selective fluorescence 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 AFM1 (7 min). The calibration curve was linear over the concentration range of 0.05e1 mg l1, with satisfactory coefficient of determination (R2 ¼ 0.99907). The regression line was y ¼ 0.073x 0.006. The LOD and LOQ values, accuracy and precision of analytical method for AFM1 in milk and supernatant samples are summarised in Table 2. The LODs (S/N ¼ 3), defined as the lowest concentration of AFM1 that can be clearly detected above the baseline signal, were 0.004 and 0.005 mg l1 for milk and biological fluid, respectively. The LOQs (S/N ¼ 10), defined as the lowest concentration of analyte that can be determined with acceptable precision and accuracy
Fig. 1. A schematic diagram representing the in vitro digestion experiment. The in vitro digestion model consists of three compartments: mouth, stomach and small intestine.
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341
Table 2 The accuracy, precision, LOD and LOQ for AFM1 in UHT milk and biological fluid samples. Matrix
Spiking level (mg l1)
Milk
0.05 0.5 1 0.05 0.5 1
Biological fluid
a b c d
Intra-daya (n ¼ 3)
Inter-dayb (n ¼ 9)
Recovery (%)
RSD (%)
Recovery (%)
RSD (%)
88.0 92.1 92.9 86.6 84.3 85.9
5.6 4.2 4.0 6.6 7.8 4.9
90.6 88.7 88.4 85.7 87.6 83.5
7.3 9.4 6.9 11.2 10.5 9.7
LODc (mg l1)
LOQd (mg l1)
0.004
0.014
0.005
0.016
Intra-day repeatability was estimated by analysis of three replicate samples on the same day. Inter-day repeatability was estimated by analysis of three replicate samples on the consecutive days. LOD, limit of detection of analytical method (S/N ¼ 3). LOQ, limit of quantification of analytical method (S/N ¼ 10).
were 0.014 and 0.016 mg l1 for milk and biological fluid, respectively. The intra-day and inter-day accuracy varied from 84.3 to 92.9% and from 83.5 to 90.6%, respectively, for both matrices, and the intra-day and inter-day precision were satisfactory, with RSD values always lower than 12%. There were no significant differences (P > 0.05) in AFM1 recoveries between the intra-day and inter-day studies. These recovery values (within the range 60e120% for concentration of 0.05 mg l1, and 70e110% for concentration above 0.05 mg l1) meet the requirements of the Commission Regulation (EC). No. 401/2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs.
The natural occurrence of AFM1 in milk and dairy products is a global problem. In Portugal, Martins and Martins (2000) analysed 31 raw milk and 70 UHT milk samples for AFM1, of which 25 raw milk samples (range ¼ 0.005e0.05 mg l1) and 59 UHT milk samples (range ¼ 0.005e0.061 mg l1) contained AFM1. Galvano et al. (2001) found 78% of UHT milk samples collected in Italy, with a maximum concentration of 0.024 mg l1. In another survey conducted in Brazil, 7 of 12 pasteurized milk and 10 of 12 UHT milk samples were contaminated with AFM1, with mean values of 0.072 and 0.048 mg l1, respectively (Oliveira & Ferraz, 2007). Cano-Sancho, Marin, Ramos, Peris-Vicente, and Sanchis (2010) detected AFM1 in 94.4% (68/72) of whole UHT milk samples from Spain at levels ranging from <0.005 to 0.030 mg l1. In Iran, AFM1 was detected in all samples analysed (49 samples) and 83.67% of them contained AFM1 greater than EU limit of 0.05 mg l1 (Movassagh, 2011).
3.2. Occurrence of AFM1 in UHT milk The incidence and concentration of AFM1 in whole UHT milk samples are summarised in Table 3. Eight out of 40 samples (20%) contained AFM1. The contamination level of AFM1 in milk samples ranged from <0.004 to 0.076 mg l1. A chromatogram of naturally contaminated UHT milk sample with AFM1 (0.076 mg l1) is shown in Fig. 2. Only 2 samples contained AFM1 higher than permissible limit of 0.05 mg kg1 set by EU regulation (European Commission, 2006b). There are many studies on the occurrence of AFM1 in milk and dairy products. In a survey on pasteurized and UHT milk in Turkey, AFM1 was found 59.3% of samples with levels ranging from <0.01 to 0.05 mg l1 (Gürbay, Aydın, Girgin, Engin, & S¸ahin, 2006). Unusan (2006) found AFM1 in 75 out of 129 UHT milk samples in Central Anatolia, Turkey, with a mean concentration of 0.11 mg l1. In another survey, 67 of 100 of UHT milk samples were contaminated with AFM1 at concentrations between 0.01 and 0.63 mg l1 (Tekins¸en & Eken, 2008). More recently, Atasever, Adıgüzel, Atasever, Özlü, and Özturhan (2010) found AFM1 in 59% of UHT milk samples from Turkey ranging from 0.005 to 0.185 mg l1. A comparison of the results of our study with those of other studies conducted in Turkey shows a rather low incidence of AFM1 in UHT milk analysed in present work. The variations in AFM1 levels among studies could be attributed to forage and feed quality, cows’ diet, genetic variation in dairy cows, geographical and seasonal variations, and analytical method procedures.
3.3. Bioaccessibility of AFM1 and efficacy of probiotic bacteria in reducing AFM1 bioaccessibility The results of the AFM1 bioaccessibility from spiked and naturally contaminated UHT milk are shown in Table 4. AFM1 concentration in the spiked milks ranged from 0.047 to 0.939 mg l1. The bioaccessibility of AFM1 from spiked milk varied from 80.5 to 83.8%, depending on contamination level. In addition, the bioaccessibility of AFM1 ranged from 81.7 to 86.3% for naturally contaminated UHT milk samples containing 0.011e0.076 mg l1 AFM1. This is the first 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 bioavailability. 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).
Table 3 Occurrence and level of AFM1 in whole UHT milk marketed in Turkey. Samples Analysed samples
Positive samples (%)
1
40 a b
8 (20)
Minemax. Mean of positive samples SD.
AFM1 levels (mg l1)
Frequency distribution, n (%) 1
1
1
<0.005 mg l
0.005e0.015 mg l
0.015e0.05 mg l
>0.05 mg l
Rangea
Averageb
32
1 (12.5)
5 (62.5)
2 (25)
<0.004e0.076
0.029 0.02
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AFM1 /7 .0 4 3
0.75
mV Detector A:Ex:365nm,Em:435nm
0.50 0.25 0.00 0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
min
Fig. 2. HPLC chromatogram of a naturally contaminated UHT milk sample with AFM1 (0.076 mg l1).
Regarding the bioaccessibility of AFB1, 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 AFB1 in the duodenal compartment to be 94%. In another study, the total intestinal absorption of AFB1 was 44% in the 6 h of digestion of the contaminated feeds (Avantaggiato, Havenaar, & Visconti, 2007). More recently, Kabak et al. (2009) showed that the bioaccessibilities of AFB1 from 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 (83e100%) 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 AFB1. Concerning Fusarium toxins, it was shown that 32% of ZEA intake through artificially contaminated wheat (4.1 mg kg1) was released from the food matrix to the bioaccessible fraction during 6 h 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 170 mg DON and 230 mg 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 1.5 and 3 mmol g1 concentrations, their transepithelial transport and bioavailability using Caco-2 cells as a model of the human intestinal epithelium. The overall ENs bioaccessibility was 80%. It has been also demonstrated 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, Table 4 Bioaccessibility of AFM1 from spiked and naturally contaminated UHT milk samples. Matrix
AFM1 level (mg l1)
Amount of AFM1 in digestion model (ng)
Bioaccessibility (%, mean SD)
Spiked milk Spiked milk Spiked milk Sample 6 Sample 8 Sample 11 Sample 19 Sample 27 Sample 32 Sample 35 Sample 38
0.047 0.464 0.939 0.029 0.044 0.038 0.076 0.046 0.058 0.011 0.027
0.21 2.09 4.22 0.13 0.20 0.17 0.34 0.21 0.26 0.05 0.12
83.75 81.35 80.53 85.45 83.13 85.29 83.34 81.67 83.12 86.26 84.64
4.99 3.49 5.06 4.72 4.28 5.53 4.30 6.43 4.77 6.53 3.82
compound and type of contamination (spiked versus naturally contaminated food materials) (Kabak et al., 2009). According to present study, there was no significant variation in AFM1 bioaccessibility (P > 0.05) among the spiked and naturally contaminated milk samples. Similarly, the amount of AFM1 in the digestion model did not influence (P > 0.05) the percentage of its bioaccessibility. These results are consistent with that reported by Versantvoort et al. (2005) who showed that different amounts of OTA (4e51 ng) in the digestion model had no effect on the bioaccessibility of this compound. Moreover, He and Wang (2011) observed no significant difference in bioaccessibility of mercury (61e62%) and methylmercury (68%) between the two concentration treatments. Fig. 3 shows the reductions in the AFM1 bioaccessibility in milk following addition of probiotic bacteria to the digestion model. The use of probiotic bacteria resulted in reductions in AFM1 bioaccessibility ranging from 15.5 to 31.6%, in comparison to the control. These results showed that when AFM1 are released from its matrix into the biological fluid, they are partially bound by probiotic bacteria. Among test bacteria, Lactobacillus acidophilus NCFM 150B was the most efficient binder for AFM1, 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 AFM1 in aqueous solution and reconstituted milk with ranging from 10.22 to 26.65% and from 7.85 to 25.94%, respectively (Kabak & Var, 2008). In another work, Pierides, ElNezami, Peltonen, Salminen, and Ahokas (2000) found that Lactobacillus strains decreased AFM1 level in the range from 18.1 to 53.8% in aqueous solution. In agreement with the in vitro screening test, the intestinal absorption of AFB1 was 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 AFM1 in spiked milk in the digestion model had no effect (P > 0.05) in the reductions of AFM1 bioaccessibility 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 AFB1 by bacteria was independent of toxin concentration. 3.4. Estimated daily intake of AFM1 The mean daily intake of AFM1 was 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 of
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Fig. 3. Bioaccessibility of AFM1 from spiked milk at 0.042, 0.439 and 0.882 mg l1 in the presence of probiotic bacteria.
mean was obtained by setting all
and dairy products should be conducted by the government agencies. This is also the first time that bioaccessibility of AFM1 from UHT milk has been examined using an in vitro digestion model. The results show that AFM1 is highly bioaccessible (80.5e86.3%) in milk. The bacteria used in this study showed only partially reduction (15.5e31.6%) in the bioaccessibility of AFM1 under simulated gastrointestinal conditions. However, more non-pathogenic bacteria must be tested for their ability in reducing AFM1 bioaccessibility either with in vivo experiments or with the digestion models. Acknowledgements This work was funded by the Scientific and Technological Research Council of Turkey (TUBITAK, project no: TOVAG108O502). References Atasever, M. A., Adıgüzel, G., Atasever, M., Özlü, H., & Özturhan, K. (2010). Occurrence of aflatoxin M1 in UHT milk in Erzurum-Turkey. Kafkas Universitesi Veteriner Fakultesi Dergisi, 16, 119e122. Avantaggiato, G., Havenaar, R., & Visconti, A. (2003). Assessing the zearalenonebinding activity of adsorbent materials during passage through a dynamic in vitro gastrointestinal model. Food and Chemical Toxicology, 41, 1283e1290. Avantaggiato, G., Havenaar, R., & Visconti, A. (2004). Evaluation of the intestinal absorption of deoxynivalenol and nivalenol by an in vitro gastrointestinal model, and the binding efficacy of activated carbon and other adsorbent materials. Food and Chemical Toxicology, 42, 817e824. Avantaggiato, G., Havenaar, R., & Visconti, A. (2007). Assessment of the multimycotoxin-binding efficacy of a carbon/aluminosilicate-based product in an in vitro gastrointestinal model. Journal of Agricultural and Food Chemistry, 55, 4810e4819. Bognanno, M., La Fauci, L., Ritieni, A., Tafuri, A., De Lorenzo, A., Micari, P., et al. (2006). Survey of the occurrence of aflatoxin M1 in ovine milk by HPLC and its confirmation by MS. Molecular Nutrition and Food Research, 50, 300e305. Cabanero, A. I., Madrid, Y., & Camara, C. (2004). Selenium and mercury bioaccessibility in fish samples: an in vitro digestion method. Analytica Chimica Acta, 526, 51e61. Cano-Sancho, G., Marin, S., Ramos, A. J., Peris-Vicente, J., & Sanchis, V. (2010). Occurrence of aflatoxin M1 and exposure assessment in Catalonia (Spain). Revista Iberoamericana de Micología, 27, 130e135. Coffey, R., Cummins, E., & Ward, S. (2009). Exposure assessment of mycotoxins in dairy milk. Food Control, 20, 239e249. El-Nezami, H., Kankaanpää, P., Salminen, S., & Ahokas, J. (1998). Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1. Food and Chemical Toxicology, 36, 321e326. El-Nezami, H., Mykkänen, K., Kankaanpää, P., Salminen, S., & Ahokas, J. (2000). Ability of Lactobacillus and Propionibacterium strains to remove aflatoxin B1 from the chicken duodenum. Journal of Food Protection, 63, 549e552. European Commission. (2006a). Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union, L364, 5e24.
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