- Email: [email protected]
Assessment of aflatoxin M1 levels in pasteurized and UHT milk consumed in Prishtina, Kosovo
Accepted Manuscript Assessment of Aflatoxin M1 levels in pasteurized and UHT milk consumed in Prishtina, Kosovo A. Rama, F. Latifi, D. Bajraktari, N. Ramadani PII:
S0956-7135(15)00238-8
DOI:
10.1016/j.foodcont.2015.04.021
Reference:
JFCO 4418
To appear in:
Food Control
Received Date: 15 August 2014 Revised Date:
2 April 2015
Accepted Date: 11 April 2015
Please cite this article as: Rama A., Latifi F, Bajraktari D. & Ramadani N., Assessment of Aflatoxin M1 levels in pasteurized and UHT milk consumed in Prishtina, Kosovo, Food Control (2015), doi: 10.1016/ j.foodcont.2015.04.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
A. Rama1,∗, F.Latifi1, D. Bajraktari2, N.Ramadani3, 1
University of Prishtina, Faculty of Agriculture and Veterinary, Prishtina, Kosovo; 2Food and Veterinary Agency, Prishtina, Kosovo; 3National Institute of Public Health of Kosovo
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Abstract To assess public health hazards associated with the occurrence of AFM1 residues in pasteurized milk and UHT milk a survey was carried out, in Prishtina, capital city of Kosovo. In the present study, a total of 178 samples, 84 pasteurized milk and 94 UHT milk were collected during 6 months (July to December 2013). They were obtained from retail outlets in Prishtina city (Kosovo). The occurrence and concentration range of AFM1 in the samples were investigated by competitive enzyme-linked immunosorbent assay (ELISA) method. There was a high incidence of AFM1 (81.0%) in both pasteurized and UHT milk samples. Eighty three percent (83.3%) of the pasteurized milk samples and seventy eight percent (78.7%) of the UHT milk samples contained AFM1. The positive incidence of AFM1 in the pasteurized milk and the UHT milk samples ranged from 5.16 to 110 ng/L and from 5.02 to 62 ng/L, respectively. AFM1 levels in 18(21.4%) pasteurized milk samples and 4 (4.2%) UHT milk samples exceeded the maximum tolerable limit of the EC according to the European Union regulation limits of 50 ng/L. AFM1 levels in the samples show that there is a presence of high AFM1 level that constitutes a human health risk in Kosovo. The results of this study imply that more emphasis should be given to the routine AFM1 inspection of milk and dairy products in the Prishtina region. Key words: milk, aflatoxin M1, ELISA, Kosovo.
Introduction
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*Corresponding author: Adem Rama, Department of Veterinary sciences, Faculty of Agriculture and Veterinary, University of Prishtina, Prishtina, Kosovo. Tel (+377)44265265 Email: [email protected]
Moulds can grow and produce mycotoxins in plant material during the whole chain from field to table. Mycotoxins are ubiquitous and produced by different Aspergillus species such as Aspergillus flavus and Aspergillus parasiticus. Aflatoxin M1 in milk arises from the metabolic conversion in the animal of Aflatoxin B1, present in contaminated feed.(Van Egmond, 1989, Prandini et al. 2009). Among all of the mycotoxins, aflatoxin is the most problematic in dairy due to its derivative aflatoxin M1 (AFM1), present in milk, and its potential health hazard for human consumption.(Van Egmond, 1991; Wood, 1991). Aflatoxin can be present in several forms in feedstuffs, aflatoxin B1, B2, G1, and G2, with aflatoxin B1 (AFB1)being the most biologically active and toxic to animals (and humans). The AFB1 is rapidly absorbed in the digestive tract and primarily metabolized by liver enzymes, converting it to AFM1, which is then excreted in milk and urine. (Pettersson et al. 1990; Veldman et al., 1992). Although, AFM1 is less mutagenic and carcinogenic than AFB1, it exhibits high genotoxic activity and has been recently evaluated by the International Agency for Research on Cancer (IARC) as a class 2B, possibly carcinogenic to humans (IARC, 1993) but in next classification from 2002 year this toxin was moved to the first group, as a proved carcinogen (IARC, 2002). Aflatoxins exert acute and chronic effects in animals (Aydin et al., 2008). It may cause liver damage, drop in milk production, immune suppression
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Assessment of Aflatoxin M1 levels in pasteurized and UHT milk consumed in Prishtina, Kosovo
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Corresponding: E-Mail: [email protected]; Tel: +377(0)44 265 265; Fax: +381 38 603 102
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and anemia. Furthermore, it is also associated with reduced feed consumption and overall retarded growth and development in dairy cattle (Akande et al., 2006). The presence of AFM1 in milk and dairy products may pose a threat, therefore to reduce the risk of exposure many countries have established maximum levels of permissible AFM1 in milk, varies from one country to another and is dependent on economic considerations and regulatory systems (Stoloff, Van Egmond and Parks, 1991). According to the European Union regulations, the maximum level of AFM1 in milk should not exceed 50 ng/L. (European Commission, 2006b). Regulation for MRL of AFM1 in milk in Kosovo was recently adopted and harmonized with European Union Regulation (Official journal of RK, 2013). While the USA regulations prescribe that the maximum level should not be higher than 500 ng/L (FDA, 2011). Due to lack of data regarding AFM1 presence in milk from Kosovo, the aim of this survey was to evaluate milk safety for consumer consumption and to underline the importance of continuous surveillance for AM1 in milk to ensure consumer health.
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A total of 178 pasteurized milk and UHT milk samples from different supermarkets in Prishtina city were collected randomly during 6 months (July to December 2013). Immediately after collection samples were transported to the laboratory and analyzed. Sampling was performed according to EU requirements (European Commission, 2006a).
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2.2. Aflatoxin analysis by ELISA
65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89
Determination of AFM1 was done by Enzyme Linked Immunosorbent Assay (ELISA) method using I ‘screen AFLAM1 test kit (Tecna S. r. l., Trieste, Italy). Analyses were performed according to the test kits instructions. Procedure is based on binding of free AFM1 from samples and standard solutions to the anti-AFM1 antibodies during first incubation. Any unbound substance is removed in a washing step. A second incubation is performed with an aflatoxin-HRP conjugate, which covers all the remaining free binding sites of the antibody. The bound enzyme activity is determined by adding a fixed amount of a chromogenic substrate. The enzyme converts the colorless chromogen into a blue product during the third incubation. The addition of the stop reagent leads to a color change from blue to yellow. The absorbance is measured by a microplate reader (Bio-Tek, USA) at 450 nm. The color development is inversely proportional to the AFM1 concentration in the sample. Concentration of AFM1 was calculated from calibration curve which was obtained using 7 standards with the following concentrations: 0, 5, 10, 25, 50, 100 and 250 ng/L. The analytical quality of ELISA method was assured by the use of certified reference material (CRM) as well by participation in interlaboratory study. Partially defatted raw lyophilized milk with certified AFM1 content of 11.11 ng/kg was used as CRM ( MI1043A-1), Progeto Trieste. Test Veritas, Padova, Italy for validation of I’ screen AFLAM1 test kit. The validation parameters (Table 1) were calculated and expressed using Europian official Decision procedure for screening methods (European Commission,2002b) and their values were in accordance with recommendations given in Commission Decision (European Commission 2006a). Further, obtained Z value of 0.32 in interlaboratory study confirmed analytical quality of the results obtained by ELISA method ( Progeto Trieste 2010- Mycotoxins - Italy).
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2. Materials and methods
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Table 1 Validation parameters
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Results
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Table 2 Occurrence of Aflatoxin M1 in commercial liquid milk in Kosovo
I ‘screen AFLAM1 test kit 9 25 14.84 91.89 94
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LOD LOQ Standard deviation Coefficient of Variation Recovery LOD: limit of detection (ng/kg) LOQ: limit of quantification (ng/kg)
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2.3. Sample preparation Milk samples were prepared according to the manufacturer’s instructions. Samples were centrifuged at 3000 g for 10 min. The upper creamy layer was removed by Pasteur pipette and 100 ml from the lower phase was used for the analysis.
Sample tested n
Pasteurized milk UHT milk Total
84 94 178
Positive samples n (%) Concentrations (ng/l)___________________________________ Total samples mean±SD Positive samples___________ mean (SD) Min-Max 70 (83.3) 27.8±27.0 32.4±27.4 5.16-110.93 74(78.7) 17.9±14.0 21.4±13.9 5.02-62.26 144 (80.8) 22.6±21.7 26.8±22.1 5.02-110.93
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The occurrence and levels of AFM1 in Kosovo commercial liquid milk samples are presented in Tables 2 and 3. AFM1 was found above measurable level (5 ng/L) in 83.3% (70/84) and 78.7% (74/94) of pasteurized and UHT milk samples, respectively. Altogether, the toxin was detected in 144 commercial liquid milk samples, corresponding to 81.0% of the total samples examined, ranging from 5.0 to 110.9 ng/l. Considering the European Commission limit, 18 (21.4%) and 4 (4.2%) contaminated pasteurized and UHT milk samples had AFM1 in concentrations in excess of the maximum tolerance limit, respectively. As shown in Table 3, most of the contaminated samples (68.5%) contained AFM1 at the level of 5–50 ng/L. The mean concentration of AFM1 in pasteurized milk was 27.8±27.0 while in UHT milk it was 17.9±14.0.
Table 3 Levels of aflatoxin M1 in commercial liquid milk in Kosovo Milk Products
Distribution of samples n (%)______________________________________________________________ ˂5 ng/la 5-10 ng/l 11-25 ng/l 26-50 ng/l 50-80 ng/l ˃80 ng/l
Pasteurized milk 14 (16.6) UHT milk 20 (21.2) Total 34 (19.1) a Distribution of negative samples.
13 (15.4) 16 (17.0) 29 (16.2)
27 (32.1) 37 (39.3) 64 (35.9)
12 (14.2) 17 (18.0) 29 (16.2)
12 (14.2) 4 (4.2) 16 (8.9)
6 (7.1) 0 (0.0) 6 (3.3)
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Discussion
Location Spain Albania Greece Turkey(Ankara) Turkey Italy Kosovo Serbia Serbia
Percent of contaminated milk samples ˃50 ng/l 4.26 8.3 0 3.7 47 0.6 0 76 86
References Blanco et al.(1988) Panariti (2001) Roussi et al.(2002) Gurbay et al(2006) Unusan et al.(2006) Nachtmann et al.(2007) Gallina et al.(2013) Skrbic et al.(2014) Kos et al.(2014)
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There is little information about the occurrence of AFM1 in milk products in Kosovo. Studies undertaken by (Gallina et al. 2013) showed that 1 of 39 UHT samples were found contaminated with AFM1 but lower than the maximum tolerance limit (50 ng/L). Comparing previous data with present results obtained in Kosovo milk contamination with AFM1 in Prishtina shoved to be increased more than standard levels. Our results showed that contamination with AFM1 exceeding allowable limits and comparing to other reports from the region contamination is lower (25.6%), most probably because of the time period when samples were collected (milk contamination happened in the entire region during first half of the year 2013 and our sampling has been done in second half of the year 2013), and due to media coverage of the problem food safety measures were taken by the government and producers to lower presence of AFM1 in milk prior our investigation. According to results obtained in Kosovo, the incidence and contamination levels of AFM1 seem to be a problem for public health, especially infants and children.
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155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178
No. of samples UHT Pasteurized ng/l 47 120 17 3 27 129 316 39 50 150
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Table 4 The occurrence of AFM1 in milk samples in other countries
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In our study, AFM1 was determined in 83.3% of the pasteurized milk samples and 78.7% of the UHT milk samples. These results are in parallel with the findings of some previous reports (Unusan,2006; Tekinsen K et al.,2008; Fonseca et al., 2008; Skrbic et al., 2014; Kos et al.2014) which pointed out the presence of AFM1 in more than 80% of the UHT milk samples (Table 4). However, as seen on Table 4 a number of researchers (Blanco et al., 1988; Panariti, 2001; Gurbay et al., 2006; Gallina et al., 2013; Roussi et al, 2002; Nachtmann et al, 2007) reported a lower incidence or not detected of AFM1 in UHT milk and pasteurized milk. The variations in AFM1 levels among studies could be related to geographic and climatic differences but also to differences in feeding systems, and farm management practices. (Blanco et al., 1988; Tekins_en and Ucar 2008).
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Conclusions
The present investigation is the second performed in Kosovo to evaluate the presence of AFM1 residues in foodstuffs, especially in milk. The commercial milk samples analyzed in this study were selected from the markets generally distributed to all markets in Kosovo. Therefore, it can be considered that our results might give an idea about contamination profile with AFM1 for these milk samples in Kosovo. Since there is not enough study in Kosovo about the AFM1 content of milk and dairy products, more studies are required to be done. Ultimately, surveillance should be continuous, widespread, and must be conducted by government and related ministry.
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Acknowledgments: The project was funded with grants from Institute for international Education the Agency of the US Department of State.
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References
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Akande, K. E., Abubakar, M. M., Adegbola, T. A., & Bogoro, S.E.(2006). Nutritional and health implications of mycotoxin in animal feed. Pakistan Journal of Nutrition. 5(5), 398-403.
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Aydin, A., Gunsen, U., & Demirel, S. (2008). Total aflatoxin B1 and ochratoxin A levels in Turkish wheat flour. Journal Food and Drug Analysis, 16(2), 48-53.
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Blanco, JL., Dominguez, L., Gomez-Lucia, E., Garayzabal, JFF., Garcia, JA., & Suarez, G.(1988). Presence of aflatoxin M1 in commercial ultra-high-temperature-treated milk. applied environmental microbiology. 54, 1622-1623.
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European Commission. (2006a). Commission Regulation 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 Union, L 70, 12e34. European Commission. (2006b). Commission Regulation 1881/2006 of 19 December2006 setting maximum levels for certain contaminants in foodstuffs as regards Fusarium toxins in maize and maize products. Official Journal of the European Union, L 364, 5e18.
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FDA U.S. Food and Drug Administration. (2011). Guidance for industry: Action levels for poisonous or deleterious substances in human food and animal feed. Available from http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/ GuidanceDocuments/ChemicalContaminantsandPesticides/ucm077969.htm. Fonseca, GP., Cruzi, AG., Faria, AF., Silvia, R., Moura, MRL, & Carvalho, LMJ.(2009). Antibiotic residues in Brazilian UHT milk. a screening study. Ciência e Tecnologia de Alimentos. 29, 451-453.
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Gallina, G., Rama, A., Lucatello, L., Benetti, C., Bajraktari, D., Uka, K, Montesissa C.(2013). Aflatoxin M1 Contamination and antibacterial residues in milk in Kosovo. Trends in Veterinary Sciences. 2013, pp
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Gurbay, A., Aydin, S., Girgin, G., Engin, AB, & Sahin, G.(2006). Assessment of aflatoxin M1 levels in milk in Ankara, Turkey. Food Control, 17, 1-4. IARC, International Agency for Research on Cancer. (1993). Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC, International Agency for Research on Cancer. (2002). Monograph on the evaluation of carcinogenic risk to humans (Vol. 82 (pp. 171). Lyon, France: World Health Organization, IARC. Kos, A., Levic, J., Djuragic, O., Kokic, B., & Miladinovic, I.(2014). Occurrence and estimation of aflatoxin M1 exposure in milk in Serbia. Food Control, 38, 41-46.
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Nachtmann, C.S., Gallina, M,. Rastelli, G.L., Decastelli, Ferro, & Decastelli, L.(2007). Regional monitoring plan regarding the presence of aflatoxin M1 in pasteurized and UHT milk in Italy. Food Control, 18 6, pp. 623–629.
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Official journal of Republic of Kosovo, 2013 (GRK). No. 43/2013 laying down maximum levels of certain contaminants in foodstuffs and undesirable substances in animal feeding stuffs. Panariti, E.(2001). Seasonal variations of aflatoxin m1 in the farm milk in albania Arhiv za Higijenu Rada i Toksikologiju. 52, pp. 37–41
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Pettersson, H., Bertilsson, J., & Wennberg, O.(1989). Carry-over of aflatoxin from dairy cattle feed to milk. Healthy animals, safe foods, healthy man. World association of veterinary food hygienists Xth jubilee international symposium, pp. 97–102. Prandini, A., Tansini, G., Sigolo, S., Filippi, L., Laporta, M., & Piva, G.(2009). On occurrence of aflatoxin M1 in milk and dairy products. Food and Chemical Toxicology. 47, 984-991.
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Roussi, V., Govaris, A., Varagouli, A., & Botsoglou, N. A.(2002). Occurrence of aflatoxin M1 in raw and market milk commercialized in Greece. Food Additives and Contaminants, London, v. 19, n. 9, p. 863868. Skrbic, B., Zivancev, J., Antic, I., & Godula, M.(2014). Levels of aflatoxin M1 in different types of milk collected in Serbia: Assessment of human and animal exposure. Food Control, 40, 113-119 Stoloff, L., Van Egmond, H.P., & Parks, D.L.(1991). Rationales for the establishment of limits and regulations for mycotoxins. Food Additives & Contaminants. 8, 213–221.
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Tekinsen, KK., Ucar, G.(2008). Aflatoxin M1 levels in butter and cream cheese consumed in Turkey. Food Control, 19, 27-30. Tekinsen, KK., Eken, S.(2008). Aflatoxin M1 levels in UHT milk and kashar cheese consumed in Turkey. Food and Chemical Toxicology. 46, 3287-3289.
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Unusan, N. (2006). Occurrence of aflatoxin M1 in UHT milk in Turkey, Food and Chemical Toxicology. 44, 1897–1900. Van Egmond, H. P. (1989). Introduction. In H.P. Van Egmond (Ed.) Mycotoxins in dairy products (pp. 110). London and New York: Elsevier Applied Science.
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Van Egmond, H. P. (1991). Mycotoxins. international dairy federation special issue. 101, 131-135. Veldman, A., Meijs J.A.C, Borggreve, G.J., & Heeres van der Tol J.J.(1992). Carry-over of aflatoxin from cows' food to milk. Animal Production. 55, 163-168. Wood, G. E. (1991). Aflatoxin M1, Mycotoxins and Phytoalexins (pp.145-163) (Chapter 6). London: CRC In.
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Highlights
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Presence and levels of AFM1 in pasteurized milk was examined implementing ELISA method High percentage of contaminated samples was detected, 25.6% exceeded MRL Our study might give an idea about contamination profile of milk with AFM1. Further systemic monitoring and investigation of AFM1 contamination are necessary.
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1. 2. 3. 4.
S0956-7135(15)00238-8
DOI:
10.1016/j.foodcont.2015.04.021
Reference:
JFCO 4418
To appear in:
Food Control
Received Date: 15 August 2014 Revised Date:
2 April 2015
Accepted Date: 11 April 2015
Please cite this article as: Rama A., Latifi F, Bajraktari D. & Ramadani N., Assessment of Aflatoxin M1 levels in pasteurized and UHT milk consumed in Prishtina, Kosovo, Food Control (2015), doi: 10.1016/ j.foodcont.2015.04.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
A. Rama1,∗, F.Latifi1, D. Bajraktari2, N.Ramadani3, 1
University of Prishtina, Faculty of Agriculture and Veterinary, Prishtina, Kosovo; 2Food and Veterinary Agency, Prishtina, Kosovo; 3National Institute of Public Health of Kosovo
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Abstract To assess public health hazards associated with the occurrence of AFM1 residues in pasteurized milk and UHT milk a survey was carried out, in Prishtina, capital city of Kosovo. In the present study, a total of 178 samples, 84 pasteurized milk and 94 UHT milk were collected during 6 months (July to December 2013). They were obtained from retail outlets in Prishtina city (Kosovo). The occurrence and concentration range of AFM1 in the samples were investigated by competitive enzyme-linked immunosorbent assay (ELISA) method. There was a high incidence of AFM1 (81.0%) in both pasteurized and UHT milk samples. Eighty three percent (83.3%) of the pasteurized milk samples and seventy eight percent (78.7%) of the UHT milk samples contained AFM1. The positive incidence of AFM1 in the pasteurized milk and the UHT milk samples ranged from 5.16 to 110 ng/L and from 5.02 to 62 ng/L, respectively. AFM1 levels in 18(21.4%) pasteurized milk samples and 4 (4.2%) UHT milk samples exceeded the maximum tolerable limit of the EC according to the European Union regulation limits of 50 ng/L. AFM1 levels in the samples show that there is a presence of high AFM1 level that constitutes a human health risk in Kosovo. The results of this study imply that more emphasis should be given to the routine AFM1 inspection of milk and dairy products in the Prishtina region. Key words: milk, aflatoxin M1, ELISA, Kosovo.
Introduction
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*Corresponding author: Adem Rama, Department of Veterinary sciences, Faculty of Agriculture and Veterinary, University of Prishtina, Prishtina, Kosovo. Tel (+377)44265265 Email: [email protected]
Moulds can grow and produce mycotoxins in plant material during the whole chain from field to table. Mycotoxins are ubiquitous and produced by different Aspergillus species such as Aspergillus flavus and Aspergillus parasiticus. Aflatoxin M1 in milk arises from the metabolic conversion in the animal of Aflatoxin B1, present in contaminated feed.(Van Egmond, 1989, Prandini et al. 2009). Among all of the mycotoxins, aflatoxin is the most problematic in dairy due to its derivative aflatoxin M1 (AFM1), present in milk, and its potential health hazard for human consumption.(Van Egmond, 1991; Wood, 1991). Aflatoxin can be present in several forms in feedstuffs, aflatoxin B1, B2, G1, and G2, with aflatoxin B1 (AFB1)being the most biologically active and toxic to animals (and humans). The AFB1 is rapidly absorbed in the digestive tract and primarily metabolized by liver enzymes, converting it to AFM1, which is then excreted in milk and urine. (Pettersson et al. 1990; Veldman et al., 1992). Although, AFM1 is less mutagenic and carcinogenic than AFB1, it exhibits high genotoxic activity and has been recently evaluated by the International Agency for Research on Cancer (IARC) as a class 2B, possibly carcinogenic to humans (IARC, 1993) but in next classification from 2002 year this toxin was moved to the first group, as a proved carcinogen (IARC, 2002). Aflatoxins exert acute and chronic effects in animals (Aydin et al., 2008). It may cause liver damage, drop in milk production, immune suppression
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Assessment of Aflatoxin M1 levels in pasteurized and UHT milk consumed in Prishtina, Kosovo
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Corresponding: E-Mail: [email protected]; Tel: +377(0)44 265 265; Fax: +381 38 603 102
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and anemia. Furthermore, it is also associated with reduced feed consumption and overall retarded growth and development in dairy cattle (Akande et al., 2006). The presence of AFM1 in milk and dairy products may pose a threat, therefore to reduce the risk of exposure many countries have established maximum levels of permissible AFM1 in milk, varies from one country to another and is dependent on economic considerations and regulatory systems (Stoloff, Van Egmond and Parks, 1991). According to the European Union regulations, the maximum level of AFM1 in milk should not exceed 50 ng/L. (European Commission, 2006b). Regulation for MRL of AFM1 in milk in Kosovo was recently adopted and harmonized with European Union Regulation (Official journal of RK, 2013). While the USA regulations prescribe that the maximum level should not be higher than 500 ng/L (FDA, 2011). Due to lack of data regarding AFM1 presence in milk from Kosovo, the aim of this survey was to evaluate milk safety for consumer consumption and to underline the importance of continuous surveillance for AM1 in milk to ensure consumer health.
60 61 62 63
A total of 178 pasteurized milk and UHT milk samples from different supermarkets in Prishtina city were collected randomly during 6 months (July to December 2013). Immediately after collection samples were transported to the laboratory and analyzed. Sampling was performed according to EU requirements (European Commission, 2006a).
64
2.2. Aflatoxin analysis by ELISA
65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89
Determination of AFM1 was done by Enzyme Linked Immunosorbent Assay (ELISA) method using I ‘screen AFLAM1 test kit (Tecna S. r. l., Trieste, Italy). Analyses were performed according to the test kits instructions. Procedure is based on binding of free AFM1 from samples and standard solutions to the anti-AFM1 antibodies during first incubation. Any unbound substance is removed in a washing step. A second incubation is performed with an aflatoxin-HRP conjugate, which covers all the remaining free binding sites of the antibody. The bound enzyme activity is determined by adding a fixed amount of a chromogenic substrate. The enzyme converts the colorless chromogen into a blue product during the third incubation. The addition of the stop reagent leads to a color change from blue to yellow. The absorbance is measured by a microplate reader (Bio-Tek, USA) at 450 nm. The color development is inversely proportional to the AFM1 concentration in the sample. Concentration of AFM1 was calculated from calibration curve which was obtained using 7 standards with the following concentrations: 0, 5, 10, 25, 50, 100 and 250 ng/L. The analytical quality of ELISA method was assured by the use of certified reference material (CRM) as well by participation in interlaboratory study. Partially defatted raw lyophilized milk with certified AFM1 content of 11.11 ng/kg was used as CRM ( MI1043A-1), Progeto Trieste. Test Veritas, Padova, Italy for validation of I’ screen AFLAM1 test kit. The validation parameters (Table 1) were calculated and expressed using Europian official Decision procedure for screening methods (European Commission,2002b) and their values were in accordance with recommendations given in Commission Decision (European Commission 2006a). Further, obtained Z value of 0.32 in interlaboratory study confirmed analytical quality of the results obtained by ELISA method ( Progeto Trieste 2010- Mycotoxins - Italy).
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2. Materials and methods
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Table 1 Validation parameters
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Results
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Table 2 Occurrence of Aflatoxin M1 in commercial liquid milk in Kosovo
I ‘screen AFLAM1 test kit 9 25 14.84 91.89 94
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LOD LOQ Standard deviation Coefficient of Variation Recovery LOD: limit of detection (ng/kg) LOQ: limit of quantification (ng/kg)
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2.3. Sample preparation Milk samples were prepared according to the manufacturer’s instructions. Samples were centrifuged at 3000 g for 10 min. The upper creamy layer was removed by Pasteur pipette and 100 ml from the lower phase was used for the analysis.
Sample tested n
Pasteurized milk UHT milk Total
84 94 178
Positive samples n (%) Concentrations (ng/l)___________________________________ Total samples mean±SD Positive samples___________ mean (SD) Min-Max 70 (83.3) 27.8±27.0 32.4±27.4 5.16-110.93 74(78.7) 17.9±14.0 21.4±13.9 5.02-62.26 144 (80.8) 22.6±21.7 26.8±22.1 5.02-110.93
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The occurrence and levels of AFM1 in Kosovo commercial liquid milk samples are presented in Tables 2 and 3. AFM1 was found above measurable level (5 ng/L) in 83.3% (70/84) and 78.7% (74/94) of pasteurized and UHT milk samples, respectively. Altogether, the toxin was detected in 144 commercial liquid milk samples, corresponding to 81.0% of the total samples examined, ranging from 5.0 to 110.9 ng/l. Considering the European Commission limit, 18 (21.4%) and 4 (4.2%) contaminated pasteurized and UHT milk samples had AFM1 in concentrations in excess of the maximum tolerance limit, respectively. As shown in Table 3, most of the contaminated samples (68.5%) contained AFM1 at the level of 5–50 ng/L. The mean concentration of AFM1 in pasteurized milk was 27.8±27.0 while in UHT milk it was 17.9±14.0.
Table 3 Levels of aflatoxin M1 in commercial liquid milk in Kosovo Milk Products
Distribution of samples n (%)______________________________________________________________ ˂5 ng/la 5-10 ng/l 11-25 ng/l 26-50 ng/l 50-80 ng/l ˃80 ng/l
Pasteurized milk 14 (16.6) UHT milk 20 (21.2) Total 34 (19.1) a Distribution of negative samples.
13 (15.4) 16 (17.0) 29 (16.2)
27 (32.1) 37 (39.3) 64 (35.9)
12 (14.2) 17 (18.0) 29 (16.2)
12 (14.2) 4 (4.2) 16 (8.9)
6 (7.1) 0 (0.0) 6 (3.3)
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Discussion
Location Spain Albania Greece Turkey(Ankara) Turkey Italy Kosovo Serbia Serbia
Percent of contaminated milk samples ˃50 ng/l 4.26 8.3 0 3.7 47 0.6 0 76 86
References Blanco et al.(1988) Panariti (2001) Roussi et al.(2002) Gurbay et al(2006) Unusan et al.(2006) Nachtmann et al.(2007) Gallina et al.(2013) Skrbic et al.(2014) Kos et al.(2014)
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There is little information about the occurrence of AFM1 in milk products in Kosovo. Studies undertaken by (Gallina et al. 2013) showed that 1 of 39 UHT samples were found contaminated with AFM1 but lower than the maximum tolerance limit (50 ng/L). Comparing previous data with present results obtained in Kosovo milk contamination with AFM1 in Prishtina shoved to be increased more than standard levels. Our results showed that contamination with AFM1 exceeding allowable limits and comparing to other reports from the region contamination is lower (25.6%), most probably because of the time period when samples were collected (milk contamination happened in the entire region during first half of the year 2013 and our sampling has been done in second half of the year 2013), and due to media coverage of the problem food safety measures were taken by the government and producers to lower presence of AFM1 in milk prior our investigation. According to results obtained in Kosovo, the incidence and contamination levels of AFM1 seem to be a problem for public health, especially infants and children.
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155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178
No. of samples UHT Pasteurized ng/l 47 120 17 3 27 129 316 39 50 150
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Table 4 The occurrence of AFM1 in milk samples in other countries
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In our study, AFM1 was determined in 83.3% of the pasteurized milk samples and 78.7% of the UHT milk samples. These results are in parallel with the findings of some previous reports (Unusan,2006; Tekinsen K et al.,2008; Fonseca et al., 2008; Skrbic et al., 2014; Kos et al.2014) which pointed out the presence of AFM1 in more than 80% of the UHT milk samples (Table 4). However, as seen on Table 4 a number of researchers (Blanco et al., 1988; Panariti, 2001; Gurbay et al., 2006; Gallina et al., 2013; Roussi et al, 2002; Nachtmann et al, 2007) reported a lower incidence or not detected of AFM1 in UHT milk and pasteurized milk. The variations in AFM1 levels among studies could be related to geographic and climatic differences but also to differences in feeding systems, and farm management practices. (Blanco et al., 1988; Tekins_en and Ucar 2008).
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140 141 142 143 144 145 146 147 148 149 150 151 152 153 154
Conclusions
The present investigation is the second performed in Kosovo to evaluate the presence of AFM1 residues in foodstuffs, especially in milk. The commercial milk samples analyzed in this study were selected from the markets generally distributed to all markets in Kosovo. Therefore, it can be considered that our results might give an idea about contamination profile with AFM1 for these milk samples in Kosovo. Since there is not enough study in Kosovo about the AFM1 content of milk and dairy products, more studies are required to be done. Ultimately, surveillance should be continuous, widespread, and must be conducted by government and related ministry.
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Acknowledgments: The project was funded with grants from Institute for international Education the Agency of the US Department of State.
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Highlights
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Presence and levels of AFM1 in pasteurized milk was examined implementing ELISA method High percentage of contaminated samples was detected, 25.6% exceeded MRL Our study might give an idea about contamination profile of milk with AFM1. Further systemic monitoring and investigation of AFM1 contamination are necessary.
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1. 2. 3. 4.