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Fate of aflatoxin M1 during production and storage of parmesan cheese
Food Control 60 (2016) 478e483
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Fate of aflatoxin M1 during production and storage of parmesan cheese Amedeo Pietri, Annalisa Mulazzi, Gianfranco Piva, 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
a b s t r a c t
Article history: Received 7 July 2015 Received in revised form 20 August 2015 Accepted 25 August 2015 Available online 29 August 2015
The fate of AFM1 during production of a long maturing cheese (Parmesan cheese) was assessed. Different levels of AFM1 contamination and of the fat/casein (F/C) ratio of milk were considered, in order to evaluate if these factors can influence the enrichment factor (EF) of AFM1 in cheese. For this purpose, 24 cheese-makings were carried out using naturally contaminated milk at 3 different AFM1 levels and at 2 F/ C ratios. AFM1 analysis was performed by HPLC in raw milk, cream, cauldron milk, liquid cattle rennet, whey, curd and cheese at 3, 9, 16 and 24 months of ageing. The mass balances of the cheese-making processes were close to 100%; in whey, AFM1 concentration was about 40% less than the concentration in cauldron milk. The EF in curd was between 4.0 and 5.2, with an average value of 4.7 ± 0.4; this factor was not significantly affected by either AFM1 contamination level or F/C ratio. During maturation, AFM1 concentration and consequently EF increased from curd to 16 ageing months; successively, AFM1 slightly decreased at 24 months and consequently the EF. At 3, 9, 16 months of maturation, the EF was significantly higher for cheeses prepared using milk with low F/C than those with high F/C milk; on the contrary, EF was not significantly influenced by the AFM1 contamination level. In cheeses, EF values were between 4.7 and 6.3; from these results, the maximum admissible level for AFM1 in Parmesan cheese should be about 0.275 mg kg1. © 2015 Elsevier Ltd. All rights reserved.
Keywords: AFM1 Parmesan cheese Enrichment factor
1. Introduction Lactating animals that have consumed feedstuffs contaminated with aflatoxin B1 (AFB1), excrete the 4-hydroxylated metabolite aflatoxin M1 (AFM1) into the milk. This toxin has shown acute toxic effects similar to AFB1; long term studies of toxicity and carcinogenicity in trout and rats showed that AFM1 is a hepatic carcinogen, but with a potency 2e10% that of AFB1. Further studies showed 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). AFM1 occurs in cheese when AFM1 contaminated milk is used for cheese-making; the partition between curd and whey is generally in the range 40e60%. AFM1 is associated with the protein fraction of milk by a hydrophobic interaction (Mendonca & Venancio, 2005; Van Egmond,
* Corresponding author. E-mail address: [email protected] (T. Bertuzzi). http://dx.doi.org/10.1016/j.foodcont.2015.08.032 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
1989); consequently, the concentration is higher in cheese than in the milk from which the cheese is made. The ratio between the concentration of AFM1 in cheese and the concentration in milk is called the enrichment factor (EF). The EF is generally higher for hard than for soft cheeses; this difference is mainly caused by the drying process of the cheese and by the AFM1 stability during maturation. On the basis of several studies, it was concluded that the EF is 2.5e3.3 in many soft cheeses and 3.9e5.8 in hard cheeses (Yousef & Marth, 1989). These ranges were generally confirmed in more recent studies (Cattaneo et al. 2008; Colak, 2007; Deveci, ^a, Rosim, Kobashigawa, & Oliveira, 2012; 2007; Fernandes, Corre Iha, Barbosa, Okada, & Trucksess, 2013; Kamkar, Karim, Aliabadi, & Khaksar, 2008; Oruc, Cibik, Yilmaz, & Kalkanli, 2006, 2007). However, EF values of 1.4, 2.2 and 6.7 were calculated for two soft and one hard cheeses, respectively (Cavallarin, Antoniazzi, Giaccone, Tabacco, & Borreani, 2014). The different EF values found mainly depend on the type of cheese-making process; but the origin (natural or artificial) of milk contamination and the methods of analysis may also have influenced the results. The Commission of the European Communities (Commission of the European Communities, 2006a) fixed an AFM1 maximum level
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in milk (0.050 mg/kg); for dairy products, the limit must be calculated taking into account the changes of AFM1 concentration caused by processing. The specific EF must be provided and justified by the food business operator. In Italy, the production of long maturing cheeses is of considerable economic importance in the food chain; in the Po valley (northern Italy), about 70% of cow's milk produced is used to make Grana Padano and Parmigiano Reggiano cheeses (about 7,350,000 cheese wheels/year; the weight of each wheel is about 38 kg and approximately 550 kg of partially skimmed milk is used for each wheel). Grana Padano and Parmigiano Reggiano are PDO cheeses produced in different areas using very similar technology. They must be matured for at least 9 and 12 months, respectively; however, the ageing period is often longer. Two Consortia supervise the production of the two cheeses. In recent years, but particularly in 2003 and 2012, remarkable AFB1 contamination occurred in maize produced in northern Italy, caused by high temperatures and drought during the summer; since maize grain is used in dairy rations, AFM1 levels in milk increased. In 2004, the Italian Ministry of Health fixed a provisional limit of 0.45 mg kg1 for hard and long maturing cheeses (Italian Ministry of Health, 2004); in 2013, the same Ministry suggested an EF of 3.0 and 5.5 for soft and hard cheeses, respectively (Italian Ministry of Health, 2013). In the scientific literature, there are only two studies on AFM1 distribution during parmesan-type cheese production. In the first, the authors (Zarenghi et al., 2008) analysed 29 wheels of Grana Padano aged 9e24 months and produced with AFM1 contaminated milk ranging from 0.056 to 0.28 mg/kg; they found very variable EFs, ranging from 0.7 to 7.7, with a mean value of 2.8. In the second, the authors (Manetta et al., 2009), in a two month period, collected in some commercial dairies 25 samples of contaminated milk (range 30e98 mg/kg of AFM1) destined for Grana Padano production and the same number of the respective whey, curd and cheese samples (after 12 months of ageing); they calculated an EF of about 4.5 for cheese. However, in both studies the mass balance of AFM1 during cheese-making was not controlled and consequently the calculated EF may be inaccurate. The present study investigated the fate of AFM1 during cheesemaking for the production of parmesan-type cheese and during the ageing time. For this purpose, 24 cheese-makings were carried out in an experimental cheese factory using naturally contaminated milk at 3 different AFM1 levels, (0.030, 0.065 and 0.080 mg kg1) and at 2 fat/casein (F/C) ratios, high (H) and low (L) [about 1.10e1.15 (H) and 0.85e0.90 (L)], in order to evaluate if these factors may influence the EF. The mass balance and the EF of the cheese-making were calculated; the EF was also calculated after 3, 9, 16 and 24 months of ageing.
2. Materials and methods 2.1. Production of naturally contaminated maize Maize grain (20 kg) was moistened by the addition of distilled water to obtain a moisture value close to 40% (v/w), distributed in several 1000 ml beakers and contaminated with a high aflatoxin producing strain of Aspergillus flavus; then, maize was incubated in a thermostatic chamber at 35 C for 2 weeks. Successively, maize was desiccated at 65 C overnight, ground (taking particular precautions) using a cyclone hammer mill (1 mm sieve; Pulverisette, Fritsch GmbH, Idar-Oberstein, Germany) and homogenised. Four aliquots were analysed for AFB1 content using HPLC with fluorescence detection; the level was very high, close to 100 mg kg1.
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2.2. Production of naturally contaminated milk The production of naturally contaminated milk, the cheesemaking and the ageing of wheels were carried out at ERSAF experimental farm (Ente regionale per i Servizi all'Agricoltura e alle Foreste, Mantova, Lombardia). The herd consisted of 42 milking cows; they were milked twice a day and the total mixed ration was distributed every day after morning milking. The contaminated maize was added and homogenised first with the concentrates and then with all the ingredients of the total mixed ration, to give the required AFB1 level. The animals were fed rations containing the AFB1 naturally contaminated maize flour for 21 days. In order to obtain the desired milk contamination levels, the experimental period was divided into 3 sub-periods of 7 days; increasing amounts of the maize flour were added to the daily rations, so that each cow could receive a calculated AFB1 amount of about 25, 45 and 65 mg day1 in the 1st, 2nd and 3rd sub-period, respectively. The aim was to obtain a herd bulk milk at AFM1 levels close to 0.030, 0.055 and 0.080 mg kg1, during the 3 sub-periods, respectively. The formula: AFM1 (ng kg1 milk) ¼ 1.19 AFB1 intake (mg per cow per day) þ 1.9 (Veldman, Meijs, Borggreve, & Heeres-van der Tol, 1992) was used to calculate the required amount of AFB1 in the ration, in order to have the desired AFM1 level in the bulk milk. 2.3. Cheese-making In each 7-day sub-period, starting from the 4th day, milk from evening milking was mixed with that of the following morning and used for cheese-making. Milk (about 950 kg) was poured into two holding basins provided with refrigerating coils and refrigerated at 11e12 C; separation of cream took place naturally (10e12 h) in the basins. The milk was partially skimmed in order to obtain a level of fat in the range 2.8e3.0% (high F/C ratio, close to 1.10e1.15) in one basin and 2.2e2.4% (low F/C ratio, close to 0.85e0.90) in the other one; 0.80e1.15 is the allowed F/C range for the production of Grana Padano. Then, the partially skimmed milk from the two basins was separately poured into two copper cauldrons for cheese-making. Eight cheese-makings were carried out in each sub-period and a total of 24 wheels were produced in the experimental period. After warming the milk in the cauldron at 34 C, naturally fermented whey (obtained from the cheese-making of the previous day, 13e16 kg), natural calf rennet [35 mg kg1, title 1:125,000 with high chymosin content (96%)] and lysozyme (18 mg kg1) were added to allow the milk to curdle. The cheese making process was carried out using traditional methods by technicians of the two Consortia (Grana Padano and Parmigiano Reggiano). 2.4. Analysis of samples AFM1 analysis was performed in raw milk, cream, cauldron milk, liquid cattle rennet, whey, curd and cheese at 3, 9, 16 and 24 months of ageing; the weight (kg) of these products was recorded to calculate the mass balance of AFM1. Milk was also analysed for fat, lactose and casein content; curd and cheese were analysed for moisture and fat (Milkoscan FT6000, Foss Italy). The sampling of cheese, made after scraping of the rind (1e2 mm), was carried out by means of a small corer (length 14 cm). In a previous study, we demonstrated that AFM1 is homogeneously distributed within the wheel (Pecorari et al., 2009). All samples were kept at 20 C until analysis. 2.5. Reagents Chemicals and solvents used for AFM1 extraction and clean-up solutions were ACS grade or equivalent (Carlo Erba, Milan, Italy).
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All 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, 800e2500 units/mg protein, cod P7000) was obtained from SigmaeAldrich (St. Louis, MO, USA).
using the post-hoc Tukey Test (a ¼ 0.05). For the cheese-making process, the mean AFM1 concentrations in curd were evaluated considering as fixed factors the F/C ratio and the AFM1 contamination level in milk; for the cheese ripening process, the mean EF values, calculated also on a dry matter basis, were compared considering as fixed factors the F/C ratio, the ripening time and the AFM1 contamination level in milk.
2.6. Standard solution AFM1 standard (0.01 mg, purity degree higher than 98%) was obtained from SigmaeAldrich (St. Louis, MO, USA). A stock solution of 4e6 mg ml1 was prepared in acetonitrile (1.5e2 ml) and stored at 20 C (AOAC 2005a). The solution was calibrated spectrophotometrically at 350 nm using the value 19,000 l mol1 cm1 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 0.05 and 5 mg l1. All standard solutions were stored at 20 C when not in use. 2.7. Analysis for aflatoxin M1 AFM1 analysis was carried out by HPLC after purification through an immunoaffinity (IA) column (Aflatoxin Easi-extract, rbiopharm, Glasgow, UK). Liquid samples (milk, whey, natural whey starter) were analysed according to the method described by Pietri, Bertuzzi, Moschini, and Piva (2003): 20 g of sample, previously defatted by means of centrifugation at 4 C for 10 min and filtered on filter paper, was passed through an IA column. After washing of the column with water (5 ml), AFM1 was slowly eluted with 3 ml methanol in a graduated glass vial. Curd and cheese were analysed according to the method developed by Pietri et al. (2016), that proved to be very accurate both for soft and for hard cheeses. Five g of grated sample was dispersed in 50 ml 0.1 N HCl containing 0.2% pepsin and incubated at 42 C for 16 h under magnetic stirring. The solution was then neutralised with 1 N NaOH, centrifuged at 4500 g for 15 min at 4 C, filtered on filter paper and purified (30 ml) through an IA column. AFM1 in cream was determined after extraction using chloroform and purification through an IA column, as reported by Pietri, Bertuzzi, Bertuzzi, and Piva (1997). The purified extracts were evaporated under a gentle flow of nitrogen and re-dissolved with 1 ml water:acetonitrile 75 þ 25 (v/ v) by ultrasonication. Analysis was performed using an HPLC instrument consisting of a Jasco PU 1580 pump (Jasco Corp., Tokyo, Japan), an AS 1555 sampling system and a FP 1520 fluorescence detector set at 365 nm excitation and 440 nm emission wavelength. The system was controlled by Borwin 1.5 software (Jasco). AFM1 was separated on a Lichrospher RP-18 column (5 mm particle size, 125 4 mm i.d., Merck, Darmstadt, Germany) at ambient temperature, with a mobile phase water:acetonitrile 75 þ 25 (v/v); the flow rate was 1.0 ml min1. The injection volume for both standard solutions and sample extracts was 30 ml. 2.8. Mass balance For AFM1, mass balances of skimming {[(AFM1 in cauldron milk þ cream)/(AFM1 in raw milk)] 100} and cheese-making {[(AFM1 in curd þ whey)/(AFM1 in cauldron milk þ fermented whey)] 100} were calculated. 2.9. Statistical analysis Statistical comparisons were performed using the GLM procedure (IBM SPSS 20.0, Inc., Chicago, IL); mean values were compared
3. Results and discussion 3.1. Performances of the methods For liquid samples, recovery tests were performed by spiking uncontaminated (blank) samples of milk and whey with an appropriate volume of AFM1 standard solution, to obtain a level of 0.050 mg kg1; for solid samples, blank samples of curd and cheese (aged 3, 9, 16 and 24 months) were spiked, in order to have a contamination level of 0.300 mg kg1. Three replicates were analysed for each matrix; for cheese, the recovery was checked at every time of sampling. The average recoveries were 98.6 ± 1.3, 84.8 ± 3.1, 97.6 ± 2.4, 94.9 ± 2.8, 96.3 ± 1.9, 96.8 ± 2.0, 96.4 ± 2.2, 95.1 ± 1.9 for milk, cream, whey, curd, and cheese aged 3, 9, 16, 24 months, respectively; the recoveries completely fulfilled the performance criteria fixed by Regulation (EC) 401/2006 of the Commission of the European Communities (Commission of the European Communities, 2006b). All the results were corrected for recovery. The limits of detection (LOD) and quantification (LOQ) 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 purified blank sample extract (1 ml) to which between 40 and 100 ml of an AFM1 solution (0.428 mg l1) had been added. The LOD and LOQ were 0.015 and 0.045 mg kg1 in solid samples and 0.002 and 0.006 mg kg1 in liquid samples, respectively.
3.2. Partition of aflatoxin M1 during cheese-making During the experimental period, the chemical composition of raw milk was constant; the average percentage of fat, protein and lactose was 4.18 ± 0.09%, 3.44 ± 0.05% and 4.80 ± 0.06%, respectively. The AFM1 levels in raw milk were close to the expected values, in the range 0.028e0.031, 0.055e0.068 and 0.077e0.083 mg kg1 in the 1st, 2nd and 3rd 7-day sub-period, respectively. These values are close to the maximum limit of 0.050 mg kg1 and represent the levels that can be found in bulk milk produced in northern Italy; concentrations higher than 0.100 mg kg1 are rare. The fate of AFM1 during natural creaming and cheese-making is shown in Table 1; the mass balances of these processes resulted close to 100%. Because of its lipophobicity, only a small amount of AFM1 carried-over to cream; consequently, the concentration in raw and partially skimmed milk was very similar. No significant difference between AFM1 levels in cauldron milk with high and low F/C ratio was found. The EF in curd resulted between 4.0 and 5.2, with a mean value of 4.7 ± 0.4; this factor was not significantly affected by either AFM1 contamination level or F/C ratio. The value found resulted higher than that reported by Manetta et al. (2009), that calculated an EF of about 3. Around 41% (mean 41.2 ± 3.8%) of the AFM1 amount occurring in the milk (mg AFM1 in partially skimmed milk þ mg AFM1 in fermented whey) carried-over into the curd, while about 59% (mean 58.6 ± 3.6%) remained in the whey.
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Table 1 Concentration (mg kg1) of AFM1 in raw milk, cream, cauldron milk, whey and curd; mass balances of skimming and cheese-making; enrichment factor curd/cauldron milk. day AFM1 in raw milk 1 1st level (0.028 e0.031 mg kg1) 2
0.030 0.029
3
0.028
4
0.031
Mean 2nd level 1 (0.055 1 e0.068 mg kg ) 2
0.056
3
0.061
4
0.068
0.055
Mean 3rd level 1 (0.077 e0.083 mg kg1) 2
0.077
3
0.083
4
0.083
0.079
Fat/ casein
AFM1 conc. in cream
AFM1 conc. in cauldron Mass balance AFM1 conc. in milk (%)a whey
AFM1 conc. in curd
Mass balance Enrichment (%)b factor
0.017 0.018 0.017 0.018 0.015 0.016 0.019 0.020
0.143 0.140 0.143 0.153 0.148 0.137 0.133 0.131
0.033 0.033 0.033 0.034 0.038 0.040 0.043 0.043
0.251 0.269 0.276 0.267 0.250 0.293 0.332 0.339
0.052 0.050 0.046 0.046 0.056 0.055 0.053 0.055
0.307 0.388 0.360 0.370 0.337 0.404 0.330 0.335
95.2 94.6 102.1 101.7 97.1 97.9 95.5 94.7 97.3 ± 3.0 99.4 100.0 104.5 101.4 97.7 103.1 105.2 104.7 102.0 ± 2.8 101.0 105.2 97.7 97.0 100.7 104.9 95.7 96.7 99.9 ± 3.7 99.7 ± 3.6
1.05 0.83 1.10 0.87 1.06 0.79 1.13 0.88
0.013 0.019 0.021 0.017 0.013 0.019 0.014 0.014
0.030 0.030 0.028 0.029 0.028 0.027 0.031 0.031
1.08 0.87 1.14 0.90 1.08 0.89 1.13 0.91
0.030 0.031 0.027 0.028 0.029 0.029 0.031 0.044
0.054 0.054 0.054 0.054 0.059 0.060 0.067 0.066
1.17 0.90 1.09 0.89 1.14 0.90 1.16 0.96
0.044 0.046 0.042 0.041 0.038 0.038 0.044 44
0.076 0.076 0.076 0.075 0.081 0.080 0.081 0.081
97.8 97.3 95.5 97.0 98.5 94.7 98.2 96.8 97.0 96.4 95.6 94.8 93.6 94.9 95.2 97.0 94.4 95.2 94.9 94.0 97.3 94.8 95.4 93.1 95.7 94.8 95.0 95.7
Mean Overall mean a b
± 1.3
± 1.1
± 1.2 ± 1.5
4.7 4.6 5.0 5.2 5.2 5.0 4.2 4.2 4.8 4.7 5.0 5.1 5.0 4.2 4.9 5.0 5.1 4.9 4.0 5.1 4.7 4.9 4.2 5.0 4.1 4.1 4.5 4.7
± 0.4
± 0.3
± 0.5 ± 0.4
[(AFM1 in cauldron milk þ cream)/(AFM1 in raw milk)]*100. [(AFM1 in curd þ whey)/(AFM1 in cauldron milk þ fermented whey)]*100.
The AFM1 concentration and EF increased by 10e12% during the first 3 months of ageing, mainly because in this period the curd lost a nearly equivalent amount of moisture in the drain/dry phase; successively, moisture loss was much lower and consequently AFM1 concentration and EF increased much less. With a view to eliminating the effect of moisture loss during cheese ageing, the EFs were also calculated on a dry matter basis. In this case, the EF was not significantly influenced by the ripening time in cheese produced using milk with a high F/C ratio; however, in cheese produced using milk with a low F/C ratio, the EF after 24 months was significantly lower than those after 9 and 16 months of ageing (but not different from curd and cheese aged 3 months). These results
3.3. Fate of AFM1 during ripening Table 2 (high F/C ratio) and 3 (low F/C ratio) show the AFM1 concentrations and the EFs in curd and cheese after 3, 9, 16 and 24 months of maturation. The EF was significantly higher in cheese produced using milk with a low, rather than with a high F/C ratio; this is consistent with the fact that a higher fat level entails a higher weight of the wheel and a lower casein concentration in which AFM1 is bound. The EF was also significantly lower at the highest AFM1 level; this result might be explained by a proportionally reduced bonding capacity of casein at increasing AFM1 concentrations (see Table 3).
Table 2 Concentration (mg kg1) and enrichment factor (EF) of AFM1 in curd and cheese after 3, 9, 16 and 24 months of ripening; milk with high fat/casein ratio (1.05e1.17). Curd
1st level (0.028e0.031 mg kg1) Mean 2nd level (0.054e0.067 mg kg1) mean 3rd level (0.076e0.082 mg kg1) Mean Overall mean Overall mean (on dry matter basis) a,b
Cheese aged 3 months
Cheese aged 9 months
Cheese aged 16 months
AFM1 conc.
EF
AFM1 conc.
EF
AFM1 conc.
EF
AFM1 conc.
EF
AFM1 conc.
EF
0.143 0.143 0.148 0.133
4.7 5.0 5.2 4.2 4.8 4.7 5.1 4.3 5.0 4.8 4.0 4.7 4.2 4.1 4.2 4.6 7.8
0.159 0.148 0.165 0.151
5.2 5.2 5.8 4.8 5.3 5.3 5.0 4.8 5.4 5.1 4.5 5.3 4.7 4.7 4.8 5.1 7.8
0.155 0.152 0.173 0.161
5.1 5.4 6.1 5.1 5.4 5.5 5.6 5.4 5.6 5.5 4.7 5.5 4.9 4.9 5.0 5.3 7.9
0.166 0.162 0.180 0.158
5.5 5.7 6.3 5.0 5.6 5.7 5.9 5.4 5.6 5.7 4.9 5.6 5.0 5.0 5.1 5.5 8.0
0.164 0.162 0.174 0.152
5.4 5.7 6.1 4.8 5.5 5.3 5.4 5.4 4.8 5.2 4.9 5.7 4.9 4.9 5.1 5.3 7.6
0.251 0.276 0.250 0.332 0.307 0.360 0.337 0.330
± 0.4 0.283 0.268 0.283 0.362 ± 0.4 0.345 0.404 0.380 0.387 ± 0.3 ± 0.4a ± 0.7a
± 0.4 0.302 0.308 0.319 0.372 ± 0.3 0.358 0.425 0.396 0.398 ± 0.4 ± 0.4a,b ± 0.6a
Values marked by different letters within a line are significantly different (P < 0.05).
± 0.5 0.306 0.316 0.324 0.374 ± 0.1 0.372 0.429 0.406 0.408 ± 0.3 ± 0.4b ± 0.6a
Cheese aged 24 months
± 0.5 0.286 0.288 0.320 0.323 ± 0.2 0.369 0.433 0.403 0.399 ± 0.3 ± 0.4b ± 0.6a
± 0.5
± 0.3
± 0.4 ± 0.4a,b ± 0.6a
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Table 3 Concentration (mg kg1) and enrichment factor (EF) of AFM1 in curd and cheeses at 3, 9, 16 and 24 months of ripening; milk with low fat/casein ratio (0.79e0.96). Curd
1st level (0.028e0.031 mg kg1) mean 2nd level (0.054e0.067 mg kg1) mean 3rd level (0.076e0.082 mg kg1) Mean Overall mean Overall mean (on dry matter basis) a,b,c
Cheese aged 3 months
Cheese aged 9 months
Cheese aged 16 months
Cheese aged 24 months
AFM1 conc.
EF
AFM1 conc.
EF
AFM1 conc.
EF
AFM1 conc.
EF
AFM1 conc.
EF
0.140 0.153 0.137 0.131
4.6 5.2 5.0 4.2 4.7 5.0 5.0 4.9 5.1 5.0 5.1 4.9 5.0 4.1 4.8 4.8 8.3
0.154 0.171 0.161 0.159
5.0 5.8 5.8 5.1 5.4 5.7 5.7 5.2 5.5 5.5 5.5 5.6 5.5 4.9 5.4 5.4 8.4
0.170 0.176 0.165 0.168
5.6 6.0 6.0 5.4 5.7 5.9 5.7 5.4 5.8 5.7 5.8 6.0 5.9 5.3 5.8 5.7 8.6
0.181 0.180 0.172 0.174
5.9 6.1 6.2 5.6 6.0 6.1 6.2 5.7 5.9 6.0 5.9 6.1 5.9 5.3 5.8 5.9 8.7
0.165 0.149 0.159 0.167
5.4 5.1 5.8 5.3 5.4 5.6 5.8 5.8 5.8 5.8 5.1 5.9 5.0 5.1 5.3 5.5 8.0
0.269 0.267 0.293 0.339 0.388 0.370 0.404 0.335
± 0.5 0.308 0.306 0.310 0.366 ± 0.1 0.418 0.420 0.439 0.397 ± 0.5 ± 0.4a ± 0.6a,b
± 0.4 0.321 0.312 0.324 0.384 ± 0.2 0.439 0.450 0.475 0.431 ± 0.3 ± 0.3b ± 0.5a,b
± 0.3 0.328 0.333 0.344 0.391 ± 0.2 0.445 0.459 0.472 0.432 ± 0.3 ± 0.3b,c ± 0.4b
± 0.3 0.300 0.310 0.349 0.386 ± 0.2 0.390 0.446 0.401 0.415 ± 0.3 ± 0.3c ± 0.4b
± 0.3
± 0.1
± 0.4 ± 0.3b ± 0.5a
Values marked by different letters within a line are significantly different (P < 0.05).
showed that AFM1 is quite stable during a long cheese maturation, in agreement with previous studies (Anfossi et al., 2012; Deveci, 2007; Iha et al., 2013; Kamkar et al., 2008; Oruc et al., 2006); some observed variations could be due to the fact that the AFM1 levels are in the part-per-trillion range. In cheese aged 9 months (minimum length of time for Grana Padano), the EF values were between 4.7 and 6.1; EFs of 5.0 and above were calculated in 87.5% of the wheels. Zarenghi et al. (2008) calculated EF values in long maturing cheeses, aged from 9 to 24 months, between 0.7 and 7.7 (mean 2.8) and lower than 5.1 in 90.7% of the cheeses. Manetta et al. (2009) reported that AFM1 concentration in Grana Padano cheese, aged 12 months, was about 4.5 fold higher than in the respective milk. Our results demonstrate that the EF for aged grana-type cheese produced using milk with high and low F/C ratio is about 5.4 and 5.7, respectively. 4. Conclusions This study evaluated the fate of AFM1 during cheese-making for the preparation of parmesan cheese and during the following maturation, using naturally contaminated milk at levels close to the maximum admissible level. The mass balances was close to 100%, showing that no loss of AFM1 occurred during the cheese-making process. The average EF was 4.7 for curd, 5.5 for aged cheese. Considering these values and EU legislation, the maximum admissible level of AFM1 in grana-type cheese should be about 0.275 mg kg1. Acknowledgements The present work was supported by the Consorzio per la tutela del Formaggio Grana Padano and the Consorzio del Formaggio Parmigiano Reggiano. The authors thank Dr Gianni Colombari, from ERSAF Lombardia, for his valuable assistance. References Anfossi, L., Baggiani, C., Giovannoli, C., D'Arco, G., Passini, C., & Girardi, G. (2012). Occurrence of aflatoxin M1 in Italian cheese: results of a survey conducted in 2010 and correlation with manufacturing, production season, milking animals, and maturation of cheese. Food Control, 25, 125e130. AOAC International. (2005a). Official methods of analysis of the international
association of official analytical chemists. Natural toxins. Aflatoxins, 49.3.07: 46e47. Method 2000.08 (18th ed.) Gaithersburg, MD. AOAC International. (2005b). Official methods of analysis of the international association of official analytical chemists. Natural toxins. Aflatoxins, 49.2.03: 4e5. Method 971.22 (18th ed.) Gaithersburg, MD. Cattaneo, T. M., Monti, L., Panarelli, E., Francolino, S., Bertuzzi, T., & Pietri, A. (2008). Fate of aflatoxin M1 during production and storage of Crescenza cheese. Italian Journal of Food Science, 20(4), 463e470. Cavallarin, L., Antoniazzi, S., Giaccone, D., Tabacco, E., & Borreani, G. (2014). Transfer of aflatoxin M1 from milk to ripened cheese in three Italian traditional production methods. Food Control, 38, 174e177. Colak, H. (2007). Determination of aflatoxin M1 levels in Turkish White and Kashar cheeses made of experimentally contaminated raw milk. Journal of Food and Drug Analysis, 15(2), 163e168. Commission of the European Communities. (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, L 364, 5e24. Commission of the European Communities. (2006b). 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 of the European Communities, L 70, 12e34. Deveci, O. (2007). Changes in the concentration of aflatoxin M1 during manufacture and storage of White pickled cheese. Food Control, 18, 1103e1107. ^a, B., Rosim, R. E., Kobashigawa, E., & Oliveira, C. (2012). Fernandes, A. M., Corre Distribution and stability of aflatoxin M1 during processing and storage of Minas Frescal cheese. Food Control, 24(1e2), 104e108. IARC. (1993). Aflatoxins. IARC monograph on the evaluation of carcinogenic risks to humans (Vol. 56, p. 489). Lyon, France: World Health Organization. IARC. IARC. (2012b). http://monographs.iarc.fr/ENG/Classification/index.php. IARC. (2002). Aflatoxins. IARC monograph on the evaluation of carcinogenic risks to humans (Vol. 82, p. 171). Lyon, France: World Health Organization. IARC. IARC. (2012a). Aflatoxins. A review of human Carcinogens: Chemical agents and related occupations. IARC monograph on the evaluation of carcinogenic risks to humans (Vol. 100F, p. 225). Lyon, France: World Health Organization. IARC. Iha, M., 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, 1e6. Italian Ministry of Health, 2004, D.G.V.A/IX/25664/f.5.b.b.2/P. Italian Ministry of Health, 2013, H1 2013 0019815. Kamkar, A., Karim, G., Aliabadi, F., & Khaksar, R. (2008). Fate of aflatoxin M1 in Iranian white cheese processing. Food and Chemical Toxicology, 46, 2236e2238. Manetta, A., Giammarco, M., Di Giuseppe, L., Fusaro, I., Gramenzi, A., Formigoni, A., et al. (2009). Distribution of aflatoxin M1 during Grana Padano cheese production from naturally contaminated milk. Food Chemistry, 113, 595e599. Mendonca, C., & Venancio, A. (2005). Fate of aflatoxin M1 in cheese whey processing. Journal of the Science of Food and Agriculture, 85(12), 2067e2070. 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), 190e195. Oruc, H., Cibik, R., Ylmaz, E., & Gunes, E. (2007). Fate of aflatoxin M1 in Kashar cheese. Journal of Food Safety, 27(1), 82e90. Pecorari, A., Gambini, G., Nocetti, M., Bertuzzi, T., Mulazzi, A., & Pietri, A. (2009). Aflatoxin M1 and Parmigiano-Reggiano cheese: distribution within the wheel and evolution throughout ripening. Scienza e Tecnica LattieroeCasearia, 60(2), 125e129.
A. Pietri et al. / Food Control 60 (2016) 478e483 Pietri, A., Bertuzzi, T., Bertuzzi, P., & Piva, G. (1997). Aflatoxin M1 occurrence in samples of Grana Padano cheese. Food Additives and Contaminants, 14(4), 341e344. Pietri, A., Bertuzzi, T., Moschini, M., & Piva, G. (2003). Aflatoxin M1 occurrence in milk samples destined for Parmigiano Reggiano cheese production. Italian Journal Food Science, 15(2), 301e306. Pietri, A., Fortunati, P., Mulazzi, A., & Bertuzzi, T. (2016). Enzyme-assisted extraction for the HPLC determination of aflatoxin M1 in cheese. Food Chemistry, 192, 235e241. Van Egmond, H. P. (1989). Stability and degradation of aflatoxins M1. In H. P. Van Egmond (Ed.), Mycotoxins in dairy products (Vol. 11). London: Elsevier Science,
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London. Veldman, A., Meijs, J. A. C., Borggreve, G. J., & Heeres-van der Tol, J. J. (1992). Carryover of aflatoxin from cow's food to milk. Animal production, 55, 163e168. Yousef, A., & Marth, E. (1989). Stability and degradation of aflatoxins M1. In H. P. Van Egmond (Ed.), Mycotoxins in dairy products (pp. 127e161). London: Elsevier Science, London. Zarenghi, L., Fedrizzi, G., Masselli, M., Caprai, E., Ungari, D., Gorreri, M., et al. (2008). Aflatoxin M1 concentration levels inside cheese samples and evaluation of its distribution between whey and cheese during production. Scienza e Tecnica Lattiero e Casearia, 59(2), 133e140.
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Fate of aflatoxin M1 during production and storage of parmesan cheese Amedeo Pietri, Annalisa Mulazzi, Gianfranco Piva, 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
a b s t r a c t
Article history: Received 7 July 2015 Received in revised form 20 August 2015 Accepted 25 August 2015 Available online 29 August 2015
The fate of AFM1 during production of a long maturing cheese (Parmesan cheese) was assessed. Different levels of AFM1 contamination and of the fat/casein (F/C) ratio of milk were considered, in order to evaluate if these factors can influence the enrichment factor (EF) of AFM1 in cheese. For this purpose, 24 cheese-makings were carried out using naturally contaminated milk at 3 different AFM1 levels and at 2 F/ C ratios. AFM1 analysis was performed by HPLC in raw milk, cream, cauldron milk, liquid cattle rennet, whey, curd and cheese at 3, 9, 16 and 24 months of ageing. The mass balances of the cheese-making processes were close to 100%; in whey, AFM1 concentration was about 40% less than the concentration in cauldron milk. The EF in curd was between 4.0 and 5.2, with an average value of 4.7 ± 0.4; this factor was not significantly affected by either AFM1 contamination level or F/C ratio. During maturation, AFM1 concentration and consequently EF increased from curd to 16 ageing months; successively, AFM1 slightly decreased at 24 months and consequently the EF. At 3, 9, 16 months of maturation, the EF was significantly higher for cheeses prepared using milk with low F/C than those with high F/C milk; on the contrary, EF was not significantly influenced by the AFM1 contamination level. In cheeses, EF values were between 4.7 and 6.3; from these results, the maximum admissible level for AFM1 in Parmesan cheese should be about 0.275 mg kg1. © 2015 Elsevier Ltd. All rights reserved.
Keywords: AFM1 Parmesan cheese Enrichment factor
1. Introduction Lactating animals that have consumed feedstuffs contaminated with aflatoxin B1 (AFB1), excrete the 4-hydroxylated metabolite aflatoxin M1 (AFM1) into the milk. This toxin has shown acute toxic effects similar to AFB1; long term studies of toxicity and carcinogenicity in trout and rats showed that AFM1 is a hepatic carcinogen, but with a potency 2e10% that of AFB1. Further studies showed 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). AFM1 occurs in cheese when AFM1 contaminated milk is used for cheese-making; the partition between curd and whey is generally in the range 40e60%. AFM1 is associated with the protein fraction of milk by a hydrophobic interaction (Mendonca & Venancio, 2005; Van Egmond,
* Corresponding author. E-mail address: [email protected] (T. Bertuzzi). http://dx.doi.org/10.1016/j.foodcont.2015.08.032 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
1989); consequently, the concentration is higher in cheese than in the milk from which the cheese is made. The ratio between the concentration of AFM1 in cheese and the concentration in milk is called the enrichment factor (EF). The EF is generally higher for hard than for soft cheeses; this difference is mainly caused by the drying process of the cheese and by the AFM1 stability during maturation. On the basis of several studies, it was concluded that the EF is 2.5e3.3 in many soft cheeses and 3.9e5.8 in hard cheeses (Yousef & Marth, 1989). These ranges were generally confirmed in more recent studies (Cattaneo et al. 2008; Colak, 2007; Deveci, ^a, Rosim, Kobashigawa, & Oliveira, 2012; 2007; Fernandes, Corre Iha, Barbosa, Okada, & Trucksess, 2013; Kamkar, Karim, Aliabadi, & Khaksar, 2008; Oruc, Cibik, Yilmaz, & Kalkanli, 2006, 2007). However, EF values of 1.4, 2.2 and 6.7 were calculated for two soft and one hard cheeses, respectively (Cavallarin, Antoniazzi, Giaccone, Tabacco, & Borreani, 2014). The different EF values found mainly depend on the type of cheese-making process; but the origin (natural or artificial) of milk contamination and the methods of analysis may also have influenced the results. The Commission of the European Communities (Commission of the European Communities, 2006a) fixed an AFM1 maximum level
A. Pietri et al. / Food Control 60 (2016) 478e483
in milk (0.050 mg/kg); for dairy products, the limit must be calculated taking into account the changes of AFM1 concentration caused by processing. The specific EF must be provided and justified by the food business operator. In Italy, the production of long maturing cheeses is of considerable economic importance in the food chain; in the Po valley (northern Italy), about 70% of cow's milk produced is used to make Grana Padano and Parmigiano Reggiano cheeses (about 7,350,000 cheese wheels/year; the weight of each wheel is about 38 kg and approximately 550 kg of partially skimmed milk is used for each wheel). Grana Padano and Parmigiano Reggiano are PDO cheeses produced in different areas using very similar technology. They must be matured for at least 9 and 12 months, respectively; however, the ageing period is often longer. Two Consortia supervise the production of the two cheeses. In recent years, but particularly in 2003 and 2012, remarkable AFB1 contamination occurred in maize produced in northern Italy, caused by high temperatures and drought during the summer; since maize grain is used in dairy rations, AFM1 levels in milk increased. In 2004, the Italian Ministry of Health fixed a provisional limit of 0.45 mg kg1 for hard and long maturing cheeses (Italian Ministry of Health, 2004); in 2013, the same Ministry suggested an EF of 3.0 and 5.5 for soft and hard cheeses, respectively (Italian Ministry of Health, 2013). In the scientific literature, there are only two studies on AFM1 distribution during parmesan-type cheese production. In the first, the authors (Zarenghi et al., 2008) analysed 29 wheels of Grana Padano aged 9e24 months and produced with AFM1 contaminated milk ranging from 0.056 to 0.28 mg/kg; they found very variable EFs, ranging from 0.7 to 7.7, with a mean value of 2.8. In the second, the authors (Manetta et al., 2009), in a two month period, collected in some commercial dairies 25 samples of contaminated milk (range 30e98 mg/kg of AFM1) destined for Grana Padano production and the same number of the respective whey, curd and cheese samples (after 12 months of ageing); they calculated an EF of about 4.5 for cheese. However, in both studies the mass balance of AFM1 during cheese-making was not controlled and consequently the calculated EF may be inaccurate. The present study investigated the fate of AFM1 during cheesemaking for the production of parmesan-type cheese and during the ageing time. For this purpose, 24 cheese-makings were carried out in an experimental cheese factory using naturally contaminated milk at 3 different AFM1 levels, (0.030, 0.065 and 0.080 mg kg1) and at 2 fat/casein (F/C) ratios, high (H) and low (L) [about 1.10e1.15 (H) and 0.85e0.90 (L)], in order to evaluate if these factors may influence the EF. The mass balance and the EF of the cheese-making were calculated; the EF was also calculated after 3, 9, 16 and 24 months of ageing.
2. Materials and methods 2.1. Production of naturally contaminated maize Maize grain (20 kg) was moistened by the addition of distilled water to obtain a moisture value close to 40% (v/w), distributed in several 1000 ml beakers and contaminated with a high aflatoxin producing strain of Aspergillus flavus; then, maize was incubated in a thermostatic chamber at 35 C for 2 weeks. Successively, maize was desiccated at 65 C overnight, ground (taking particular precautions) using a cyclone hammer mill (1 mm sieve; Pulverisette, Fritsch GmbH, Idar-Oberstein, Germany) and homogenised. Four aliquots were analysed for AFB1 content using HPLC with fluorescence detection; the level was very high, close to 100 mg kg1.
479
2.2. Production of naturally contaminated milk The production of naturally contaminated milk, the cheesemaking and the ageing of wheels were carried out at ERSAF experimental farm (Ente regionale per i Servizi all'Agricoltura e alle Foreste, Mantova, Lombardia). The herd consisted of 42 milking cows; they were milked twice a day and the total mixed ration was distributed every day after morning milking. The contaminated maize was added and homogenised first with the concentrates and then with all the ingredients of the total mixed ration, to give the required AFB1 level. The animals were fed rations containing the AFB1 naturally contaminated maize flour for 21 days. In order to obtain the desired milk contamination levels, the experimental period was divided into 3 sub-periods of 7 days; increasing amounts of the maize flour were added to the daily rations, so that each cow could receive a calculated AFB1 amount of about 25, 45 and 65 mg day1 in the 1st, 2nd and 3rd sub-period, respectively. The aim was to obtain a herd bulk milk at AFM1 levels close to 0.030, 0.055 and 0.080 mg kg1, during the 3 sub-periods, respectively. The formula: AFM1 (ng kg1 milk) ¼ 1.19 AFB1 intake (mg per cow per day) þ 1.9 (Veldman, Meijs, Borggreve, & Heeres-van der Tol, 1992) was used to calculate the required amount of AFB1 in the ration, in order to have the desired AFM1 level in the bulk milk. 2.3. Cheese-making In each 7-day sub-period, starting from the 4th day, milk from evening milking was mixed with that of the following morning and used for cheese-making. Milk (about 950 kg) was poured into two holding basins provided with refrigerating coils and refrigerated at 11e12 C; separation of cream took place naturally (10e12 h) in the basins. The milk was partially skimmed in order to obtain a level of fat in the range 2.8e3.0% (high F/C ratio, close to 1.10e1.15) in one basin and 2.2e2.4% (low F/C ratio, close to 0.85e0.90) in the other one; 0.80e1.15 is the allowed F/C range for the production of Grana Padano. Then, the partially skimmed milk from the two basins was separately poured into two copper cauldrons for cheese-making. Eight cheese-makings were carried out in each sub-period and a total of 24 wheels were produced in the experimental period. After warming the milk in the cauldron at 34 C, naturally fermented whey (obtained from the cheese-making of the previous day, 13e16 kg), natural calf rennet [35 mg kg1, title 1:125,000 with high chymosin content (96%)] and lysozyme (18 mg kg1) were added to allow the milk to curdle. The cheese making process was carried out using traditional methods by technicians of the two Consortia (Grana Padano and Parmigiano Reggiano). 2.4. Analysis of samples AFM1 analysis was performed in raw milk, cream, cauldron milk, liquid cattle rennet, whey, curd and cheese at 3, 9, 16 and 24 months of ageing; the weight (kg) of these products was recorded to calculate the mass balance of AFM1. Milk was also analysed for fat, lactose and casein content; curd and cheese were analysed for moisture and fat (Milkoscan FT6000, Foss Italy). The sampling of cheese, made after scraping of the rind (1e2 mm), was carried out by means of a small corer (length 14 cm). In a previous study, we demonstrated that AFM1 is homogeneously distributed within the wheel (Pecorari et al., 2009). All samples were kept at 20 C until analysis. 2.5. Reagents Chemicals and solvents used for AFM1 extraction and clean-up solutions were ACS grade or equivalent (Carlo Erba, Milan, Italy).
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All 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, 800e2500 units/mg protein, cod P7000) was obtained from SigmaeAldrich (St. Louis, MO, USA).
using the post-hoc Tukey Test (a ¼ 0.05). For the cheese-making process, the mean AFM1 concentrations in curd were evaluated considering as fixed factors the F/C ratio and the AFM1 contamination level in milk; for the cheese ripening process, the mean EF values, calculated also on a dry matter basis, were compared considering as fixed factors the F/C ratio, the ripening time and the AFM1 contamination level in milk.
2.6. Standard solution AFM1 standard (0.01 mg, purity degree higher than 98%) was obtained from SigmaeAldrich (St. Louis, MO, USA). A stock solution of 4e6 mg ml1 was prepared in acetonitrile (1.5e2 ml) and stored at 20 C (AOAC 2005a). The solution was calibrated spectrophotometrically at 350 nm using the value 19,000 l mol1 cm1 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 0.05 and 5 mg l1. All standard solutions were stored at 20 C when not in use. 2.7. Analysis for aflatoxin M1 AFM1 analysis was carried out by HPLC after purification through an immunoaffinity (IA) column (Aflatoxin Easi-extract, rbiopharm, Glasgow, UK). Liquid samples (milk, whey, natural whey starter) were analysed according to the method described by Pietri, Bertuzzi, Moschini, and Piva (2003): 20 g of sample, previously defatted by means of centrifugation at 4 C for 10 min and filtered on filter paper, was passed through an IA column. After washing of the column with water (5 ml), AFM1 was slowly eluted with 3 ml methanol in a graduated glass vial. Curd and cheese were analysed according to the method developed by Pietri et al. (2016), that proved to be very accurate both for soft and for hard cheeses. Five g of grated sample was dispersed in 50 ml 0.1 N HCl containing 0.2% pepsin and incubated at 42 C for 16 h under magnetic stirring. The solution was then neutralised with 1 N NaOH, centrifuged at 4500 g for 15 min at 4 C, filtered on filter paper and purified (30 ml) through an IA column. AFM1 in cream was determined after extraction using chloroform and purification through an IA column, as reported by Pietri, Bertuzzi, Bertuzzi, and Piva (1997). The purified extracts were evaporated under a gentle flow of nitrogen and re-dissolved with 1 ml water:acetonitrile 75 þ 25 (v/ v) by ultrasonication. Analysis was performed using an HPLC instrument consisting of a Jasco PU 1580 pump (Jasco Corp., Tokyo, Japan), an AS 1555 sampling system and a FP 1520 fluorescence detector set at 365 nm excitation and 440 nm emission wavelength. The system was controlled by Borwin 1.5 software (Jasco). AFM1 was separated on a Lichrospher RP-18 column (5 mm particle size, 125 4 mm i.d., Merck, Darmstadt, Germany) at ambient temperature, with a mobile phase water:acetonitrile 75 þ 25 (v/v); the flow rate was 1.0 ml min1. The injection volume for both standard solutions and sample extracts was 30 ml. 2.8. Mass balance For AFM1, mass balances of skimming {[(AFM1 in cauldron milk þ cream)/(AFM1 in raw milk)] 100} and cheese-making {[(AFM1 in curd þ whey)/(AFM1 in cauldron milk þ fermented whey)] 100} were calculated. 2.9. Statistical analysis Statistical comparisons were performed using the GLM procedure (IBM SPSS 20.0, Inc., Chicago, IL); mean values were compared
3. Results and discussion 3.1. Performances of the methods For liquid samples, recovery tests were performed by spiking uncontaminated (blank) samples of milk and whey with an appropriate volume of AFM1 standard solution, to obtain a level of 0.050 mg kg1; for solid samples, blank samples of curd and cheese (aged 3, 9, 16 and 24 months) were spiked, in order to have a contamination level of 0.300 mg kg1. Three replicates were analysed for each matrix; for cheese, the recovery was checked at every time of sampling. The average recoveries were 98.6 ± 1.3, 84.8 ± 3.1, 97.6 ± 2.4, 94.9 ± 2.8, 96.3 ± 1.9, 96.8 ± 2.0, 96.4 ± 2.2, 95.1 ± 1.9 for milk, cream, whey, curd, and cheese aged 3, 9, 16, 24 months, respectively; the recoveries completely fulfilled the performance criteria fixed by Regulation (EC) 401/2006 of the Commission of the European Communities (Commission of the European Communities, 2006b). All the results were corrected for recovery. The limits of detection (LOD) and quantification (LOQ) 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 purified blank sample extract (1 ml) to which between 40 and 100 ml of an AFM1 solution (0.428 mg l1) had been added. The LOD and LOQ were 0.015 and 0.045 mg kg1 in solid samples and 0.002 and 0.006 mg kg1 in liquid samples, respectively.
3.2. Partition of aflatoxin M1 during cheese-making During the experimental period, the chemical composition of raw milk was constant; the average percentage of fat, protein and lactose was 4.18 ± 0.09%, 3.44 ± 0.05% and 4.80 ± 0.06%, respectively. The AFM1 levels in raw milk were close to the expected values, in the range 0.028e0.031, 0.055e0.068 and 0.077e0.083 mg kg1 in the 1st, 2nd and 3rd 7-day sub-period, respectively. These values are close to the maximum limit of 0.050 mg kg1 and represent the levels that can be found in bulk milk produced in northern Italy; concentrations higher than 0.100 mg kg1 are rare. The fate of AFM1 during natural creaming and cheese-making is shown in Table 1; the mass balances of these processes resulted close to 100%. Because of its lipophobicity, only a small amount of AFM1 carried-over to cream; consequently, the concentration in raw and partially skimmed milk was very similar. No significant difference between AFM1 levels in cauldron milk with high and low F/C ratio was found. The EF in curd resulted between 4.0 and 5.2, with a mean value of 4.7 ± 0.4; this factor was not significantly affected by either AFM1 contamination level or F/C ratio. The value found resulted higher than that reported by Manetta et al. (2009), that calculated an EF of about 3. Around 41% (mean 41.2 ± 3.8%) of the AFM1 amount occurring in the milk (mg AFM1 in partially skimmed milk þ mg AFM1 in fermented whey) carried-over into the curd, while about 59% (mean 58.6 ± 3.6%) remained in the whey.
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481
Table 1 Concentration (mg kg1) of AFM1 in raw milk, cream, cauldron milk, whey and curd; mass balances of skimming and cheese-making; enrichment factor curd/cauldron milk. day AFM1 in raw milk 1 1st level (0.028 e0.031 mg kg1) 2
0.030 0.029
3
0.028
4
0.031
Mean 2nd level 1 (0.055 1 e0.068 mg kg ) 2
0.056
3
0.061
4
0.068
0.055
Mean 3rd level 1 (0.077 e0.083 mg kg1) 2
0.077
3
0.083
4
0.083
0.079
Fat/ casein
AFM1 conc. in cream
AFM1 conc. in cauldron Mass balance AFM1 conc. in milk (%)a whey
AFM1 conc. in curd
Mass balance Enrichment (%)b factor
0.017 0.018 0.017 0.018 0.015 0.016 0.019 0.020
0.143 0.140 0.143 0.153 0.148 0.137 0.133 0.131
0.033 0.033 0.033 0.034 0.038 0.040 0.043 0.043
0.251 0.269 0.276 0.267 0.250 0.293 0.332 0.339
0.052 0.050 0.046 0.046 0.056 0.055 0.053 0.055
0.307 0.388 0.360 0.370 0.337 0.404 0.330 0.335
95.2 94.6 102.1 101.7 97.1 97.9 95.5 94.7 97.3 ± 3.0 99.4 100.0 104.5 101.4 97.7 103.1 105.2 104.7 102.0 ± 2.8 101.0 105.2 97.7 97.0 100.7 104.9 95.7 96.7 99.9 ± 3.7 99.7 ± 3.6
1.05 0.83 1.10 0.87 1.06 0.79 1.13 0.88
0.013 0.019 0.021 0.017 0.013 0.019 0.014 0.014
0.030 0.030 0.028 0.029 0.028 0.027 0.031 0.031
1.08 0.87 1.14 0.90 1.08 0.89 1.13 0.91
0.030 0.031 0.027 0.028 0.029 0.029 0.031 0.044
0.054 0.054 0.054 0.054 0.059 0.060 0.067 0.066
1.17 0.90 1.09 0.89 1.14 0.90 1.16 0.96
0.044 0.046 0.042 0.041 0.038 0.038 0.044 44
0.076 0.076 0.076 0.075 0.081 0.080 0.081 0.081
97.8 97.3 95.5 97.0 98.5 94.7 98.2 96.8 97.0 96.4 95.6 94.8 93.6 94.9 95.2 97.0 94.4 95.2 94.9 94.0 97.3 94.8 95.4 93.1 95.7 94.8 95.0 95.7
Mean Overall mean a b
± 1.3
± 1.1
± 1.2 ± 1.5
4.7 4.6 5.0 5.2 5.2 5.0 4.2 4.2 4.8 4.7 5.0 5.1 5.0 4.2 4.9 5.0 5.1 4.9 4.0 5.1 4.7 4.9 4.2 5.0 4.1 4.1 4.5 4.7
± 0.4
± 0.3
± 0.5 ± 0.4
[(AFM1 in cauldron milk þ cream)/(AFM1 in raw milk)]*100. [(AFM1 in curd þ whey)/(AFM1 in cauldron milk þ fermented whey)]*100.
The AFM1 concentration and EF increased by 10e12% during the first 3 months of ageing, mainly because in this period the curd lost a nearly equivalent amount of moisture in the drain/dry phase; successively, moisture loss was much lower and consequently AFM1 concentration and EF increased much less. With a view to eliminating the effect of moisture loss during cheese ageing, the EFs were also calculated on a dry matter basis. In this case, the EF was not significantly influenced by the ripening time in cheese produced using milk with a high F/C ratio; however, in cheese produced using milk with a low F/C ratio, the EF after 24 months was significantly lower than those after 9 and 16 months of ageing (but not different from curd and cheese aged 3 months). These results
3.3. Fate of AFM1 during ripening Table 2 (high F/C ratio) and 3 (low F/C ratio) show the AFM1 concentrations and the EFs in curd and cheese after 3, 9, 16 and 24 months of maturation. The EF was significantly higher in cheese produced using milk with a low, rather than with a high F/C ratio; this is consistent with the fact that a higher fat level entails a higher weight of the wheel and a lower casein concentration in which AFM1 is bound. The EF was also significantly lower at the highest AFM1 level; this result might be explained by a proportionally reduced bonding capacity of casein at increasing AFM1 concentrations (see Table 3).
Table 2 Concentration (mg kg1) and enrichment factor (EF) of AFM1 in curd and cheese after 3, 9, 16 and 24 months of ripening; milk with high fat/casein ratio (1.05e1.17). Curd
1st level (0.028e0.031 mg kg1) Mean 2nd level (0.054e0.067 mg kg1) mean 3rd level (0.076e0.082 mg kg1) Mean Overall mean Overall mean (on dry matter basis) a,b
Cheese aged 3 months
Cheese aged 9 months
Cheese aged 16 months
AFM1 conc.
EF
AFM1 conc.
EF
AFM1 conc.
EF
AFM1 conc.
EF
AFM1 conc.
EF
0.143 0.143 0.148 0.133
4.7 5.0 5.2 4.2 4.8 4.7 5.1 4.3 5.0 4.8 4.0 4.7 4.2 4.1 4.2 4.6 7.8
0.159 0.148 0.165 0.151
5.2 5.2 5.8 4.8 5.3 5.3 5.0 4.8 5.4 5.1 4.5 5.3 4.7 4.7 4.8 5.1 7.8
0.155 0.152 0.173 0.161
5.1 5.4 6.1 5.1 5.4 5.5 5.6 5.4 5.6 5.5 4.7 5.5 4.9 4.9 5.0 5.3 7.9
0.166 0.162 0.180 0.158
5.5 5.7 6.3 5.0 5.6 5.7 5.9 5.4 5.6 5.7 4.9 5.6 5.0 5.0 5.1 5.5 8.0
0.164 0.162 0.174 0.152
5.4 5.7 6.1 4.8 5.5 5.3 5.4 5.4 4.8 5.2 4.9 5.7 4.9 4.9 5.1 5.3 7.6
0.251 0.276 0.250 0.332 0.307 0.360 0.337 0.330
± 0.4 0.283 0.268 0.283 0.362 ± 0.4 0.345 0.404 0.380 0.387 ± 0.3 ± 0.4a ± 0.7a
± 0.4 0.302 0.308 0.319 0.372 ± 0.3 0.358 0.425 0.396 0.398 ± 0.4 ± 0.4a,b ± 0.6a
Values marked by different letters within a line are significantly different (P < 0.05).
± 0.5 0.306 0.316 0.324 0.374 ± 0.1 0.372 0.429 0.406 0.408 ± 0.3 ± 0.4b ± 0.6a
Cheese aged 24 months
± 0.5 0.286 0.288 0.320 0.323 ± 0.2 0.369 0.433 0.403 0.399 ± 0.3 ± 0.4b ± 0.6a
± 0.5
± 0.3
± 0.4 ± 0.4a,b ± 0.6a
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Table 3 Concentration (mg kg1) and enrichment factor (EF) of AFM1 in curd and cheeses at 3, 9, 16 and 24 months of ripening; milk with low fat/casein ratio (0.79e0.96). Curd
1st level (0.028e0.031 mg kg1) mean 2nd level (0.054e0.067 mg kg1) mean 3rd level (0.076e0.082 mg kg1) Mean Overall mean Overall mean (on dry matter basis) a,b,c
Cheese aged 3 months
Cheese aged 9 months
Cheese aged 16 months
Cheese aged 24 months
AFM1 conc.
EF
AFM1 conc.
EF
AFM1 conc.
EF
AFM1 conc.
EF
AFM1 conc.
EF
0.140 0.153 0.137 0.131
4.6 5.2 5.0 4.2 4.7 5.0 5.0 4.9 5.1 5.0 5.1 4.9 5.0 4.1 4.8 4.8 8.3
0.154 0.171 0.161 0.159
5.0 5.8 5.8 5.1 5.4 5.7 5.7 5.2 5.5 5.5 5.5 5.6 5.5 4.9 5.4 5.4 8.4
0.170 0.176 0.165 0.168
5.6 6.0 6.0 5.4 5.7 5.9 5.7 5.4 5.8 5.7 5.8 6.0 5.9 5.3 5.8 5.7 8.6
0.181 0.180 0.172 0.174
5.9 6.1 6.2 5.6 6.0 6.1 6.2 5.7 5.9 6.0 5.9 6.1 5.9 5.3 5.8 5.9 8.7
0.165 0.149 0.159 0.167
5.4 5.1 5.8 5.3 5.4 5.6 5.8 5.8 5.8 5.8 5.1 5.9 5.0 5.1 5.3 5.5 8.0
0.269 0.267 0.293 0.339 0.388 0.370 0.404 0.335
± 0.5 0.308 0.306 0.310 0.366 ± 0.1 0.418 0.420 0.439 0.397 ± 0.5 ± 0.4a ± 0.6a,b
± 0.4 0.321 0.312 0.324 0.384 ± 0.2 0.439 0.450 0.475 0.431 ± 0.3 ± 0.3b ± 0.5a,b
± 0.3 0.328 0.333 0.344 0.391 ± 0.2 0.445 0.459 0.472 0.432 ± 0.3 ± 0.3b,c ± 0.4b
± 0.3 0.300 0.310 0.349 0.386 ± 0.2 0.390 0.446 0.401 0.415 ± 0.3 ± 0.3c ± 0.4b
± 0.3
± 0.1
± 0.4 ± 0.3b ± 0.5a
Values marked by different letters within a line are significantly different (P < 0.05).
showed that AFM1 is quite stable during a long cheese maturation, in agreement with previous studies (Anfossi et al., 2012; Deveci, 2007; Iha et al., 2013; Kamkar et al., 2008; Oruc et al., 2006); some observed variations could be due to the fact that the AFM1 levels are in the part-per-trillion range. In cheese aged 9 months (minimum length of time for Grana Padano), the EF values were between 4.7 and 6.1; EFs of 5.0 and above were calculated in 87.5% of the wheels. Zarenghi et al. (2008) calculated EF values in long maturing cheeses, aged from 9 to 24 months, between 0.7 and 7.7 (mean 2.8) and lower than 5.1 in 90.7% of the cheeses. Manetta et al. (2009) reported that AFM1 concentration in Grana Padano cheese, aged 12 months, was about 4.5 fold higher than in the respective milk. Our results demonstrate that the EF for aged grana-type cheese produced using milk with high and low F/C ratio is about 5.4 and 5.7, respectively. 4. Conclusions This study evaluated the fate of AFM1 during cheese-making for the preparation of parmesan cheese and during the following maturation, using naturally contaminated milk at levels close to the maximum admissible level. The mass balances was close to 100%, showing that no loss of AFM1 occurred during the cheese-making process. The average EF was 4.7 for curd, 5.5 for aged cheese. Considering these values and EU legislation, the maximum admissible level of AFM1 in grana-type cheese should be about 0.275 mg kg1. Acknowledgements The present work was supported by the Consorzio per la tutela del Formaggio Grana Padano and the Consorzio del Formaggio Parmigiano Reggiano. The authors thank Dr Gianni Colombari, from ERSAF Lombardia, for his valuable assistance. References Anfossi, L., Baggiani, C., Giovannoli, C., D'Arco, G., Passini, C., & Girardi, G. (2012). Occurrence of aflatoxin M1 in Italian cheese: results of a survey conducted in 2010 and correlation with manufacturing, production season, milking animals, and maturation of cheese. Food Control, 25, 125e130. AOAC International. (2005a). Official methods of analysis of the international
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