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Enzyme-assisted extraction for the HPLC determination of aflatoxin M1 in cheese
Food Chemistry 192 (2016) 235–241
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Analytical Methods
Enzyme-assisted extraction for the HPLC determination of aflatoxin M1 in cheese Amedeo Pietri, Paola Fortunati, Annalisa Mulazzi, Terenzio Bertuzzi ⇑ Feed & Food Science and Nutrition Institute, Faculty of Agriculture, UCSC, Via Emilia Parmense, 84, 29122 Piacenza, Italy
a r t i c l e
i n f o
Article history: Received 24 February 2014 Received in revised form 24 March 2015 Accepted 5 July 2015 Available online 6 July 2015 Keywords: Aflatoxin M1 Cheese Enzyme-assisted extraction Pepsin Pancreatin
a b s t r a c t The extraction of aflatoxin M1 (AFM1) from cheese is generally carried out using chlorinated organic solvents. In this study, two innovative methods were developed, based on an enzyme-assisted (EA) extraction using proteolytic enzymes (pepsin or pepsin–pancreatin). After purification through an immunoaffinity column, AFM1 is determined by HPLC–FLD. A comparison between the new EA methods and an established chloroform (CH) method was carried out on 24 cheese samples. The results showed that the extraction efficiency of the EA methods was independent of ripening time of cheese, whereas the CH method was not able to fully recover AFM1 from ripened cheeses. The simpler (pepsin) of the two methods has been adopted by our laboratory for routine analysis of AFM1 in cheese. In comparison with the CH method, the pepsin-HCl (P-HCl) method is simpler, avoiding solvent evaporation, dissolution and partition in a separating funnel; moreover, it gives higher recoveries, comparable LOD and LOQ and more accurate results. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Lactating animals that have consumed feedstuffs contaminated with aflatoxin B1 (AFB1) excrete the 4-hydroxylated metabolite aflatoxin M1 (AFM1) into the milk. Aflatoxins are potent toxic and carcinogenic agents. Studies on the acute toxicity of aflatoxins in one-day old ducklings suggest that AFM1 and AFB1 act by a similar mechanism in causing subcellular alterations in liver parenchymal cells. Long term studies of toxicity and carcinogenicity in trout and rats have shown that AFM1 is a hepatic carcinogen, but with a potency 2–10% that of AFB1. Further studies have shown that AFM1 is genotoxic in mammalian systems in vivo. The International Agency for Research on Cancer (IARC) stated that there is sufficient evidence for the carcinogenicity of aflatoxins in humans and for the carcinogenicity of aflatoxin B1, G1 and M1 in experimental animals. AFM1 has been categorised as a class 2B, possible human carcinogen (IARC, 1993, 2002, 2012a, 2012b; Van Egmond, 1989). The toxin is associated with the casein fraction of milk by a hydrophobic interaction (Brackett & Marth, 1982; Van Egmond, 1989); hence it is present in cheese when AFM1 contaminated milk is used for cheese-making. AFM1 tends to partition in the range 40–60% between curd and whey; this partitioning depends upon which kind of cheese-making procedure is used ⇑ Corresponding author. E-mail address: [email protected] (T. Bertuzzi). http://dx.doi.org/10.1016/j.foodchem.2015.07.006 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
(Yousef & Marth, 1989). On account of this, the Commission of the European Communities (Commission of the European Communities, 2010) fixed a limit for AFM1 in milk and milk products (0.050 lg kg 1 for milk and a variable limit for milk products, depending on the concentration caused by the drying process or processing into cheese). Specific limits for AFM1 in cheese have been set by other countries (Food, 2004). Quantitative analytical methods for AFM1 in milk have been largely simplified by the introduction of immunoaffinity (IA) columns. An aliquot of milk is passed through an IA column and AFM1 is extracted by specific antibodies immobilised on a solid support material; a subsequent clean-up by water washing removes all other matrix components of the sample. AFM1 is then eluted from the column with a small volume of acetonitrile. Differently, quantitative analytical methods for AFM1 in cheese involve solvent extraction, filtration and then some clean-up stages prior to HPLC determination. In the clean-up step IA columns are widely used and have replaced adsorption or partition chromatography on pre-packed or prepared columns. Generally, the toxin is extracted with chloroform or dichloromethane (Bijl, Van Peteghem, & Dekeyser, 1987; Cavaliere et al., 2006; Colak, Hampikyan, Ulusoy, & Ergun, 2006; Dragacci, Gleizes, Fremy, & Candlish, 1995; Elgerbi, Aidoo, Candlish, & Tester, 2004; Piva, Pietri, Galazzi, & Curto, 1988; Sharman, Patey, & Gilbert, 1989). Then the chloroform or dichloromethane extract is evaporated to dryness under vacuum, and methanol, water and hexane are
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added to the residue. The mixture is transferred to a separating funnel and the lower layer is collected and purified through an IA column (Sharman et al., 1989). This method has been widely used and has been adopted as an official AOAC method (AOAC, 2005a). It showed a detection limit as low as 0.005 lg kg 1 (Pietri, Bertuzzi, Bertuzzi, & Piva, 1997) and sufficient repeatability, but AFM1 recovery values were generally lower in comparison with milk, ranging from 51% to 88% (Cattaneo, Marinoni, Barzaghi, Cremonesi, & Monti, 2011; Dragacci et al., 1995; Govaris, Roussi, Koidis, & Botsoglou, 2001; Iha, Barbosa, Okada, & Trucksess, 2013; Oruc, Cibik, Yilmaz, & Kalkanli, 2006), probably because of the more complex clean-up step. Finally, this method is time consuming and uses a chlorinated solvent. This method was used for many years in our laboratory; however, we noticed that hard cheeses, even if grated, did not completely lose their texture in chlorinated solvents and sometimes formed small aggregates (little balls) during shaking; this detail raised some doubts about a complete AFM1 extraction. But above all, we wondered whether chlorinated solvents were able to break up all the AFM1-casein hydrophobic interactions in cheese. Therefore, taking into account these considerations, we tried to develop a new method, based on an enzyme-assisted (EA) extraction of AFM1 and subsequent clean-up of the extract by an IA column, avoiding solvent evaporation, dissolution and partition in a separating funnel. Furthermore, as proteolytic enzymes cleave proteins, we hypothesised that casein digestion may facilitate the (hidden) toxin release into an aqueous solution. To this end, we applied two well-established in vitro digestibility methods, routinely used in our laboratory. A comparison between the new EA extraction and chloroform (CH) extraction methods was performed on naturally contaminated cheese samples. Finally, two laboratory-scale cheesemakings were carried out using naturally contaminated milk, in order to evaluate the accuracy of the methods from the AFM1 mass balance of the process. 2. Materials and methods 2.1. Samples In recent years, numerous samples of soft, semi-hard and hard cheeses collected from several cheese factories and retail outlets located in northern Italy, have been analysed in our laboratory for AFM1 determination. Considerable AFM1 concentrations were found in several samples, while some were uncontaminated; 300 g of cheese portions from contaminated and blank samples were collected and kept at 20 °C for this study. In detail, six hard cheese (parmesan) samples were used to compare the extraction efficiency of solvents (chloroform vs. dichloromethane) and apparatus (rotary-shaking stirrer vs. homogeniser). Two blank samples, one hard (parmesan) and one soft (crescenza) cheese, were used for recovery tests (the hard one) and for AFM1 stability kinetics during EA extraction (both samples). Two semi-hard (fontina and parmesan) and one hard (parmesan) cheese samples were used to determine the best length of time for EA extraction. Furthermore, 24 cheese samples (6 soft fresh, 8 semi-hard, 10 hard cheeses) were analysed to compare the EA and the CH extraction methods (Table 2). Finally, one blank and two naturally contaminated milk samples were used to compare the EA extraction with the official AOAC method. 2.2. Reagents Chemicals and solvents used for the extraction and clean-up solutions were ACS grade or equivalent (Carlo Erba, Milan, Italy).
Water used was deionised and, for HPLC, purified through a Milli-Q treatment system (Millipore, London, U.K.). For HPLC analysis, methanol and acetonitrile were HPLC grade (Merck, Darmstadt, Germany). Pepsin (from porcine gastric mucosa, 800– 2500 units/mg protein, cod P7000) and pancreatin (from porcine pancreas, activity equivalent to 4 USP, cod P1750) were obtained from Sigma–Aldrich (St. Louis, MO, USA). 2.3. Standard solution AFM1 standard (0.01 mg, purity degree > 98%) was obtained from Sigma–Aldrich. A stock solution of 4–6 lg ml 1 was prepared in acetonitrile (1.5–2 ml) and stored at 20 °C. The solution was calibrated spectrophotometrically at 350 nm using the value 19,000 L mol 1cm 1 for the absorption coefficient (AOAC, 2005b). For HPLC calibration or spiking purposes, the stock solution was diluted with the HPLC mobile phase, to obtain 7 working standard solutions at AFM1 concentration between 0.1 and 5 lg l 1. All standard solutions were stored at 20 °C when not in use. 2.4. Analysis of AFM1 in cheese The samples of semi-hard and hard cheeses were grated before analysis. 2.4.1. Chloroform (CH) standard method AFM1 was extracted according to the method reported by Pietri et al. (1997). Briefly, chloroform (75 ml), saturated sodium chloride (1 ml) and Celite 545 (5 g) were added to 20 g of cheese and the mixture was shaken for 45 min (160 rpm), using a rotary-shaking stirrer (Shaker 709, ASAL, Milan, Italy). Then, the mixture was filtered through a folded filter paper (5892 S&S, Whatman LTD, Kent, UK) and the volume was recorded. After evaporation of chloroform, the residue was re-dissolved in methanol (1.5 ml) water (50 ml) and hexane (50 ml). The mixture was transferred into a separating funnel and an aliquot (20 ml) of the lower layer was collected and purified through an IA column. In two preliminary experiments, we replaced chloroform with dichloromethane and the rotary-shaking stirrer with an Ultraturrax T-25 homogeniser (14,000 rpm for 5 min). 2.4.2. Pepsin–HCl (P–HCl) method In a 250 ml plastic centrifuge bottle, 50 ml of pepsin solution (0.2% pepsin dissolved in 0.075 M HCl), was added to 5 g of cheese. The pH was measured and eventually adjusted to between 2.6 and 2.8 with 0.1 M NaOH or 0.1 M HCl; then, the mixture was stirred with a magnetic stirrer in a thermostatic chamber at 42 °C for 16 h (overnight). After cooling at ambient temperature, the extract was adjusted to pH 7.4 ± 0.2 with 1 M NaOH (3–5 ml) and the volume was recorded; then, the mixture was centrifuged at 11,700g for 10 min at 4 °C and filtered through a folded filter paper. An aliquot of the sample extract (20 ml) was purified through an IA column. 2.4.3. Pepsin–pancreatin (PP) method In a 250 ml plastic centrifuge bottle, 40 ml of pepsin solution (0.2% pepsin dissolved in 0.075 M HCl), was added to 3 g of cheese. The pH was controlled and eventually adjusted to between 2.6 and 2.8 with 0.1 M NaOH or 0.1 M HCl; then, the mixture was stirred with a magnetic stirrer in a thermostatic chamber at 37 °C for 3 h and 45 min. After cooling at ambient temperature, the extract was adjusted to pH 7.4 ± 0.2 with 1 M NaOH (3–5 ml) and 40 ml of pancreatin solution [1% pancreatin dissolved in 0.2 M phosphate buffer (27.60 g NaH2PO4 H2O + 28.38 g Na2HPO4 in 1 l, pH 7.4)] was added. Then, the mixture was again stirred in a thermostatic chamber at 37 °C for 3 h and 45 min. After cooling at ambient
A. Pietri et al. / Food Chemistry 192 (2016) 235–241
temperature, the volume was recorded; then, the mixture was centrifuged at 11,700g for 10 min at 4 °C and filtered through a folded filter paper. An aliquot of the sample extract (30 ml) was purified through an IA column. 2.5. Analysis of AFM1 in milk
2.5.1. AOAC method AFM1 was extracted and purified as in the AOAC Official Method (AOAC, 2005c). 2.5.2. Pepsin–HCl method An aliquot (100 ml) of milk was centrifuged at 7000g for 10 min at 4 °C; then, the thin upper fat layer was removed and the skimmed milk was filtered through a folded filter paper. In a 50 ml plastic centrifuge bottle, 20 ml of pepsin solution (0.2% pepsin dissolved in 0.075 M HCl), was added to 20 g of filtered milk. The pH was adjusted to between 2.6 and 2.8 with 3 M HCl and the solution was stirred with a magnetic stirrer in a thermostatic chamber at 42 °C for 16 h. After cooling at ambient temperature, the mixture was adjusted to pH 7.4 ± 0.2 with 1 M NaOH and the volume was recorded; then, the mixture was centrifuged (11,700g for 10 min at 4 °C) and filtered through a folded filter paper. An aliquot (20 ml) of the extract was purified through an IA column. 2.6. Clean-up by immunoaffinity column The IA column (Easy-Extract Aflatoxin, R-Biopharm Rhône LTD, Glasgow, Scotland, UK) was placed on a SPE vacuum manifold (Visiprep, Supelco, Bellefonte, PE, USA). The sample extract, prepared as described above, was applied to the column, followed by a washing with distiled water (5 ml). Then, AFM1 was slowly eluted (0.5 ml min 1) from the column with methanol (2.5 ml) into a graduated glass vial; the eluate was evaporated under a gentle stream of nitrogen and re-dissolved in 1 ml acetonitrile:water (25 + 75, v/v) by ultrasonication. The extract was filtered (HV 0.45 lm, Millipore Corporation, Bedford, MA, USA) before HPLC analysis.
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Initially, a recovery test on cheese was performed for CH, P–HCl and PP methods; the recovery values were determined by spiking a blank sample of hard cheese (parmesan) with an appropriate volume of AFM1 standard solution, to obtain contamination levels of 0.25, 0.50 and 1.00 lg kg 1. Successively, a recovery test on milk was also performed (only for CH and P–HCl methods), spiking a blank sample of milk to obtain levels of 0.050, 0.100 and 0.250 lg kg 1. Three replicates were analysed for each level and matrix. The recovery values were compared using the GLM procedure and HSD Tukey post hoc test (SPSS 18.0) considering as factors: method (CH, P-HCl and PP) and level (0.25, 0.50 and 1.00 lg kg 1). Finally, the CH, P-HCl and PP extraction methods were tested on 6 fresh soft, 8 semi-hard (1–3 months of ripening) and 10 hard (8– 24 months of ripening) naturally contaminated cheese samples (Table 2). Each sample was analysed using the 3 methods on the same day and 3 replicate analyses were carried out on different days. For soft, semi-hard and hard cheeses, the results were compared separately using the paired t-test (SPSS 18.0). 2.9. Cheese-making Two cheese-makings were carried out. After pasteurisation at 70 °C for 2 min and subsequent cooling up to 30 °C, 20 kg of naturally contaminated milk was treated with Streptococcus thermofilus, bulgaricus and helveticus (0.6 g) and rennet (chimosine 80%, 5 ml). After complete curdling, the curd was collected and kept at 45 °C for 10 h and successively at 25 °C for 14 h. Then, the curd was placed in a NaCl saturated aqueous solution for 1 h and finally ripened at 4–6 °C. The ripening time was different: 3 months and 1 month for the curd obtained from the first and the second cheese-making, respectively. Milk and whey were analysed using the AOAC method; curd and cheese were analysed using CH and EA extraction methods. All the analyses were carried out in triplicate. The AFM1 concentrations in curd and cheese obtained with the three methods were compared by ANOVA analysis (SPSS 18.0). For each cheese-making, the weight of milk, whey, curd and cheese was recorded and the AFM1 mass balance of the process was calculated. 3. Results and discussion
2.7. HPLC analysis 3.1. Performances of the standard method The HPLC system consisted of a Jasco PU 1580 pump (Jasco Corp., Tokyo, Japan) equipped with an AS 1555 sampling system and a FP 1520 fluorescence detector set at 365 nm excitation and 440 nm emission wavelength. The system was governed by Borwin 1.5 software (Jasco). AFM1 was separated on a Lichrospher RP-18 column (5 lm particle size, 125 4 mm i.d., Merck, Darmstadt, Germany) at ambient temperature (22 °C), with a mobile phase water:acetonitrile (75 + 25, v/v); the flow rate was 1.0 ml min 1. The injection volume for both standard solutions and sample extracts was 100 ll, corresponding to about 0.64, 0.18 and 0.11 g of cheese sample using the CH, P–HCl and PP methods, respectively. 2.8. Performances of the methods The limits of detection (LOD) and quantification (LOQ) of the methods were determined by the signal-to-noise approach, defined at those levels resulting in signal-to-noise ratios of 3 and 10, respectively. The analytical response and the chromatographic noise were both measured from the chromatogram of a blank sample extract (1 ml) to which between 40 and 100 ll of an AFM1 solution (0.428 lg l 1) had been added.
The AFM1 extraction efficiency of chloroform and dichloromethane, tested on four hard cheese samples, was very similar (0.365 vs. 0.373, 0.180 vs. 0.172, 0.017 vs. 0.019, 0.405 vs. 0.414 lg kg 1, respectively); likewise, the extraction efficiency of the stirrer and the homogeniser, tested on two hard cheese samples using chloroform as solvent, was very close (0.981 vs. 0.964, 0.496 vs. 0.480 lg kg 1, respectively). Therefore the CH method, as described in Section 2.4.1, was used in the comparison trials, 3.2. Development of a new extraction method In order to develop an enzyme-assisted extraction of AFM1 from cheese, the AOAC 971.09 method – Pepsin Digestibility of Animal Protein Feeds – (AOAC, 2005d), was first considered. This method involves an enzymatic digestion with pepsin for 16 h; pepsin is the principal digestive enzyme of gastric juice and is distinctive among enzymes because it exhibits maximal activity at pH 2.2; however, at pH 4.5 it still shows about 35% of its maximal activity (Sigma-Aldrich, 2012). AFM1 is slightly soluble in water (about 10– 30 lg ml 1, Cole & Cox, 1981), but this concentration is a thousand times higher than the levels occurring in naturally contaminated
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Fig. 1. Aflatoxin M1 levels (3 replicates) in 3 naturally contaminated cheese samples analysed using the pepsin–HCl extraction method at different times (h) of enzymatic digestion.
milk and in the digested cheese solutions obtained with this method. However, it is known that at very low pH values AFM1 is converted to the emiacetal AFM2a. In order to verify if this reaction can occur during the EA extraction, two AFM1 standard solutions (0.8 and 8 lg l 1) were prepared in the pepsin–HCl solution (pH 1.1); then, a stability kinetics of the standard solutions was performed in a thermostatic chamber at 37 and 42 °C. The AFM1 concentration decreased (consequently AFM2a increased) in the solutions; the loss was 4% after 4 h at both temperatures, 15% and 22% after 16 h at 37 and 42 °C, respectively. The stability kinetics was repeated adding 5 g of uncontaminated soft (crescenza) or hard (parmesan) cheese to 50 ml of AFM1 standard solutions prepared as above. The pH values were slightly higher and depended on the type of cheese; the values were 2.0 ± 0.2 and 2.4 ± 0.2 for the soft cheese, 2.6 ± 0.2 and 3.1 ± 0.2 for the hard cheese, after 0 and 16 h at 42 °C, respectively. The AFM1 concentrations after 16 h decreased by 11% and 0% for the soft and the hard cheese, respectively. The experiment was repeated for the soft cheese after adjusting the pH between 2.6 and 2.8 and no AFM1 loss was observed after 16 h. Therefore, it is important that the pH value of the extraction solution be initially not less than 2.6–2.8. However, in the case of hard cheeses ripened more than 20 months, the pH value of the solution was always higher than 3.0 and for some samples close to 4.5. Consequently, we decided to adjust the pH between 2.6 and 2.8 for all samples, to be near the optimum pH of pepsin activity and to avoid AFM1 losses. Successively, a study was performed to choose the best length of time for EA extraction; the AFM1 concentration in two semi hard
cheeses, ripened for 3 months (fontina and parmesan) and in one hard cheese (parmesan) ripened for 16 months, was determined after 2, 4, 6, 8 and 16 h (Fig. 1). Fontina and parmesan were chosen because they are produced with whole and partly skimmed milk, respectively. The AFM1 recovery after 2 h, with respect to the final level, was 84% for the fontina cheese, and 90% and 70%, for the parmesan cheeses ripened for 3 and 16 months, respectively. Moreover, the solutions after centrifugation were cloudy and the purification step through the IA column was very slow. After 8 h, the results were similar to those after 16 h, but in the case of fontina the solution was still cloudy. As a consequence, a digestion time of 16 h (overnight) was maintained for the P–HCl method. Successively, AFM1 was extracted from the same samples using only 0.075 M HCl (without pepsin); the values were very low compared to those obtained by the P-HCl method. In a further trial, the amount of cheese for analysis was increased to10 g; the final extract was cloudy and the AFM1 concentration resulted lower than that obtained weighing 5 g. The P–HCl method was also tested on milk, in comparison with the AOAC 2000.08 method. Two naturally contaminated milk samples were analysed; the results (0.083 vs. 0.076, 0.131 vs. 0.124 lg kg 1 using the AOAC and the P–HCl method, respectively) were similar, only slightly higher for the AOAC method. Besides the P–HCl method, another EA procedure (PP method) based on the method of Vervaeke, Dierick, Demeyer, and Decuypere (1989) was tested. The method entails two subsequent EA extractions (both for 3 h and 45 min), the first using pepsin at pH between 2.6 and 2.8, the second using pancreatin at pH 7.4 ± 0.2. Pancreatin is a substance containing many enzymes, including amylase, trypsin, protease, ribonuclease and lipase; its greatest activity is exhibited in neutral or slightly alkaline media (The Merck Index, 1983). The PP method was compared to the P– HCl and the CH methods for AFM1 analysis in 24 cheese samples (Table 2 and paragraph 3.4 for results). Finally, AFM1 was extracted from 6 cheese samples by a digestion using only pancreatin (16 h at 37 °C, pH 7.4 ± 0.2). After EA extraction and centrifugation, the solution was cloudy and the purification step through the IA column was not possible for 2 samples (clogged column); for the other 4 samples, the results were lower than those obtained by the PP method.
3.3. Performances of the chloroform and the enzyme-assisted extraction methods The average recoveries of the CH, P–HCl and PP methods were between 87.1% and 96.3% for cheese and those of the AOAC and P–HCl methods were higher than 96.9% for milk (Table 1), with satisfactory relative standard deviation (RSD) values; the performance
Table 1 Average recovery (3 replicates) and relative standard deviation (RSD) of AFM1 from blank hard cheese and milk samples spiked at different levels, using chloroform (CH) and enzyme-assisted [pepsin–HCl (P–HCl) and pepsin–pancreatin (PP)] extraction methods. Spiking level (lg kg
1
)
0.25
0.50
1.00
Method
Recovery %
RSD
Recovery %
RSD
Recovery %
RSD
Cheese CH extraction P–HCl extraction PP extraction
87.5 96.3 94.3
2.0 1.5 1.4
88.5 95.4 93.5
1.8 1.4 1.7
87.1 95.5 94.0
2.3 1.6 1.5
Method
0.050 Recovery %
RSD
0.100 Recovery %
RSD
0.250 Recovery %
RSD
Milk AOAC 2000.08 P–HCl extraction
98.9 97.6
0.8 1.1
98.6 96.9
1.0 1.2
97.4 97.0
0.9 0.9
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Fig. 2. Chromatograms (HPLC; fluorescence detection) of: (a) an AFM1 standard solution (1.23 lg L 1) equivalent to 123 pg of AFM1 injected; (b) a naturally contaminated cheese sample extracted by pepsin–HCl method, containing 0.776 lg kg 1 (equivalent to 155.2 pg of AFM1 injected); (c) a naturally contaminated cheese sample extracted by chloroform method, containing 0.496 lg kg 1 (equivalent to 238.1 pg of AFM1 injected). Injection volume 100 ll.
criteria fixed by Regulation (EC) 401/2006 of the Commission of the European Communities, i.e., recovery in the range 50–120% and 70–110% for levels <1 lg kg 1 and in the range 1–10 lg kg 1, respectively (Commission of the European Communities, 2006), were completely fulfilled. The statistical analysis showed that significantly higher mean recoveries from cheese were obtained by the EA methods compared to the CH method (95.7% vs. 87.7% for P–HCl vs. CH, P < 0.001; 93.9% vs. 87.7% for PP vs. CH, P < 0.001); a small but significant difference was also found between the P– HCl and the PP method (P = 0.041). No significant difference between AFM1 levels and no interaction among factors were observed. The LOD and LOQ in cheese resulted 0.005 and 0.015 lg kg 1 for the CH method, 0.015 and 0.040 lg kg 1 for the P–HCl method, 0.020 and 0.060 lg kg 1 for the PP method, respectively; the LOD and LOQ in milk were 0.001 and 0.003 lg l 1 for the AOAC method, 0.002 and 0.006 lg l 1 for the P–HCl method, respectively. Concerning HPLC analysis, Fig. 2 shows the chromatograms of an AFM1 standard solution and of a naturally contaminated hard cheese sample, extracted by both the P–HCl and the CH method. In the chromatogram of some cheese samples, irrespective of the extraction method used, we observed an interfering peak near the AFM1 elution time; the problem can be overcome using gradient elution as follows: 23–48% acetonitrile in 9 min, then isocratic for 5 min; 48–70% acetonitrile in 1 min, then return to 23% and equilibrate for 7 min. The suitability of the P–HCl method was also tested by participating in a collaborative study for AFM1 determination in soft fresh cheese (Cattaneo et al., 2011). In this study, most of the participants used chloroform or dichloromethane to extract the toxin; however, the P–HCl method gave accurate results and the highest recovery values. 3.4. AFM1 extraction from naturally contaminated cheeses by chloroform and enzyme-assisted methods Twenty-four naturally contaminated cheese samples (13 parmesan, 5 caciotta, 4 crescenza and 2 mozzarella) were analysed for AFM1 using the CH and the EA extraction methods; the results
(Table 2) were corrected for recoveries. The values obtained by the two EA methods were similar for all the samples and, applying the paired t-test, no significant difference was observed (P = 0.88). Considering the hard cheeses (8–24 months of ripening), the statistical analysis showed that both the EA methods gave significantly higher results with respect to the CH method (P < 0.001); the same resulted for the semi-hard cheeses (1–3 months of ripening, Table 2 AFM1 values (lg kg 1, 3 replicates) and relative standard deviation (RSD) in naturally contaminated cheeses analysed using chloroform (CH) and enzyme-assisted [pepsin– HCl (P–HCl) and pepsin–pancreatin (PP)] extraction methods. CH extraction Mean (RSD)
P–HCl extraction Mean (RSD)
PP extraction Mean (RSD)
0.320 0.294 1.042 0.555 0.981 0.496 0.172 0.373 1.207 1.069
(3.3) (3.5) (2.2) (2.5) (1.9) (2.4) (3.5) (2.4) (2.3) (2.1)
0.469 0.424 1.520 0.881 1.439 0.776 0.235 0.462 1.572 1.380
(2.7) (3.2) (2.0) (2.3) (1.2) (1.4) (3.4) (1.7) (1.6) (1.4)
0.460 0.418 1.535 0.881 1.425 0.780 0.230 0.455 1.577 1.396
(3.4) (2.9) (1.9) (2.5) (1.5) (1.8) (3.9) (2.4) (1.9) (1.7)
Semi-hard cheese (parmesan and caciotta) 1 3 0.240 (5.0) 2 3 0.519 (2.9) 3 3 0.197 (3.5) 4 3 0.428 (2.6) 5 1 0.794 (2.1) 6 1 0.161 (3.1) 7 1 0.115 (3.8) 8 1 0.146 (3.3)
0.260 0.564 0.228 0.513 0.986 0.191 0.129 0.160
(4.2) (2.3) (2.6) (2.7) (2.6) (2.6) (2.3) (2.5)
0.258 0.558 0.225 0.515 0.972 0.187 0.130 0.157
(6.2) (3.2) (3.5) (2.9) (2.3) (3.2) (3.1) (3.2)
Soft fresh cheese (mozzarella and crescenza) 1 / 0.438 (2.7) 2 / 0.977 (2.6) 3 / 0.378 (3.0) 4 / 0.270 (3.2) 5 / 0.131 (3.4) 6 / 0.203 (3.1)
0.452 0.944 0.386 0.284 0.129 0.201
(2.6) (2.6) (2.9) (3.0) (3.2) (3.4)
0.458 0.962 0.366 0.289 0.121 0.213
(2.4) (2.9) (2.8) (3.0) (3.3) (3.0)
Cheese
Ripening time (months)
Hard cheese (parmesan) 1 24 2 24 3 18 4 18 5 18 6 18 7 12 8 12 9 8 10 8
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Table 3 Aflatoxin M1 (lg kg 1, 3 replicates) distribution and mass balance in two laboratory scale cheese-makings. Milk and whey were analysed using the AOAC method; curd and cheese were analysed using the chloroform (CH), the pepsin–HCl (P–HCl) and the pepsin–pancreatin (PP) extraction methods. 1st cheese-making Weight (kg) Milk Whey
*
Moisture (%)
20.0 17.7
Curd
CH PHCl PP
2.04
43.0
Cheese (3 months)
CH PHCl PP
1.80
35.4
Cheese (1 month)
CH PHCl PP
2nd cheese-making AFM1 (RSD)*
Amount (lg)
0.114 (2.4) 0.073 (2.2)
2.280 1.292
0.451 (2.7) 0.460 (2.8)
0.920 0.938
97.0 97.8
0.465 (2.6)
0.949
98.3
0.418 (2.8) 0.513 (2.7)
0.752 0.924
89.7 97.2
0.515 (2.7)
0.927
97.3
Mass balance (%)
Weight (kg)
Moisture (%)
20.0 17.5 2.01
1.92
41.1
38.5
AFM1 (RSD)*
Amount (lg)
0.230 (1.8) 0.138 (2.0)
4.600 2.415
1.007 (2.2) 1.011 (2.6)
2.024 2.032
96.5 96.7
1.014 (3.0)
2.038
96.8
0.834 (3.0) 1.026 (1.9)
1.601 1.970
87.3 95.3
1.012 (2.0)
1.943
94.7
Mass balance (%)
RSD = relative standard deviation.
P = 0.039 for P–HCl vs. CH method; P = 0.041 for PP vs. CH method). No difference between methods was found for the fresh soft cheeses. For the hard cheeses ripened for 18 months or more, the AFM1 results obtained by the EA methods were almost 50% higher than those by the CH method. These data indicated that chloroform does not completely extract AFM1 from ripened cheeses. 3.5. Evaluation of extraction efficiency in two cheese-making processes The results of two laboratory scale cheese-makings are reported in Table 3. For both curds, the AFM1 concentrations obtained by the 3 extraction methods were not significantly different (P = 0.397 and P = 0.948 for the 1st and the 2nd cheese making, respectively). Conversely, for both cheeses the values obtained by the EA methods were significantly higher than those by the CH method (P < 0.001 for both cheese-makings). Then, the AFM1 concentrations in cheese and curd were calculated on a dry matter basis and the values were compared. The data obtained by the CH method in cheese were significantly lower than those in curd (P < 0.001) in both cheese makings; on the contrary, no significant difference was found comparing the AFM1 concentrations obtained by the EA methods. The mass balance from milk to curd proved to be very good (>96%), independently of the AFM1 extraction method; the mass balance from milk to cheese was close to the previous one if calculated using data from the EA methods (94.7–97.3%), but lower using data from the CH method (87.3–89.7%). These results confirmed that the extraction efficiency of the EA methods was independent of ripening time of cheese, whereas the CH method was not able to fully recover AFM1 from ripened cheese. 4. Conclusions In our laboratory, we routinely use the P–HCl method, because it entails a single enzymatic step, that can be carried out overnight. In comparison with the standard CH method, the P–HCl method is simpler, avoiding solvent evaporation, dissolution and partition in a separating funnel; moreover, it gives higher recoveries, comparable LOD and LOQ and more accurate results in ripened cheeses. The new method simulates part of the digestion process, therefore the AFM1 released and then quantified is probably closer to the amount really available for in vivo absorption. Finally, the P–HCl
method does not use chlorinated solvents, resulting in both considerable environmental and economic advantage. References AOAC International. (2005a). Official Methods of Analysis of the International Association of Official Analytical Chemists. Natural Toxins. Aflatoxins, 49.3.02: 40–41. Method 980.21. 18th Edition, Gaithersburg, MD. AOAC International. (2005b). Official Methods of Analysis of the International Association of Official Analytical Chemists. Natural Toxins. Aflatoxins, 49.2.03: 4–5. Method 971.22. 18th Edition, Gaithersburg, MD. AOAC International. (2005c). Official Methods of Analysis of the International Association of Official Analytical Chemists. Natural Toxins. Aflatoxins, 49.3.07: 46–47. Method 2000.08. 18th Edition, Gaithersburg, MD. AOAC International. (2005d). Official Methods of Analysis of the International Association of Official Analytical Chemists. Animal Feed. Nitrogen, 4.4.04: 15– 16. Method 971.09. 18th Edition, Gaithersburg, MD. Bijl, J., Van Peteghem, C., & Dekeyser, D. (1987). Fluorimetric determination of aflatoxin M1 in cheese. Journal of the Association of Official Analytical Chemists, 70(3), 472–475. Brackett, R., & Marth, E. (1982). Fate of aflatoxins M1 in Parmesan and Mozzarella cheese. Journal of Protection, 45, 597–600. Cattaneo, T. M. P., Marinoni, L., Barzaghi, S., Cremonesi, K., & Monti, L. (2011). Testing the suitability of different high-performance liquid chromatographic methods to determine AFM1 in a soft fresh Italian cheese. Journal of Chromatography A, 1218, 4738–4745. Cavaliere, C., Foglia, P., Guarino, C., Marzioni, F., Nazzari, M., Samperi, R., et al. (2006). Aflatoxin M1 determination in cheese by liquid chromatography– tandem mass spectrometry. Journal of Chromatography A, 1135(2), 135–141. Colak, H., Hampikyan, H., Ulusoy, B., & Ergun, O. (2006). Comparison of a competitive ELISA with an HPLC method for the determination of aflatoxin M1 in Turkish White, Kasar and Tulum cheeses. European Food Research Technology, 223(6), 719–723. Cole, R. J., & Cox, R. H. (1981). Handbook of Toxic Fungal Metabolites. New York: Academic Press. 1–66. Commission of the European Communities. (2006). Commission Regulation (EC) No. 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal European Union, L70: 12-34. Commission of the European Communities. (2010). Commission Regulation (EC) No. 165/2010 of 26 February 2010 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards aflatoxins. Official Journal of the European Communities, L 50: 8-12. Dragacci, S., Gleizes, E., Fremy, J., & Candlish, A. (1995). Use of immunoaffinity chromatography as a purification step for the determination of aflatoxins M1 in cheeses. Food Additives and Contaminants, 12(1), 59–65. Elgerbi, A., Aidoo, K., Candlish, A., & Tester, R. (2004). Occurrence of aflatoxin M1 in randomly selected North African milk and cheese samples. Food Additives and Contaminants, 21(6), 592–597. Food and Agriculture Organization of the United Nations. (2004). Worldwide regulations for mycotoxins in food and feed in 2003. FAO Food and Nutrition Paper 81. www.fao.org/docrep/007/y5499e/y5499e00.htm. Govaris, A., Roussi, V., Koidis, P., & Botsoglou, N. (2001). Distribution and stability of aflatoxin M1 during processing, ripening and storage of Telemes cheese. Food Additives and Contaminants, 18(5), 437–443.
A. Pietri et al. / Food Chemistry 192 (2016) 235–241 IARC. (1993). Aflatoxins. IARC Monograph on the Evaluation of Carcinogenic Risks to Humans, 56: 489. IARC, Lyon, France: World Health Organization. IARC. (2002). Aflatoxins. IARC Monograph on the Evaluation of Carcinogenic Risks to Humans, 82: 171. IARC, Lyon, France: World Health Organization. IARC. (2012a). Aflatoxins. A Review of Human Carcinogens: Chemical Agents and Related Occupations. IARC Monograph on the Evaluation of Carcinogenic Risks to Humans, 100F: 225. IARC, Lyon, France: World Health Organization. IARC. (2012b).. Iha, M. H., Barbosa, C., Okada, I., & Trucksess, M. (2013). Aflatoxin M1 in milk and distribution and stability of aflatoxin M1 during production and storage of yoghurt and cheese. Food Control, 29, 1–6. Oruc, H., Cibik, R., Yilmaz, E., & Kalkanli, O. (2006). Distribution and stability of Aflatoxin M1 during processing and ripening of traditional white pickled cheese. Food Additives and Contaminants, 23(2), 190–195. Pietri, A., Bertuzzi, T., Bertuzzi, P., & Piva, G. (1997). Aflatoxin M1 occurrence in samples of Grana Padano cheese. Food Additives and Contaminants, 14(4), 341–344.
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Piva, G., Pietri, A., Galazzi, L., & Curto, O. (1988). Aflatoxin M1 occurrence in dairy products marketed in Italy. Food Additives and Contaminants, 5(2), 133–139. Sharman, M., Patey, A., & Gilbert, J. (1989). Application of an immunoaffinity column sample clean-up to the determination of aflatoxins M1 in cheese. Journal of Chromatography, 474, 457–461. Sigma-Aldrich. (2012). www.sigmaaldrich.com/life-science/metabolomics/enzymeexplorer/analytical-enzymes/pepsin.html. The Merck Index. (1983). Pancreatin, 6868: 1005. 10 ed., Published by Merck & Co., Inc., Rahway, NJ, USA. Van Egmond, H. P. (1989). Stability and degradation of aflatoxins M1. Mycotoxins in Dairy Products (11). London: Elsevier Science. Vervaeke, I., Dierick, N., Demeyer, D., & Decuypere, J. (1989). Approach to the energetic importance of fibre digestion in pigs. II. An experimental approach to hindgut digestion. Animal Feed Science and Technology, 23(1–3), 169–194. Yousef, A., & Marth, E. (1989). Stability and degradation of aflatoxins M1. In H. P. Van Egmond (Ed.), Mycotoxins in Dairy Products (pp. 127–161). London: Elsevier Science, London.
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Analytical Methods
Enzyme-assisted extraction for the HPLC determination of aflatoxin M1 in cheese Amedeo Pietri, Paola Fortunati, Annalisa Mulazzi, Terenzio Bertuzzi ⇑ Feed & Food Science and Nutrition Institute, Faculty of Agriculture, UCSC, Via Emilia Parmense, 84, 29122 Piacenza, Italy
a r t i c l e
i n f o
Article history: Received 24 February 2014 Received in revised form 24 March 2015 Accepted 5 July 2015 Available online 6 July 2015 Keywords: Aflatoxin M1 Cheese Enzyme-assisted extraction Pepsin Pancreatin
a b s t r a c t The extraction of aflatoxin M1 (AFM1) from cheese is generally carried out using chlorinated organic solvents. In this study, two innovative methods were developed, based on an enzyme-assisted (EA) extraction using proteolytic enzymes (pepsin or pepsin–pancreatin). After purification through an immunoaffinity column, AFM1 is determined by HPLC–FLD. A comparison between the new EA methods and an established chloroform (CH) method was carried out on 24 cheese samples. The results showed that the extraction efficiency of the EA methods was independent of ripening time of cheese, whereas the CH method was not able to fully recover AFM1 from ripened cheeses. The simpler (pepsin) of the two methods has been adopted by our laboratory for routine analysis of AFM1 in cheese. In comparison with the CH method, the pepsin-HCl (P-HCl) method is simpler, avoiding solvent evaporation, dissolution and partition in a separating funnel; moreover, it gives higher recoveries, comparable LOD and LOQ and more accurate results. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Lactating animals that have consumed feedstuffs contaminated with aflatoxin B1 (AFB1) excrete the 4-hydroxylated metabolite aflatoxin M1 (AFM1) into the milk. Aflatoxins are potent toxic and carcinogenic agents. Studies on the acute toxicity of aflatoxins in one-day old ducklings suggest that AFM1 and AFB1 act by a similar mechanism in causing subcellular alterations in liver parenchymal cells. Long term studies of toxicity and carcinogenicity in trout and rats have shown that AFM1 is a hepatic carcinogen, but with a potency 2–10% that of AFB1. Further studies have shown that AFM1 is genotoxic in mammalian systems in vivo. The International Agency for Research on Cancer (IARC) stated that there is sufficient evidence for the carcinogenicity of aflatoxins in humans and for the carcinogenicity of aflatoxin B1, G1 and M1 in experimental animals. AFM1 has been categorised as a class 2B, possible human carcinogen (IARC, 1993, 2002, 2012a, 2012b; Van Egmond, 1989). The toxin is associated with the casein fraction of milk by a hydrophobic interaction (Brackett & Marth, 1982; Van Egmond, 1989); hence it is present in cheese when AFM1 contaminated milk is used for cheese-making. AFM1 tends to partition in the range 40–60% between curd and whey; this partitioning depends upon which kind of cheese-making procedure is used ⇑ Corresponding author. E-mail address: [email protected] (T. Bertuzzi). http://dx.doi.org/10.1016/j.foodchem.2015.07.006 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
(Yousef & Marth, 1989). On account of this, the Commission of the European Communities (Commission of the European Communities, 2010) fixed a limit for AFM1 in milk and milk products (0.050 lg kg 1 for milk and a variable limit for milk products, depending on the concentration caused by the drying process or processing into cheese). Specific limits for AFM1 in cheese have been set by other countries (Food, 2004). Quantitative analytical methods for AFM1 in milk have been largely simplified by the introduction of immunoaffinity (IA) columns. An aliquot of milk is passed through an IA column and AFM1 is extracted by specific antibodies immobilised on a solid support material; a subsequent clean-up by water washing removes all other matrix components of the sample. AFM1 is then eluted from the column with a small volume of acetonitrile. Differently, quantitative analytical methods for AFM1 in cheese involve solvent extraction, filtration and then some clean-up stages prior to HPLC determination. In the clean-up step IA columns are widely used and have replaced adsorption or partition chromatography on pre-packed or prepared columns. Generally, the toxin is extracted with chloroform or dichloromethane (Bijl, Van Peteghem, & Dekeyser, 1987; Cavaliere et al., 2006; Colak, Hampikyan, Ulusoy, & Ergun, 2006; Dragacci, Gleizes, Fremy, & Candlish, 1995; Elgerbi, Aidoo, Candlish, & Tester, 2004; Piva, Pietri, Galazzi, & Curto, 1988; Sharman, Patey, & Gilbert, 1989). Then the chloroform or dichloromethane extract is evaporated to dryness under vacuum, and methanol, water and hexane are
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added to the residue. The mixture is transferred to a separating funnel and the lower layer is collected and purified through an IA column (Sharman et al., 1989). This method has been widely used and has been adopted as an official AOAC method (AOAC, 2005a). It showed a detection limit as low as 0.005 lg kg 1 (Pietri, Bertuzzi, Bertuzzi, & Piva, 1997) and sufficient repeatability, but AFM1 recovery values were generally lower in comparison with milk, ranging from 51% to 88% (Cattaneo, Marinoni, Barzaghi, Cremonesi, & Monti, 2011; Dragacci et al., 1995; Govaris, Roussi, Koidis, & Botsoglou, 2001; Iha, Barbosa, Okada, & Trucksess, 2013; Oruc, Cibik, Yilmaz, & Kalkanli, 2006), probably because of the more complex clean-up step. Finally, this method is time consuming and uses a chlorinated solvent. This method was used for many years in our laboratory; however, we noticed that hard cheeses, even if grated, did not completely lose their texture in chlorinated solvents and sometimes formed small aggregates (little balls) during shaking; this detail raised some doubts about a complete AFM1 extraction. But above all, we wondered whether chlorinated solvents were able to break up all the AFM1-casein hydrophobic interactions in cheese. Therefore, taking into account these considerations, we tried to develop a new method, based on an enzyme-assisted (EA) extraction of AFM1 and subsequent clean-up of the extract by an IA column, avoiding solvent evaporation, dissolution and partition in a separating funnel. Furthermore, as proteolytic enzymes cleave proteins, we hypothesised that casein digestion may facilitate the (hidden) toxin release into an aqueous solution. To this end, we applied two well-established in vitro digestibility methods, routinely used in our laboratory. A comparison between the new EA extraction and chloroform (CH) extraction methods was performed on naturally contaminated cheese samples. Finally, two laboratory-scale cheesemakings were carried out using naturally contaminated milk, in order to evaluate the accuracy of the methods from the AFM1 mass balance of the process. 2. Materials and methods 2.1. Samples In recent years, numerous samples of soft, semi-hard and hard cheeses collected from several cheese factories and retail outlets located in northern Italy, have been analysed in our laboratory for AFM1 determination. Considerable AFM1 concentrations were found in several samples, while some were uncontaminated; 300 g of cheese portions from contaminated and blank samples were collected and kept at 20 °C for this study. In detail, six hard cheese (parmesan) samples were used to compare the extraction efficiency of solvents (chloroform vs. dichloromethane) and apparatus (rotary-shaking stirrer vs. homogeniser). Two blank samples, one hard (parmesan) and one soft (crescenza) cheese, were used for recovery tests (the hard one) and for AFM1 stability kinetics during EA extraction (both samples). Two semi-hard (fontina and parmesan) and one hard (parmesan) cheese samples were used to determine the best length of time for EA extraction. Furthermore, 24 cheese samples (6 soft fresh, 8 semi-hard, 10 hard cheeses) were analysed to compare the EA and the CH extraction methods (Table 2). Finally, one blank and two naturally contaminated milk samples were used to compare the EA extraction with the official AOAC method. 2.2. Reagents Chemicals and solvents used for the extraction and clean-up solutions were ACS grade or equivalent (Carlo Erba, Milan, Italy).
Water used was deionised and, for HPLC, purified through a Milli-Q treatment system (Millipore, London, U.K.). For HPLC analysis, methanol and acetonitrile were HPLC grade (Merck, Darmstadt, Germany). Pepsin (from porcine gastric mucosa, 800– 2500 units/mg protein, cod P7000) and pancreatin (from porcine pancreas, activity equivalent to 4 USP, cod P1750) were obtained from Sigma–Aldrich (St. Louis, MO, USA). 2.3. Standard solution AFM1 standard (0.01 mg, purity degree > 98%) was obtained from Sigma–Aldrich. A stock solution of 4–6 lg ml 1 was prepared in acetonitrile (1.5–2 ml) and stored at 20 °C. The solution was calibrated spectrophotometrically at 350 nm using the value 19,000 L mol 1cm 1 for the absorption coefficient (AOAC, 2005b). For HPLC calibration or spiking purposes, the stock solution was diluted with the HPLC mobile phase, to obtain 7 working standard solutions at AFM1 concentration between 0.1 and 5 lg l 1. All standard solutions were stored at 20 °C when not in use. 2.4. Analysis of AFM1 in cheese The samples of semi-hard and hard cheeses were grated before analysis. 2.4.1. Chloroform (CH) standard method AFM1 was extracted according to the method reported by Pietri et al. (1997). Briefly, chloroform (75 ml), saturated sodium chloride (1 ml) and Celite 545 (5 g) were added to 20 g of cheese and the mixture was shaken for 45 min (160 rpm), using a rotary-shaking stirrer (Shaker 709, ASAL, Milan, Italy). Then, the mixture was filtered through a folded filter paper (5892 S&S, Whatman LTD, Kent, UK) and the volume was recorded. After evaporation of chloroform, the residue was re-dissolved in methanol (1.5 ml) water (50 ml) and hexane (50 ml). The mixture was transferred into a separating funnel and an aliquot (20 ml) of the lower layer was collected and purified through an IA column. In two preliminary experiments, we replaced chloroform with dichloromethane and the rotary-shaking stirrer with an Ultraturrax T-25 homogeniser (14,000 rpm for 5 min). 2.4.2. Pepsin–HCl (P–HCl) method In a 250 ml plastic centrifuge bottle, 50 ml of pepsin solution (0.2% pepsin dissolved in 0.075 M HCl), was added to 5 g of cheese. The pH was measured and eventually adjusted to between 2.6 and 2.8 with 0.1 M NaOH or 0.1 M HCl; then, the mixture was stirred with a magnetic stirrer in a thermostatic chamber at 42 °C for 16 h (overnight). After cooling at ambient temperature, the extract was adjusted to pH 7.4 ± 0.2 with 1 M NaOH (3–5 ml) and the volume was recorded; then, the mixture was centrifuged at 11,700g for 10 min at 4 °C and filtered through a folded filter paper. An aliquot of the sample extract (20 ml) was purified through an IA column. 2.4.3. Pepsin–pancreatin (PP) method In a 250 ml plastic centrifuge bottle, 40 ml of pepsin solution (0.2% pepsin dissolved in 0.075 M HCl), was added to 3 g of cheese. The pH was controlled and eventually adjusted to between 2.6 and 2.8 with 0.1 M NaOH or 0.1 M HCl; then, the mixture was stirred with a magnetic stirrer in a thermostatic chamber at 37 °C for 3 h and 45 min. After cooling at ambient temperature, the extract was adjusted to pH 7.4 ± 0.2 with 1 M NaOH (3–5 ml) and 40 ml of pancreatin solution [1% pancreatin dissolved in 0.2 M phosphate buffer (27.60 g NaH2PO4 H2O + 28.38 g Na2HPO4 in 1 l, pH 7.4)] was added. Then, the mixture was again stirred in a thermostatic chamber at 37 °C for 3 h and 45 min. After cooling at ambient
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temperature, the volume was recorded; then, the mixture was centrifuged at 11,700g for 10 min at 4 °C and filtered through a folded filter paper. An aliquot of the sample extract (30 ml) was purified through an IA column. 2.5. Analysis of AFM1 in milk
2.5.1. AOAC method AFM1 was extracted and purified as in the AOAC Official Method (AOAC, 2005c). 2.5.2. Pepsin–HCl method An aliquot (100 ml) of milk was centrifuged at 7000g for 10 min at 4 °C; then, the thin upper fat layer was removed and the skimmed milk was filtered through a folded filter paper. In a 50 ml plastic centrifuge bottle, 20 ml of pepsin solution (0.2% pepsin dissolved in 0.075 M HCl), was added to 20 g of filtered milk. The pH was adjusted to between 2.6 and 2.8 with 3 M HCl and the solution was stirred with a magnetic stirrer in a thermostatic chamber at 42 °C for 16 h. After cooling at ambient temperature, the mixture was adjusted to pH 7.4 ± 0.2 with 1 M NaOH and the volume was recorded; then, the mixture was centrifuged (11,700g for 10 min at 4 °C) and filtered through a folded filter paper. An aliquot (20 ml) of the extract was purified through an IA column. 2.6. Clean-up by immunoaffinity column The IA column (Easy-Extract Aflatoxin, R-Biopharm Rhône LTD, Glasgow, Scotland, UK) was placed on a SPE vacuum manifold (Visiprep, Supelco, Bellefonte, PE, USA). The sample extract, prepared as described above, was applied to the column, followed by a washing with distiled water (5 ml). Then, AFM1 was slowly eluted (0.5 ml min 1) from the column with methanol (2.5 ml) into a graduated glass vial; the eluate was evaporated under a gentle stream of nitrogen and re-dissolved in 1 ml acetonitrile:water (25 + 75, v/v) by ultrasonication. The extract was filtered (HV 0.45 lm, Millipore Corporation, Bedford, MA, USA) before HPLC analysis.
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Initially, a recovery test on cheese was performed for CH, P–HCl and PP methods; the recovery values were determined by spiking a blank sample of hard cheese (parmesan) with an appropriate volume of AFM1 standard solution, to obtain contamination levels of 0.25, 0.50 and 1.00 lg kg 1. Successively, a recovery test on milk was also performed (only for CH and P–HCl methods), spiking a blank sample of milk to obtain levels of 0.050, 0.100 and 0.250 lg kg 1. Three replicates were analysed for each level and matrix. The recovery values were compared using the GLM procedure and HSD Tukey post hoc test (SPSS 18.0) considering as factors: method (CH, P-HCl and PP) and level (0.25, 0.50 and 1.00 lg kg 1). Finally, the CH, P-HCl and PP extraction methods were tested on 6 fresh soft, 8 semi-hard (1–3 months of ripening) and 10 hard (8– 24 months of ripening) naturally contaminated cheese samples (Table 2). Each sample was analysed using the 3 methods on the same day and 3 replicate analyses were carried out on different days. For soft, semi-hard and hard cheeses, the results were compared separately using the paired t-test (SPSS 18.0). 2.9. Cheese-making Two cheese-makings were carried out. After pasteurisation at 70 °C for 2 min and subsequent cooling up to 30 °C, 20 kg of naturally contaminated milk was treated with Streptococcus thermofilus, bulgaricus and helveticus (0.6 g) and rennet (chimosine 80%, 5 ml). After complete curdling, the curd was collected and kept at 45 °C for 10 h and successively at 25 °C for 14 h. Then, the curd was placed in a NaCl saturated aqueous solution for 1 h and finally ripened at 4–6 °C. The ripening time was different: 3 months and 1 month for the curd obtained from the first and the second cheese-making, respectively. Milk and whey were analysed using the AOAC method; curd and cheese were analysed using CH and EA extraction methods. All the analyses were carried out in triplicate. The AFM1 concentrations in curd and cheese obtained with the three methods were compared by ANOVA analysis (SPSS 18.0). For each cheese-making, the weight of milk, whey, curd and cheese was recorded and the AFM1 mass balance of the process was calculated. 3. Results and discussion
2.7. HPLC analysis 3.1. Performances of the standard method The HPLC system consisted of a Jasco PU 1580 pump (Jasco Corp., Tokyo, Japan) equipped with an AS 1555 sampling system and a FP 1520 fluorescence detector set at 365 nm excitation and 440 nm emission wavelength. The system was governed by Borwin 1.5 software (Jasco). AFM1 was separated on a Lichrospher RP-18 column (5 lm particle size, 125 4 mm i.d., Merck, Darmstadt, Germany) at ambient temperature (22 °C), with a mobile phase water:acetonitrile (75 + 25, v/v); the flow rate was 1.0 ml min 1. The injection volume for both standard solutions and sample extracts was 100 ll, corresponding to about 0.64, 0.18 and 0.11 g of cheese sample using the CH, P–HCl and PP methods, respectively. 2.8. Performances of the methods The limits of detection (LOD) and quantification (LOQ) of the methods were determined by the signal-to-noise approach, defined at those levels resulting in signal-to-noise ratios of 3 and 10, respectively. The analytical response and the chromatographic noise were both measured from the chromatogram of a blank sample extract (1 ml) to which between 40 and 100 ll of an AFM1 solution (0.428 lg l 1) had been added.
The AFM1 extraction efficiency of chloroform and dichloromethane, tested on four hard cheese samples, was very similar (0.365 vs. 0.373, 0.180 vs. 0.172, 0.017 vs. 0.019, 0.405 vs. 0.414 lg kg 1, respectively); likewise, the extraction efficiency of the stirrer and the homogeniser, tested on two hard cheese samples using chloroform as solvent, was very close (0.981 vs. 0.964, 0.496 vs. 0.480 lg kg 1, respectively). Therefore the CH method, as described in Section 2.4.1, was used in the comparison trials, 3.2. Development of a new extraction method In order to develop an enzyme-assisted extraction of AFM1 from cheese, the AOAC 971.09 method – Pepsin Digestibility of Animal Protein Feeds – (AOAC, 2005d), was first considered. This method involves an enzymatic digestion with pepsin for 16 h; pepsin is the principal digestive enzyme of gastric juice and is distinctive among enzymes because it exhibits maximal activity at pH 2.2; however, at pH 4.5 it still shows about 35% of its maximal activity (Sigma-Aldrich, 2012). AFM1 is slightly soluble in water (about 10– 30 lg ml 1, Cole & Cox, 1981), but this concentration is a thousand times higher than the levels occurring in naturally contaminated
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Fig. 1. Aflatoxin M1 levels (3 replicates) in 3 naturally contaminated cheese samples analysed using the pepsin–HCl extraction method at different times (h) of enzymatic digestion.
milk and in the digested cheese solutions obtained with this method. However, it is known that at very low pH values AFM1 is converted to the emiacetal AFM2a. In order to verify if this reaction can occur during the EA extraction, two AFM1 standard solutions (0.8 and 8 lg l 1) were prepared in the pepsin–HCl solution (pH 1.1); then, a stability kinetics of the standard solutions was performed in a thermostatic chamber at 37 and 42 °C. The AFM1 concentration decreased (consequently AFM2a increased) in the solutions; the loss was 4% after 4 h at both temperatures, 15% and 22% after 16 h at 37 and 42 °C, respectively. The stability kinetics was repeated adding 5 g of uncontaminated soft (crescenza) or hard (parmesan) cheese to 50 ml of AFM1 standard solutions prepared as above. The pH values were slightly higher and depended on the type of cheese; the values were 2.0 ± 0.2 and 2.4 ± 0.2 for the soft cheese, 2.6 ± 0.2 and 3.1 ± 0.2 for the hard cheese, after 0 and 16 h at 42 °C, respectively. The AFM1 concentrations after 16 h decreased by 11% and 0% for the soft and the hard cheese, respectively. The experiment was repeated for the soft cheese after adjusting the pH between 2.6 and 2.8 and no AFM1 loss was observed after 16 h. Therefore, it is important that the pH value of the extraction solution be initially not less than 2.6–2.8. However, in the case of hard cheeses ripened more than 20 months, the pH value of the solution was always higher than 3.0 and for some samples close to 4.5. Consequently, we decided to adjust the pH between 2.6 and 2.8 for all samples, to be near the optimum pH of pepsin activity and to avoid AFM1 losses. Successively, a study was performed to choose the best length of time for EA extraction; the AFM1 concentration in two semi hard
cheeses, ripened for 3 months (fontina and parmesan) and in one hard cheese (parmesan) ripened for 16 months, was determined after 2, 4, 6, 8 and 16 h (Fig. 1). Fontina and parmesan were chosen because they are produced with whole and partly skimmed milk, respectively. The AFM1 recovery after 2 h, with respect to the final level, was 84% for the fontina cheese, and 90% and 70%, for the parmesan cheeses ripened for 3 and 16 months, respectively. Moreover, the solutions after centrifugation were cloudy and the purification step through the IA column was very slow. After 8 h, the results were similar to those after 16 h, but in the case of fontina the solution was still cloudy. As a consequence, a digestion time of 16 h (overnight) was maintained for the P–HCl method. Successively, AFM1 was extracted from the same samples using only 0.075 M HCl (without pepsin); the values were very low compared to those obtained by the P-HCl method. In a further trial, the amount of cheese for analysis was increased to10 g; the final extract was cloudy and the AFM1 concentration resulted lower than that obtained weighing 5 g. The P–HCl method was also tested on milk, in comparison with the AOAC 2000.08 method. Two naturally contaminated milk samples were analysed; the results (0.083 vs. 0.076, 0.131 vs. 0.124 lg kg 1 using the AOAC and the P–HCl method, respectively) were similar, only slightly higher for the AOAC method. Besides the P–HCl method, another EA procedure (PP method) based on the method of Vervaeke, Dierick, Demeyer, and Decuypere (1989) was tested. The method entails two subsequent EA extractions (both for 3 h and 45 min), the first using pepsin at pH between 2.6 and 2.8, the second using pancreatin at pH 7.4 ± 0.2. Pancreatin is a substance containing many enzymes, including amylase, trypsin, protease, ribonuclease and lipase; its greatest activity is exhibited in neutral or slightly alkaline media (The Merck Index, 1983). The PP method was compared to the P– HCl and the CH methods for AFM1 analysis in 24 cheese samples (Table 2 and paragraph 3.4 for results). Finally, AFM1 was extracted from 6 cheese samples by a digestion using only pancreatin (16 h at 37 °C, pH 7.4 ± 0.2). After EA extraction and centrifugation, the solution was cloudy and the purification step through the IA column was not possible for 2 samples (clogged column); for the other 4 samples, the results were lower than those obtained by the PP method.
3.3. Performances of the chloroform and the enzyme-assisted extraction methods The average recoveries of the CH, P–HCl and PP methods were between 87.1% and 96.3% for cheese and those of the AOAC and P–HCl methods were higher than 96.9% for milk (Table 1), with satisfactory relative standard deviation (RSD) values; the performance
Table 1 Average recovery (3 replicates) and relative standard deviation (RSD) of AFM1 from blank hard cheese and milk samples spiked at different levels, using chloroform (CH) and enzyme-assisted [pepsin–HCl (P–HCl) and pepsin–pancreatin (PP)] extraction methods. Spiking level (lg kg
1
)
0.25
0.50
1.00
Method
Recovery %
RSD
Recovery %
RSD
Recovery %
RSD
Cheese CH extraction P–HCl extraction PP extraction
87.5 96.3 94.3
2.0 1.5 1.4
88.5 95.4 93.5
1.8 1.4 1.7
87.1 95.5 94.0
2.3 1.6 1.5
Method
0.050 Recovery %
RSD
0.100 Recovery %
RSD
0.250 Recovery %
RSD
Milk AOAC 2000.08 P–HCl extraction
98.9 97.6
0.8 1.1
98.6 96.9
1.0 1.2
97.4 97.0
0.9 0.9
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A. Pietri et al. / Food Chemistry 192 (2016) 235–241
Fig. 2. Chromatograms (HPLC; fluorescence detection) of: (a) an AFM1 standard solution (1.23 lg L 1) equivalent to 123 pg of AFM1 injected; (b) a naturally contaminated cheese sample extracted by pepsin–HCl method, containing 0.776 lg kg 1 (equivalent to 155.2 pg of AFM1 injected); (c) a naturally contaminated cheese sample extracted by chloroform method, containing 0.496 lg kg 1 (equivalent to 238.1 pg of AFM1 injected). Injection volume 100 ll.
criteria fixed by Regulation (EC) 401/2006 of the Commission of the European Communities, i.e., recovery in the range 50–120% and 70–110% for levels <1 lg kg 1 and in the range 1–10 lg kg 1, respectively (Commission of the European Communities, 2006), were completely fulfilled. The statistical analysis showed that significantly higher mean recoveries from cheese were obtained by the EA methods compared to the CH method (95.7% vs. 87.7% for P–HCl vs. CH, P < 0.001; 93.9% vs. 87.7% for PP vs. CH, P < 0.001); a small but significant difference was also found between the P– HCl and the PP method (P = 0.041). No significant difference between AFM1 levels and no interaction among factors were observed. The LOD and LOQ in cheese resulted 0.005 and 0.015 lg kg 1 for the CH method, 0.015 and 0.040 lg kg 1 for the P–HCl method, 0.020 and 0.060 lg kg 1 for the PP method, respectively; the LOD and LOQ in milk were 0.001 and 0.003 lg l 1 for the AOAC method, 0.002 and 0.006 lg l 1 for the P–HCl method, respectively. Concerning HPLC analysis, Fig. 2 shows the chromatograms of an AFM1 standard solution and of a naturally contaminated hard cheese sample, extracted by both the P–HCl and the CH method. In the chromatogram of some cheese samples, irrespective of the extraction method used, we observed an interfering peak near the AFM1 elution time; the problem can be overcome using gradient elution as follows: 23–48% acetonitrile in 9 min, then isocratic for 5 min; 48–70% acetonitrile in 1 min, then return to 23% and equilibrate for 7 min. The suitability of the P–HCl method was also tested by participating in a collaborative study for AFM1 determination in soft fresh cheese (Cattaneo et al., 2011). In this study, most of the participants used chloroform or dichloromethane to extract the toxin; however, the P–HCl method gave accurate results and the highest recovery values. 3.4. AFM1 extraction from naturally contaminated cheeses by chloroform and enzyme-assisted methods Twenty-four naturally contaminated cheese samples (13 parmesan, 5 caciotta, 4 crescenza and 2 mozzarella) were analysed for AFM1 using the CH and the EA extraction methods; the results
(Table 2) were corrected for recoveries. The values obtained by the two EA methods were similar for all the samples and, applying the paired t-test, no significant difference was observed (P = 0.88). Considering the hard cheeses (8–24 months of ripening), the statistical analysis showed that both the EA methods gave significantly higher results with respect to the CH method (P < 0.001); the same resulted for the semi-hard cheeses (1–3 months of ripening, Table 2 AFM1 values (lg kg 1, 3 replicates) and relative standard deviation (RSD) in naturally contaminated cheeses analysed using chloroform (CH) and enzyme-assisted [pepsin– HCl (P–HCl) and pepsin–pancreatin (PP)] extraction methods. CH extraction Mean (RSD)
P–HCl extraction Mean (RSD)
PP extraction Mean (RSD)
0.320 0.294 1.042 0.555 0.981 0.496 0.172 0.373 1.207 1.069
(3.3) (3.5) (2.2) (2.5) (1.9) (2.4) (3.5) (2.4) (2.3) (2.1)
0.469 0.424 1.520 0.881 1.439 0.776 0.235 0.462 1.572 1.380
(2.7) (3.2) (2.0) (2.3) (1.2) (1.4) (3.4) (1.7) (1.6) (1.4)
0.460 0.418 1.535 0.881 1.425 0.780 0.230 0.455 1.577 1.396
(3.4) (2.9) (1.9) (2.5) (1.5) (1.8) (3.9) (2.4) (1.9) (1.7)
Semi-hard cheese (parmesan and caciotta) 1 3 0.240 (5.0) 2 3 0.519 (2.9) 3 3 0.197 (3.5) 4 3 0.428 (2.6) 5 1 0.794 (2.1) 6 1 0.161 (3.1) 7 1 0.115 (3.8) 8 1 0.146 (3.3)
0.260 0.564 0.228 0.513 0.986 0.191 0.129 0.160
(4.2) (2.3) (2.6) (2.7) (2.6) (2.6) (2.3) (2.5)
0.258 0.558 0.225 0.515 0.972 0.187 0.130 0.157
(6.2) (3.2) (3.5) (2.9) (2.3) (3.2) (3.1) (3.2)
Soft fresh cheese (mozzarella and crescenza) 1 / 0.438 (2.7) 2 / 0.977 (2.6) 3 / 0.378 (3.0) 4 / 0.270 (3.2) 5 / 0.131 (3.4) 6 / 0.203 (3.1)
0.452 0.944 0.386 0.284 0.129 0.201
(2.6) (2.6) (2.9) (3.0) (3.2) (3.4)
0.458 0.962 0.366 0.289 0.121 0.213
(2.4) (2.9) (2.8) (3.0) (3.3) (3.0)
Cheese
Ripening time (months)
Hard cheese (parmesan) 1 24 2 24 3 18 4 18 5 18 6 18 7 12 8 12 9 8 10 8
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Table 3 Aflatoxin M1 (lg kg 1, 3 replicates) distribution and mass balance in two laboratory scale cheese-makings. Milk and whey were analysed using the AOAC method; curd and cheese were analysed using the chloroform (CH), the pepsin–HCl (P–HCl) and the pepsin–pancreatin (PP) extraction methods. 1st cheese-making Weight (kg) Milk Whey
*
Moisture (%)
20.0 17.7
Curd
CH PHCl PP
2.04
43.0
Cheese (3 months)
CH PHCl PP
1.80
35.4
Cheese (1 month)
CH PHCl PP
2nd cheese-making AFM1 (RSD)*
Amount (lg)
0.114 (2.4) 0.073 (2.2)
2.280 1.292
0.451 (2.7) 0.460 (2.8)
0.920 0.938
97.0 97.8
0.465 (2.6)
0.949
98.3
0.418 (2.8) 0.513 (2.7)
0.752 0.924
89.7 97.2
0.515 (2.7)
0.927
97.3
Mass balance (%)
Weight (kg)
Moisture (%)
20.0 17.5 2.01
1.92
41.1
38.5
AFM1 (RSD)*
Amount (lg)
0.230 (1.8) 0.138 (2.0)
4.600 2.415
1.007 (2.2) 1.011 (2.6)
2.024 2.032
96.5 96.7
1.014 (3.0)
2.038
96.8
0.834 (3.0) 1.026 (1.9)
1.601 1.970
87.3 95.3
1.012 (2.0)
1.943
94.7
Mass balance (%)
RSD = relative standard deviation.
P = 0.039 for P–HCl vs. CH method; P = 0.041 for PP vs. CH method). No difference between methods was found for the fresh soft cheeses. For the hard cheeses ripened for 18 months or more, the AFM1 results obtained by the EA methods were almost 50% higher than those by the CH method. These data indicated that chloroform does not completely extract AFM1 from ripened cheeses. 3.5. Evaluation of extraction efficiency in two cheese-making processes The results of two laboratory scale cheese-makings are reported in Table 3. For both curds, the AFM1 concentrations obtained by the 3 extraction methods were not significantly different (P = 0.397 and P = 0.948 for the 1st and the 2nd cheese making, respectively). Conversely, for both cheeses the values obtained by the EA methods were significantly higher than those by the CH method (P < 0.001 for both cheese-makings). Then, the AFM1 concentrations in cheese and curd were calculated on a dry matter basis and the values were compared. The data obtained by the CH method in cheese were significantly lower than those in curd (P < 0.001) in both cheese makings; on the contrary, no significant difference was found comparing the AFM1 concentrations obtained by the EA methods. The mass balance from milk to curd proved to be very good (>96%), independently of the AFM1 extraction method; the mass balance from milk to cheese was close to the previous one if calculated using data from the EA methods (94.7–97.3%), but lower using data from the CH method (87.3–89.7%). These results confirmed that the extraction efficiency of the EA methods was independent of ripening time of cheese, whereas the CH method was not able to fully recover AFM1 from ripened cheese. 4. Conclusions In our laboratory, we routinely use the P–HCl method, because it entails a single enzymatic step, that can be carried out overnight. In comparison with the standard CH method, the P–HCl method is simpler, avoiding solvent evaporation, dissolution and partition in a separating funnel; moreover, it gives higher recoveries, comparable LOD and LOQ and more accurate results in ripened cheeses. The new method simulates part of the digestion process, therefore the AFM1 released and then quantified is probably closer to the amount really available for in vivo absorption. Finally, the P–HCl
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