Food Additives and Contaminants: Part B Vol. 4, No. 1, March 2011, 57–60
VIEW DATASET Survey of aflatoxin M1 in cheese from the North-east region of Sa˜o Paulo, Brazil C.A.F. Oliveiraa*, R.C. Francoa, R.E. Rosima and A.M. Fernandesb a
Department of Food Engineering, School of Animal Science and Food Engineering, University of Sa˜o Paulo, Pirassununga, SP, Brazil; bDepartment of Basic Sciences, School of Animal Science and Food Engineering, University of Sa˜o Paulo, Pirassununga, SP, Brazil (Received 20 September 2010; final version received 7 November 2010) In the present study, 24 samples of Minas Frescal cheese and 24 samples of Minas Padra˜o cheese produced in the North-east region of the state of Sa˜o Paulo, Brazil, were analysed for aflatoxin M1 (AFM1) by high-performance liquid chromatography (HPLC) between March and August 2008. AFM1 was detected in 13 (27.1%) samples at concentrations ranging from 0.037 to 0.313 ng g1. The mean concentrations of AFM1 in positive samples of Minas Frescal and Minas Padra˜o cheese were 0.142 0.118 and 0.118 0.054 ng g1, respectively. It is concluded that the incidence of AFM1 in Minas cheese may contribute to an increase in the overall ingestion of aflatoxins in the diet, hence indicating the need for the adoption of a tolerance limit for AFM1 in cheese in Brazil. Keywords: cheese; aflatoxins; mycotoxins; aflatoxins
Introduction Aflatoxin M1 (AFM1) is a hepatocarcinogen found in the milk of animals that consumed feeds contaminated with aflatoxin B1 (Council for Agriculture Science and Technology (CAST) 2003), the main metabolite produced by storage fungi of the genus Aspergillus, particularly A. flavus, A. parasiticus and A. nomius (Moss 1998). The occurrence of AFM1 in milk and milk products is a public health concern since the International Agency for Research on Cancer (IARC) (1993) classified AFM1 in Group 2, a probable human carcinogen. In milk, AFM1 binds to casein and remains bound to it even after thermal treatment and production of milk products, mainly cheese (Yousef and Marth 1989). The natural occurrence of AFM1 in cheese seems to be highly variable, depending on the original levels of AFM1 in milk and in differences in the manufacturing steps of the product (Lo´pez et al. 2001). Most studies on the occurrence of AFM1 in cheese were carried out in Europe, where some countries adopt tolerance limits for AFM1 in cheese between 0.250 and 0.500 ng g1 (Food and Agriculture Organization (FAO) 2004). In a survey conducted in Italy, AFM1 was detected in 19.5%, 26.5% and 53.5% of cheese samples manufactured in France, Germany and the Netherlands, respectively (Piva et al. 1998). Pietri et al. (1997) analysed 223 samples of Grana Padano cheese and found 91% samples containing AFM1 from 0.005 to 0.100 ng g1,
*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2011 Taylor & Francis DOI: 10.1080/19393210.2010.538934 http://www.informaworld.com
and 6.7% from 0.100 to 0.250 ng AFM1 g1. Barrios et al. (1996) analysed 35 cheese samples produced in Southern Spain and detected AFM1 in 16 samples (44.7%), with concentrations ranging from 0.020 to 0.200 ng g1. A Turkish study showed that the tolerance limit determined in that country (0.250 ng AFM1 g1) was exceeded in 12.28% samples, and the highest level was 0.810 ng g1 (Oruc and Sonal 2001). In Brazil, previous studies indicated a high incidence of AFM1 in fluid milk, but levels were usually below the limit (0.500 mg l1) established by Brazilian regulations (Ageˆncia Nacional de Vigilaˆncia Sanita´ria 2004). However, there is very little information on the occurrence of AFM1 in cheese manufactured in the country, except for a report by Prado et al. (2000), who observed that 74.7% Minas Frescal samples from the state of Minas Gerais showed AFM1 at levels ranging from 0.02 to 6.92 ng g1. Minas cheese is classified as a semi-fat and high-humidity cheese, obtained by the enzymatic coagulation of milk with rennet, added or not with starter cultures. In Brazil, Minas cheese accounts for nearly 10% of the 640,000 kg of cheese produced annually in the country. There are two main types of Minas cheese: the non-ripened one (Minas Frescal), which is to be consumed from up 2–4 days after processing, and the ripened cheese (Minas Padra˜o), which undergoes a ripening period of around 2 months. In the present study, AFM1 was determined both in Minas Frescal and Minas Padra˜o
58
C.A.F. Oliveira et al.
cheese produced and traded in the North-east region of the state of Sa˜o Paulo, Brazil.
0.006
0.004
Forty-eight samples of Minas Frescal and Minas Padra˜o cheese (24 samples of each) were collected. Samples belonged to the 11 brands responsible for the greatest volume of cheese produced and traded in the North-east region of state of Sa˜o Paulo. Samples were collected from March to August 2008, with no repetition of batches. According to the manufacturer’s information, fat and moisture contents in cheese samples varied between 25–30% and 46–55%, respectively. The sampling unit was the 500 g original packaging of each type of cheese. In the first week of each month four samples of cheese (one of each brand, and of each type of cheese) were randomly collected from units available in supermarkets in the cities of Leme and Pirassununga, state of Sa˜o Paulo. Samples were identified, placed in isothermal boxes and immediately sent to the laboratory, where they were ground and stored in sterile polypropylene bags at 5 C.
Analysis of aflatoxin M1 AFM1 determination was performed as recommended by Oruc et al. (2006), with some adaptations proposed by the manufacturer of the immunoaffinity columns Õ (Aflatest ; Vicam, Watertown, MA). The analytical sample (10 g of each duplicate from the same ground sample) was placed in a blender jar, and 5 g of celite, 150 ml of chloroform and 1 ml of a saturated NaCl solution were added. The mixture was blended for 2– 3 min in order to obtain a slurry, which was filtered and evaporated to near dryness. The residue was dissolved in 2 ml of methanol and the volume completed to 100 ml using phosphate buffer prepared according to Deveci (2007). The solution was transferred to a separation funnel and 75 ml of n-hexane were added to it and mixed. The upper (methanolic) phase was collected and directly passed through an immunoaffinity column connected to a glass syringe and a vacuum system (flow of 2–3 ml min1). After the sample was added the column was washed with 20 ml of an ultrapure water (Milli Q; Millipore)–methanol (75 : 25, v/v) solution. After that it was washed with 1 ml of methanol in order to elute AFM1, and the eluate was collected in an amber flask. Identification and quantification of the AFM1 residues was achieved by high-performance liquid chromatography (HPLC), using a Shimadzu 10VP liquid chromatograph with a 10 AXL fluorescence detector (excitation at 366 nm and emission at 440 nm).
AFM1
Sampling
Volts
Material and methods 0.002
0.000
0
2
4
6
8
10
Minutes
Figure 1. Chromatogram showing the retention time of aflatoxin M1 (AFM1) (nearly 6 min) in a naturally contaminated Minas Frescal cheese sample (0.313 ng g1).
A Phenomenex C18 column (4.6 150 mm, 4 mm) and a Shim-Pack pre-column (4 10 mm CLC G-ODS) were used. Calibration curves were prepared using standard solutions of AFM1 (Sigma-Aldrich, Saint Louis, MO) at concentrations of 2.38, 4.75, 9.51 and 19.01 ng ml1, as previously evaluated according to Scott (1990). A total of 20 ml of sample extracts were injected for AFM1 determination. The isocratic mobile phase consisted of methanol–water–acetonitrile (60 þ 20 þ 20, v/v/v) with a flow rate of 1.0 ml min1. Under these conditions the retention time for AFM1 was 6.1 min (Figure 1). The retention time window for peak identification was 8.0% (0.49 min).
Results and discussion The analytical methods used in this trial were evaluated using cheese samples spiked with AFM1 at levels of 0.200 and 0.500 ng g1 (n ¼ 5 per spiking level). Results are shown in Table 1. The limit of determination (LOD) and limit of quantification (LOQ) for AFM1 were determined in spiked cheese samples considering signal-to-noise ratios of 3 : 1 and 6 : 1, respectively. AFM1 levels in cheese samples are presented in Table 2. The toxin was detected in 13 of the 48 analysed samples (27.1%) at levels ranging from 0.030 to 0.313 ng g1. The number of positive samples for AFM1 in Minas Frescal and Minas Padra˜o cheese was six (25.0%) and seven (29.2%), respectively (n ¼ 24 per type of cheese). Minas Frescal cheese samples presented higher mean levels of AFM1 (0.142 0.118 ng g1) compared with Minas Padra˜o cheese (0.118 0.054 ng g1). Results obtained in this trial are different from those of Sylos et al. (1996) who did not find detectable levels of AFM1 in samples of Minas, mozzarella and cheddar cheese traded in Campinas, state of Sa˜o Paulo. However, these authors
Food Additives and Contaminants: Part B
59
Table 1. Performance characteristics of methods for the determination of aflatoxin M1 in Minas Frescal and Minas Padra˜o cheeses. Analyte
Matrix
LOD (ng g–1)
LOQ (ng g–1)
Recovery range (%)a
RSDr (%) (n ¼ 5)
Accreditation
AFM1 AFM1
Minas Frescal cheese Minas Padra˜o cheese
0.010 0.010
0.030 0.030
83.6–84.2 83.4–85.3
5.1–12.9 4.8–14.9
No No
Notes: aSpiking levels: 0.200 and 0.500 ng g–1. LOD, limit of determination; LOQ, limit of quantification; RSD, relative standard deviation.
Table 2. Aflatoxin M1 levels in Minas Frescal and Minas Padra˜o cheese samples traded in Pirassununga, Brazil. Number of samplesa Level of AFM1 (ng g–1)
Minas Frescal, n (%)
Minas Padra˜o, n (%)
Total n (%)
50.030b 0.030–0.050 0.051–0.100 0.101–0.200 40.200 Mean (ng g–1)c
18 (75.0) 1 (4.2) 3 (12.5) 0 (0) 2 (8.3) 0.142 0.118
17 (70.8) 1 (4.2) 2 (8.3) 4 (16.7) 0 (0) 0.118 0.054
35 (72.9) 2 (4.2) 5 (10.4) 4 (8.3) 2 (4.2) 0.129 0.086
Notes: aNumber of samples analysed: 24 for each type of cheese (total ¼ 48 samples). b Limit of quantification of the analytical method¼0.030 ng g–1. c Values are reported as means standard deviation (SD), for samples containing detectable levels of AFM1.
used thin-layer chromatography to identify and quantify AFM1, a technique considered to have lower sensibility when compared to] liquid chromatography. This fact may have influenced the low recovery of AFM1 in the cheese analysed by these authors. The frequency of samples positive for AFM1 in Minas cheese observed in the present study was lower than that reported by Prado et al. (2000) in Belo Horizonte, state of Minas Gerais. These authors detected 74.7% samples positive for AFM1, with levels ranging from 0.02 to 6.92 ng g1, and mean AFM1 concentrations in Minas Frescal of 0.080 ng g1 and in Minas Padra˜o of 0.62 ng g1. These results were different from those results of the present study, which showed that mean concentration of AFM1 in Minas Padra˜o cheese samples was lower than that of Minas Frescal samples. Although data of the literature on AFM1 degradation during cheese maturation are contradictory (Yousef and Marth 1989), the results of this study corroborate the hypothesis that AFM1 stability decreases in ripened cheese. In Brazil there is no legally determined limit for AFM1 levels in cheese. Limits established by Brazilian regulations are only related to powdered milk (5.0 ng g1) and fluid milk for direct human consumption (0.500 ng ml1), which is ten-fold greater than the limit adopted by the European Union (0.050 ng ml1). However, it should be considered that when compared with levels found in milk, the toxin may be more concentrated in cheese. Oruc et al. (2006) and Kamkar et al. (2008) reported that soft cheese presented AFM1
levels 1.8–4.0 times greater than the levels found in milk, which stresses the importance of regulations specifically for AFM1 for cheese. Some countries, such as Turkey and Switzerland, established a tolerance limit of 0.25 ng g1 for AFM1 in cheese (FAO 2004). In the present study two samples (8.3%) of Minas Frescal cheese would not comply with this limit, with AFM1 levels of 0.272 and 0.313 ng g1. However, 11 samples (22.9%) presented AFM1 levels above the tolerance limit for AFM1 in milk adopted by the European Union of 0.050 ng g1.
Conclusion Samples of Minas Frescal and Minas Padra˜o cheese produced in the North-east region of Sa˜o Paulo, Brazil, presented a high incidence of AFM1, with mean concentrations in positive samples of 0.142 0.118 and 0.118 0.054 ng g1, respectively. As AFM1 is one of the most potent carcinogens known, tolerance limits for AFM1 in cheese should be established by Brazilian regulations. Also, further studies are needed to evaluate the overall contribution of cheese for human exposure to this toxin in Brazil.
References Ageˆncia Nacional de Vigilaˆncia Sanita´ria. 2004. Resoluc¸a˜o RDC n 274, de 15 de outubro de 2002; [cited 2009
60
C.A.F. Oliveira et al.
Sept 10]. Available from: http://e-legis.bvs.br/leisref/public/ showAct.php?id=1653&word=Aflatoxina%20M1/ Barrios MJ, Gualda MJ, Cabanas JM, Medina LM, Jordano R. 1996. Occurrence of aflatoxin M1 in cheeses from the south of Spain. J Food Protect. 59:898–900. Council for Agriculture Science and Technology (CAST). 2003. Mycotoxins: risks in plant, animal, and human systems. Ames: CAST. Deveci O. 2007. Changes in the concentration of aflatoxin M1 during manufacture and storage of White Pickled cheese. Food Contr. 18:1103–1107. Food and Agriculture Organization (FAO). 2004. Worldwide regulations for mycotoxins in food and feed in 2003. Rome (Italy): FAO. Food and Nutrition Paper No. 81. International Agency for Research on Cancer (IARC). 1993. Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins. Lyon (France): IARC. Monographs on the Evaluation of Carcinogenic Risks to Humans No. 56; p. 489–521. Kamkar A, Karim G, Shojaee Aliabadi F, Khaksar R. 2008. Fate of aflatoxin M1 in Iranian white cheese processing. Food Chem Toxicol. 46:2236–2238. Lo´pez C, Ramos L, Ramadan S, Bulacio L, Perez J. 2001. Distribution of aflatoxin M1 in cheese obtained from milk artificially contaminated. Int J Food Microbiol. 64:211–215. Moss MO. 1998. Recent studies of mycotoxins. J Appl Microbiol Symp Suppl. 84:62S–76S.
Oruc HH, Cibik R, Yilmaz E, Kalkanli O. 2006. Distribution and stability of Aflatoxin M1 during processing and ripening of traditional white pickled cheese. Food Addit Contam A. 23:190–195. Oruc HH, Sonal S. 2001. Determination of aflatoxin M1 levels in cheese and milk consumed in Bursa, Turkey. Vet Hum Toxicol. 43:292–293. Pietri A, Bertuzzi T, Bertuzzi P, Piva G. 1997. Aflatoxin M1 occurrence in samples of Grana Padano cheese. Food Addit Contam A. 14:341–344. Piva G, Pietri A, Galazzi L, Curto O. 1988. Aflatoxin M1 occurrence in dairy products marketed in Italy. Food Addit Contam A. 5:133–139. Prado G, Oliveira MS, Pereira ML, Abrantes FM, Santos LG, Veloso T. 2000. Aflatoxin M1 in samples of ‘minas’ cheese commercialized in the city of Belo Horizonte – Minas, Minas Gerais/Brazil. Cieˆncia Tecnol Alim. 20:398–400. Scott PM. 1990. Natural poisons. In: Helrich K, editor. Official methods of analysis of the Association of Official Analytical Chemists. 15th ed. Arlington: Association of Official Analytical Chemists. p. 1184–1213. Sylos CM, Rodriguez-Amaya DB, Carvalho PRN. 1996. Occurrence of aflatoxin M1 in milk and dairy products commercialized in Campinas, Brazil. Food Addit Contam A. 13:169–172. Yousef A, Marth EH. 1989. Stability and degradation of Aflatoxin M1. In: Egmond HP van, editor. Mycotoxins in dairy products. London (UK): Elsevier Applied Science. p. 127–161.
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VIEW DATASET Survey of aflatoxin M1 in cheese from the North-east region of Sa˜o Paulo, Brazil C.A.F. Oliveiraa*, R.C. Francoa, R.E. Rosima and A.M. Fernandesb a
Department of Food Engineering, School of Animal Science and Food Engineering, University of Sa˜o Paulo, Pirassununga, SP, Brazil; bDepartment of Basic Sciences, School of Animal Science and Food Engineering, University of Sa˜o Paulo, Pirassununga, SP, Brazil (Received 20 September 2010; final version received 7 November 2010) In the present study, 24 samples of Minas Frescal cheese and 24 samples of Minas Padra˜o cheese produced in the North-east region of the state of Sa˜o Paulo, Brazil, were analysed for aflatoxin M1 (AFM1) by high-performance liquid chromatography (HPLC) between March and August 2008. AFM1 was detected in 13 (27.1%) samples at concentrations ranging from 0.037 to 0.313 ng g1. The mean concentrations of AFM1 in positive samples of Minas Frescal and Minas Padra˜o cheese were 0.142 0.118 and 0.118 0.054 ng g1, respectively. It is concluded that the incidence of AFM1 in Minas cheese may contribute to an increase in the overall ingestion of aflatoxins in the diet, hence indicating the need for the adoption of a tolerance limit for AFM1 in cheese in Brazil. Keywords: cheese; aflatoxins; mycotoxins; aflatoxins
Introduction Aflatoxin M1 (AFM1) is a hepatocarcinogen found in the milk of animals that consumed feeds contaminated with aflatoxin B1 (Council for Agriculture Science and Technology (CAST) 2003), the main metabolite produced by storage fungi of the genus Aspergillus, particularly A. flavus, A. parasiticus and A. nomius (Moss 1998). The occurrence of AFM1 in milk and milk products is a public health concern since the International Agency for Research on Cancer (IARC) (1993) classified AFM1 in Group 2, a probable human carcinogen. In milk, AFM1 binds to casein and remains bound to it even after thermal treatment and production of milk products, mainly cheese (Yousef and Marth 1989). The natural occurrence of AFM1 in cheese seems to be highly variable, depending on the original levels of AFM1 in milk and in differences in the manufacturing steps of the product (Lo´pez et al. 2001). Most studies on the occurrence of AFM1 in cheese were carried out in Europe, where some countries adopt tolerance limits for AFM1 in cheese between 0.250 and 0.500 ng g1 (Food and Agriculture Organization (FAO) 2004). In a survey conducted in Italy, AFM1 was detected in 19.5%, 26.5% and 53.5% of cheese samples manufactured in France, Germany and the Netherlands, respectively (Piva et al. 1998). Pietri et al. (1997) analysed 223 samples of Grana Padano cheese and found 91% samples containing AFM1 from 0.005 to 0.100 ng g1,
*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2011 Taylor & Francis DOI: 10.1080/19393210.2010.538934 http://www.informaworld.com
and 6.7% from 0.100 to 0.250 ng AFM1 g1. Barrios et al. (1996) analysed 35 cheese samples produced in Southern Spain and detected AFM1 in 16 samples (44.7%), with concentrations ranging from 0.020 to 0.200 ng g1. A Turkish study showed that the tolerance limit determined in that country (0.250 ng AFM1 g1) was exceeded in 12.28% samples, and the highest level was 0.810 ng g1 (Oruc and Sonal 2001). In Brazil, previous studies indicated a high incidence of AFM1 in fluid milk, but levels were usually below the limit (0.500 mg l1) established by Brazilian regulations (Ageˆncia Nacional de Vigilaˆncia Sanita´ria 2004). However, there is very little information on the occurrence of AFM1 in cheese manufactured in the country, except for a report by Prado et al. (2000), who observed that 74.7% Minas Frescal samples from the state of Minas Gerais showed AFM1 at levels ranging from 0.02 to 6.92 ng g1. Minas cheese is classified as a semi-fat and high-humidity cheese, obtained by the enzymatic coagulation of milk with rennet, added or not with starter cultures. In Brazil, Minas cheese accounts for nearly 10% of the 640,000 kg of cheese produced annually in the country. There are two main types of Minas cheese: the non-ripened one (Minas Frescal), which is to be consumed from up 2–4 days after processing, and the ripened cheese (Minas Padra˜o), which undergoes a ripening period of around 2 months. In the present study, AFM1 was determined both in Minas Frescal and Minas Padra˜o
58
C.A.F. Oliveira et al.
cheese produced and traded in the North-east region of the state of Sa˜o Paulo, Brazil.
0.006
0.004
Forty-eight samples of Minas Frescal and Minas Padra˜o cheese (24 samples of each) were collected. Samples belonged to the 11 brands responsible for the greatest volume of cheese produced and traded in the North-east region of state of Sa˜o Paulo. Samples were collected from March to August 2008, with no repetition of batches. According to the manufacturer’s information, fat and moisture contents in cheese samples varied between 25–30% and 46–55%, respectively. The sampling unit was the 500 g original packaging of each type of cheese. In the first week of each month four samples of cheese (one of each brand, and of each type of cheese) were randomly collected from units available in supermarkets in the cities of Leme and Pirassununga, state of Sa˜o Paulo. Samples were identified, placed in isothermal boxes and immediately sent to the laboratory, where they were ground and stored in sterile polypropylene bags at 5 C.
Analysis of aflatoxin M1 AFM1 determination was performed as recommended by Oruc et al. (2006), with some adaptations proposed by the manufacturer of the immunoaffinity columns Õ (Aflatest ; Vicam, Watertown, MA). The analytical sample (10 g of each duplicate from the same ground sample) was placed in a blender jar, and 5 g of celite, 150 ml of chloroform and 1 ml of a saturated NaCl solution were added. The mixture was blended for 2– 3 min in order to obtain a slurry, which was filtered and evaporated to near dryness. The residue was dissolved in 2 ml of methanol and the volume completed to 100 ml using phosphate buffer prepared according to Deveci (2007). The solution was transferred to a separation funnel and 75 ml of n-hexane were added to it and mixed. The upper (methanolic) phase was collected and directly passed through an immunoaffinity column connected to a glass syringe and a vacuum system (flow of 2–3 ml min1). After the sample was added the column was washed with 20 ml of an ultrapure water (Milli Q; Millipore)–methanol (75 : 25, v/v) solution. After that it was washed with 1 ml of methanol in order to elute AFM1, and the eluate was collected in an amber flask. Identification and quantification of the AFM1 residues was achieved by high-performance liquid chromatography (HPLC), using a Shimadzu 10VP liquid chromatograph with a 10 AXL fluorescence detector (excitation at 366 nm and emission at 440 nm).
AFM1
Sampling
Volts
Material and methods 0.002
0.000
0
2
4
6
8
10
Minutes
Figure 1. Chromatogram showing the retention time of aflatoxin M1 (AFM1) (nearly 6 min) in a naturally contaminated Minas Frescal cheese sample (0.313 ng g1).
A Phenomenex C18 column (4.6 150 mm, 4 mm) and a Shim-Pack pre-column (4 10 mm CLC G-ODS) were used. Calibration curves were prepared using standard solutions of AFM1 (Sigma-Aldrich, Saint Louis, MO) at concentrations of 2.38, 4.75, 9.51 and 19.01 ng ml1, as previously evaluated according to Scott (1990). A total of 20 ml of sample extracts were injected for AFM1 determination. The isocratic mobile phase consisted of methanol–water–acetonitrile (60 þ 20 þ 20, v/v/v) with a flow rate of 1.0 ml min1. Under these conditions the retention time for AFM1 was 6.1 min (Figure 1). The retention time window for peak identification was 8.0% (0.49 min).
Results and discussion The analytical methods used in this trial were evaluated using cheese samples spiked with AFM1 at levels of 0.200 and 0.500 ng g1 (n ¼ 5 per spiking level). Results are shown in Table 1. The limit of determination (LOD) and limit of quantification (LOQ) for AFM1 were determined in spiked cheese samples considering signal-to-noise ratios of 3 : 1 and 6 : 1, respectively. AFM1 levels in cheese samples are presented in Table 2. The toxin was detected in 13 of the 48 analysed samples (27.1%) at levels ranging from 0.030 to 0.313 ng g1. The number of positive samples for AFM1 in Minas Frescal and Minas Padra˜o cheese was six (25.0%) and seven (29.2%), respectively (n ¼ 24 per type of cheese). Minas Frescal cheese samples presented higher mean levels of AFM1 (0.142 0.118 ng g1) compared with Minas Padra˜o cheese (0.118 0.054 ng g1). Results obtained in this trial are different from those of Sylos et al. (1996) who did not find detectable levels of AFM1 in samples of Minas, mozzarella and cheddar cheese traded in Campinas, state of Sa˜o Paulo. However, these authors
Food Additives and Contaminants: Part B
59
Table 1. Performance characteristics of methods for the determination of aflatoxin M1 in Minas Frescal and Minas Padra˜o cheeses. Analyte
Matrix
LOD (ng g–1)
LOQ (ng g–1)
Recovery range (%)a
RSDr (%) (n ¼ 5)
Accreditation
AFM1 AFM1
Minas Frescal cheese Minas Padra˜o cheese
0.010 0.010
0.030 0.030
83.6–84.2 83.4–85.3
5.1–12.9 4.8–14.9
No No
Notes: aSpiking levels: 0.200 and 0.500 ng g–1. LOD, limit of determination; LOQ, limit of quantification; RSD, relative standard deviation.
Table 2. Aflatoxin M1 levels in Minas Frescal and Minas Padra˜o cheese samples traded in Pirassununga, Brazil. Number of samplesa Level of AFM1 (ng g–1)
Minas Frescal, n (%)
Minas Padra˜o, n (%)
Total n (%)
50.030b 0.030–0.050 0.051–0.100 0.101–0.200 40.200 Mean (ng g–1)c
18 (75.0) 1 (4.2) 3 (12.5) 0 (0) 2 (8.3) 0.142 0.118
17 (70.8) 1 (4.2) 2 (8.3) 4 (16.7) 0 (0) 0.118 0.054
35 (72.9) 2 (4.2) 5 (10.4) 4 (8.3) 2 (4.2) 0.129 0.086
Notes: aNumber of samples analysed: 24 for each type of cheese (total ¼ 48 samples). b Limit of quantification of the analytical method¼0.030 ng g–1. c Values are reported as means standard deviation (SD), for samples containing detectable levels of AFM1.
used thin-layer chromatography to identify and quantify AFM1, a technique considered to have lower sensibility when compared to] liquid chromatography. This fact may have influenced the low recovery of AFM1 in the cheese analysed by these authors. The frequency of samples positive for AFM1 in Minas cheese observed in the present study was lower than that reported by Prado et al. (2000) in Belo Horizonte, state of Minas Gerais. These authors detected 74.7% samples positive for AFM1, with levels ranging from 0.02 to 6.92 ng g1, and mean AFM1 concentrations in Minas Frescal of 0.080 ng g1 and in Minas Padra˜o of 0.62 ng g1. These results were different from those results of the present study, which showed that mean concentration of AFM1 in Minas Padra˜o cheese samples was lower than that of Minas Frescal samples. Although data of the literature on AFM1 degradation during cheese maturation are contradictory (Yousef and Marth 1989), the results of this study corroborate the hypothesis that AFM1 stability decreases in ripened cheese. In Brazil there is no legally determined limit for AFM1 levels in cheese. Limits established by Brazilian regulations are only related to powdered milk (5.0 ng g1) and fluid milk for direct human consumption (0.500 ng ml1), which is ten-fold greater than the limit adopted by the European Union (0.050 ng ml1). However, it should be considered that when compared with levels found in milk, the toxin may be more concentrated in cheese. Oruc et al. (2006) and Kamkar et al. (2008) reported that soft cheese presented AFM1
levels 1.8–4.0 times greater than the levels found in milk, which stresses the importance of regulations specifically for AFM1 for cheese. Some countries, such as Turkey and Switzerland, established a tolerance limit of 0.25 ng g1 for AFM1 in cheese (FAO 2004). In the present study two samples (8.3%) of Minas Frescal cheese would not comply with this limit, with AFM1 levels of 0.272 and 0.313 ng g1. However, 11 samples (22.9%) presented AFM1 levels above the tolerance limit for AFM1 in milk adopted by the European Union of 0.050 ng g1.
Conclusion Samples of Minas Frescal and Minas Padra˜o cheese produced in the North-east region of Sa˜o Paulo, Brazil, presented a high incidence of AFM1, with mean concentrations in positive samples of 0.142 0.118 and 0.118 0.054 ng g1, respectively. As AFM1 is one of the most potent carcinogens known, tolerance limits for AFM1 in cheese should be established by Brazilian regulations. Also, further studies are needed to evaluate the overall contribution of cheese for human exposure to this toxin in Brazil.
References Ageˆncia Nacional de Vigilaˆncia Sanita´ria. 2004. Resoluc¸a˜o RDC n 274, de 15 de outubro de 2002; [cited 2009
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C.A.F. Oliveira et al.
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