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Occurrence of aflatoxin M1 in some samples of UHT, raw & pasteurized milk from Indian states of Karnataka and Tamilnadu
Food and Chemical Toxicology 50 (2012) 4158–4162
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Occurrence of aflatoxin M1 in some samples of UHT, raw & pasteurized milk from Indian states of Karnataka and Tamilnadu Vinutha Siddappa, Divyashree Kallenahalli Nanjegowda, Prema Viswanath ⇑ Food Safety & Analytical Quality Control Laboratory, Central Food Technological Research Institute, Mysore 570020, India
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Article history: Received 15 June 2012 Accepted 14 August 2012 Available online 24 August 2012 Keywords: Aflatoxin M1 UHT milk Raw milk HPLC India
a b s t r a c t Aflatoxin M1 (AFM1) is a toxic metabolite found in the milk of lactating animals which have consumed feedstuffs contaminated with aflatoxin B1. Ultra high temperature treated (UHT) milk is a product which is becoming popular in developing countries like India as there is a lack of proper cold storage or refrigeration facilities. In this study, 45 samples of UHT milk of popular brands prevalent in the market were analyzed for the presence of AFM1 by reversed phase HPLC using fluorescent detector after cleanup of sample with immunoaffinity columns. All samples of plain UHT milk were positive for AFM1 and 38% of these contained levels more than 0.5 lg/kg, the maximum permitted limit prescribed by the Codex Alimentarius Commission and by the mandatory regulations of the country, the FSSAI Regulations, 2011. In 62.5% of flavored UHT milk, AFM1 was below detectable levels (0.02 lg L1). However, 12.5% of these samples also contained levels exceeding the maximum permitted limits. AFM1 was present in 61.6% of the 52 raw milk samples analyzed from the two states of Karnataka and Tamilnadu with a range of 0.1–3.8 lg L1. 17.3% of these samples also exceeded the regulatory limits of the country. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction India is the world’s largest milk producing country with an output of 110 million tonnes per annum (FAO, 2010). This country has a tropical climate with conditions favorable for the growth of aflatoxigenic fungi. Aflatoxins are toxic fungal metabolites produced by Aspergillus species, mainly byAspergillus flavus andAspergillus parasiticus, but also by Aspergillus nomius, Aspergillus pseudotamarii, Aspergillus ombycis, Aspergillus ochraceoroseus, and Aspergillus australis (IARC, 2012). Aflatoxins consist of a group of approximately 20 related metabolites, of which aflatoxins B1, B2, G1 and G2 are normally found in foods (Van Egmond, 1989). Their prevalence has been reported in a wide variety of agricultural products and animal fodder (Peraica et al., 1999; Eaton and Groopman, 1994) as a result of molds contamination before or during harvest or improper storage (Sweeney and Dobson, 1998). Aflatoxin B1 (AFB1) is metabolized by the animals consuming these contaminated feeds to AFM1 mainly by the hepatic microsomal mixed-function oxidase system (Marsi et al., 1974). The amount of AFM1 that is excreted in milk as a percentage of AFB1 is an average of 1–2%. This can vary from animal to animal and from season to season. The AFM1 is detected in milk 12–24 h after the first AFB1 ingestion, reaching a high level after a few days. If there is no further intake of AFB1 through feed, the AFM1 concentration in the milk decreases to an undetectable level after 72 h (Van Egmond, 1989). ⇑ Corresponding author. Tel.: +91 0821 2514972; fax: +91 0821 2412064. E-mail addresses: [email protected], [email protected] (P. Viswanath). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.08.034
Aflatoxins are one of the major etiological factors in the development of hepatocellular carcinoma (IARC, 2012), and more recently associations between childhood aflatoxin exposure and growth stunting have been reported (Gong et al., 2004). It has been calculated that about 27% of the hepatocellular carcinoma cases reported in Southeast Asia is aflatoxin induced (Liu and Wu, 2010). AFM1 has less mutagenic properties than AFB1 (Hsieh et al., 1984). However because of the toxic and carcinogenic effects of AFM1 IARC (International Agency for Research on Cancer) of WHO reconsidered its carcinogenic categorization and changed it from Group 2B to Group 1 (IARC, 2012). The prevalence of AFM1 in raw milk and milk products has been reported in many countries (WHO, 2010). One of the milk products is ultra high temperature treated milk (UHT) which is valuable for the fact that it is shelf stable. These products can therefore be marketed in places where there are no cold storage facilities. In India, UHT milk currently accounts for less than 1% of the total liquid milk market in India. However, International Market Analysis Research and Consulting Group (IMARC), one of the world’s leading research and advisory firms, expects the Indian UHT Milk market to more than triple its current size during 2010–11 and 2016–17 (Anonymous, 2011). ‘‘UHT (ultra high temperature) treatment of milk and liquid milk products is the application of heat to a continuously flowing product using such high temperatures for such time that renders the product commercially sterile at the time of processing. When the UHT treatment is combined with aseptic packaging, it results in a commercially sterile product. UHT treatment is normally in
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V. Siddappa et al. / Food and Chemical Toxicology 50 (2012) 4158–4162 Table 1 Occurrence of Aflatoxin M1 in ultra high temperature (UHT) treated milk. Sample category
No. of samples
No. of positive samples
UHT plain milk UHT flavored milk Total
21 24 45
21 (100) 9 (37.5) 30 (66.6)
Range of aflatoxin M1 concentration (lg L1) <0.02
0.02–0.05
0.05–0.1
0.1–0.5
>0.5
– 15(62.5) 15(62.5)
– – –
3 (14.2) 2 (8.3) 5 (11.1)
10 (47.6) 4 (16.6) 14 (31.1)
8 (38.0) 3 (12.5) 11 (24.4)
Values in parentheses indicate% samples.
the range of 135–150 °C in combination with appropriate holding times necessary to achieve commercial sterility’’ (FAO & WHO, 2007). There have been varying reports on the effect of heat either through pasteurization or sterilization on the AFM1 in milk. (Purchase et al., 1972) observed that a 32% reduction in AFM1 can be achieved by pasteurization at 62 °C for 30 min. In another report, depending on the conditions employed in heating of the milk, a decrease in the AFM1 content of the milk of between 12% and 35% was observed (Kabak, 2009). Differences in the reports of reduction of AFM1 can be attributed to the wide range of temperature, different analytical methods, and employment of both naturally and artificially contaminated milk. In general it has been observed that aflatoxins are stable during heat-treatments (Van Egmond et al., 1977; Wiseman and Marth, 1983; Yousef and Marth, 1989; Govaris et al., 2001; Prandini et al., 2009). This manuscript is the first report of the occurrence of AFM1 in UHT milk of the major brands prevalent in the Indian market. Occurrence of the toxin in raw and pasteurized milk randomly received for analysis from milk producers in the states of Karnataka and Tamil Nadu is also presented. The quantitative analysis of AFM1 in the milk samples was performed by reversed phase High Performance Liquid Chromatography with fluorometric detection after sample clean-up using immunoaffinity columns (AOAC, 2005). 2. Materials and methods 2.1. Samples A total of 45 samples of shelf stable UHT milk (Plain and flavoured) of different brands before their expiry date and with date of manufacture from April to July 2011 that is sold in the local market were purchased from retail shops in the city of Mysore, Karnataka state, India. These brands are also sold all over India. 52 samples of raw and pasteurized milk were voluntarily sent by milk producers from the states of Karnataka and Tamil Nadu for determination of the presence of AFM1. These samples were transported to the laboratory inside an ice box at temperature of less than 4 °C. 2.2. Chemicals Aflatoxin M1 (AFM1) was obtained from Sigma Chemical Co (St. Louis, MO, USA). HPLC grade solvents were purchased from Merck, Riedel (Darmstadt, Germany). AFM1 immunoaffinity columns (AFLAPREPÒ M) were obtained from R-BIOPHARM RHONE LTD, Glasgow Scotland. Other chemicals namely, methanol and acetonitrile were also obtained from Merck, Germany. All water used was purified by a MilliQ Water System (Millipore-Synergy UC, France). 2.3. Sample preparation The milk sample in the sachet or tetrapak was mixed well, warmed to about 40 °C, centrifuged at 4000 rpm for 15 min at room temperature and filtered through Whatman No. 1 filter paper. A volume of 50 ml of filtrate was collected for analysis as outlined in the AOAC Official method (AOAC, 2005). 2.4. Extraction procedure The procedure was as outlined in the AOAC Official method (AOAC, 2005). The 50 ml prepared filtrate was passed through the immunoaffinity column at a flow rate of about 2–3 ml/min. Two 10 ml portions of phosphate buffered saline were then passed through the column at approximately 5 ml/min. Air was then pressed through the column to remove any residual liquid. AFM1 was eluted with three
Fig. 1. HPLC chromatograms of (A) a negative sample, (B) a naturally contaminated milk sample, (C) milk sample spiked at 0.02 lg L1. 0.5 ml portions of a mixture of methanol:acetonitrile (2:3, v/v) into a clean glass tube at approximately one drop every 2–3 seconds. Care was taken to ensure com-
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V. Siddappa et al. / Food and Chemical Toxicology 50 (2012) 4158–4162 Table 2 Occurrence of Aflatoxin M1 in pasteurized and raw milk. Sample category
No. of samples
No. of positive samples
Pasteurized milk Raw milk Total
7 45 52
3 (42.9) 29 (64.4) 32 (61.5)
Range of aflatoxin M1 concentration (lg L1) <0.02
0.02–0.05
0.05–0.1
0.1–0.5
>0.5
4 (7.7) 16 (30.8) 20 (38.4)
– 7 (13.5) 7 (13.5)
– 5 (9.6) 5 (9.6)
– 11 (21.2) 11 (21.2)
3 (5.8) 6 (11.5) 9 (17.3)
Values in parentheses indicate% samples.
Table 3 AFM1 contamination in raw, pasteurized and UHT milk samples exceeding EC, Codex and FSSA 2006, India regulations. Sample category
Raw milk Pasteurized milk UHT milk
No. of samples analysed
45 7 45
No. of positive samples
45 (100) 3 (42.9) 29 (64.4)
Exceeding EC regulation 0.05 lg/kg
Exceeding Codex and FSSA, 2006 India regulation 0.5 lg/kg
No.
Range
No.
Range
22 (48.9) 3 (100) 29 (100)
0.1–3.8 1.8–3.8 0.06–0.7
6 (13.3) 3 (100) 10 (22.2)
0.6–3.8 1.8–3.8 0.5–2.1
Values in parentheses indicate% samples.
plete removal of the bound toxin from the antibody by leaving it in contact with the solvent mixture for at least 30 seconds and by back flushing during the final elution. The solvent in the tube was evaporated to dryness under a gentle stream of nitrogen. The residue was dissolved in 1 ml of mobile phase to inject into the HPLC column.
2.5. HPLC conditions Detection and quantification was by HPLC (Waters) fitted with a fluorescence detector. The excitation and emission wavelengths were set at 355 and 435 nm respectively. The chromatographic separation was achieved on a reversed phase C18 column (Waters Spherisorb, 5 lm particle size, 250 mm 4 mm I.D.), maintained at 40 °C. The mobile phase was isocratic, acetonitrile–water (33:67) mixture. The flow rate was 1.2 ml min1 and the injection volume was 50 ll. The system is interfaced, via network chromatographic software, to a personal computer for the control of instruments, data acquisition, and processing.
2.7. Quality control The immunoaffinity column was checked by passing a standard solution of 100 ng of AFM1 through the column, washing with PBS (20 ml) and eluting with the solvent mixture methanol:acetonitrile (2:3, v/v), evaporating the solvent and suspending the residue in 1 ml mobile phase. A recovery of 94.2% was obtained which was within the required recovery of not less than 80% as per the AOAC Official method. Milk sample found to be negative for aflatoxin M1 was spiked (n = 4) with the standard at 1.00 and 0.02 lg L1. 50 ml volumes of these samples were passed through the IAC column, the eluent evaporated to dryness and finally suspended in 1 ml mobile phase. Recoveries of 84.8 ± 2.5 and 76.5 ± 2% respectively were obtained. The aflatoxin M1 peak of the 0.02 lg L1 gave a signal to noise ratio of more than 10 and was taken as the LOQ.
3. Results and discussion 2.6. Preparation of standard and calibration curve AFM1 stock solutions of approximately 5 lg ml1 were prepared in benzene– acetonitrile (9:1, v/v). The real concentration was determined by recording the UV spectrum from 200 to 400 nm against the solvent used for solution in reference cell and by noting the absorbance at wavelength of maximum absorbance close to 350 nm. Actual concentration of the aflatoxin solution was determined using the equation given below, the values of molecular weight and molar absorptivity (e) in benzene: acetonitrile (9 + 1) is 328 and 18815 respectively (AOAC, 2005).
AFM1
lg=ml ¼
Absorbance oMolecular weight oCorrelation factor Molar absorptivity ðeÞ
An aliquot of the AFM1 in benzene:acetonitrile was transferred to a 5 ml volumetric flask, solvent evaporated to dryness under gentle stream of nitrogen, dissolved in acetonitrile and volume made up to give a solution of 20 lg L1 in acetonitrile. Aliquots of this solution were transferred to tubes, solvent evaporated to dryness and residue dissolved in 1 ml mobile phase. The concentrations of these solutions were 16, 8, 4, 2 and 1 lg L1 and the amount of AFM1 when injected into the 50 ll loop of the HPLC was 0.8, 0.4, 0.2, 0.1 and 0.05 ng respectively. Calibration curve was determined using the response of these solutions. The curve exhibited linearity with correlation of 0.998. The signal to noise ratio of a 0.2 lg L1 solution was more than three. Thus theoretically, if 50 ml of milk sample was passed through the IAC and the final residue suspended in 1 ml, the LOD is 0.004 lg L1. Aflatoxin M1 mass concentration of the test sample was determined using the following formula:
Wm ¼ Wa ðVf=ViÞ ð1=VsÞ where Wm = the numerical value of aflatoxin M1 in the test sample in lg/L; Wa = the numerical value of the amount of aflatoxin M1 corresponding to area of the aflatoxin M1 peak of the test extract (ng); Vf = the numerical value of the final volume of redissolved eluate (lL); Vi = the numerical value of the volume of injected eluate (lL). Vs = the numerical value of volume of prepared test portion passing through the column (mL).
The incidence and levels of AFM1 in UHT milk of the various brands in the Indian market are summarized in Table 1. Chromatograms of a negative sample, a positive sample and a spiked sample (0.02 lg L1) are presented in the Fig. 1. The content of AFM1 in the 45 samples of plain and flavored UHT milk ranged from below detection limit to 2.1 lg L1. All samples of plain UHT milk were positive for AFM1 and 38.0% of these contained AFM1 more than 0.5 lg L1. In 62.5% of the flavored milk samples, AFM1 was below detectable limits but 12.5% these contained the toxin in levels of more than 0.5 lg L1. The prevalence of AFM1 in shelf stable UHT milk has been reported in countries like Spain (Blanco et al., 1988), Iran (Fallah, 2010), Turkey (Unusan, 2006), Kuwait (Dashti et al., 2009), Brazil (Shundo et al., 2009) and Pakistan (Raza, 2006). In this report it is observed that AFM1 is present in UHT milk in all the popular brands available in the market. Out of a total of 52 samples of raw and pasteurized milk samples received from the southern states of Karnataka and Tamilnadu, 61.6% were positive for AFM1 within a range of 0.1 to 3.8 lg L1. The content in 17.3% of these samples was more than 0.5 lg L1 (Table 2). In India, there have been reports on the prevalence of AFM1 in raw milk. Choudhary et al. (1997) have reported AFM1 contamination of 94% of raw and pasteurized milk ranging from 0.066 to 0.763 lg kg1 in the 223 samples collected from Anand town in the western part of the country in Gujarat. Rajan et al. (1995) analyzed 504 raw milk samples from Thrissur, a town in the southern state of India in Kerala and found that 17.7% were contaminated with AFM1 at a range of 0.1–3.5 lg kg1. Paul et al. (1976) analyzed 81 raw milk samples from Ludhiana, in the north of the country and
V. Siddappa et al. / Food and Chemical Toxicology 50 (2012) 4158–4162
found 6.2% positive with one sample having a high concentration of 13.3 lg L1. Thus, the prevalence of AFM1 in raw and pasteurized milk has been reported from all parts of the country. This is directly related to the aflatoxin content of the cattle feed. Dhawan and Choudhary (1995) reported a range of 3–1734 lg kg1 with an average of 103 lg kg1 in cattle feedstuffs. Maize and peanut cake form a major part of livestock feed in India. The contamination of these ingredients with AFB1 has been found to be as high as 3300 lg kg1 (Reddy et al., 2000). Veldman et al. (1992) have derived a formula for the conversion of AFB1 to AFM1 by the cow’s metabolism, which is AFM1 (ng/kg1 of milk) = 1.19 AFB1 consumed (lg per cow per day) + 1.9. They have also indicated that in order to produce milk with AFM1 less than 0.05 lg kg1, the average daily individual intake in a herd should be limited to 40 lg/day. Presence of AFM1 in milk and dairy products is of worldwide concern since these products are frequently consumed by all sections of the population (Fallah, 2010). Because of the hazardous nature of the toxin in milk and milk derivatives (Sassahara et al., 2005; Unusan, 2006; Oveisi et al., 2007) several countries have set or proposed legal regulations for AFM1 levels in milk and dairy products. These regulations vary in different countries and are often based on economic considerations (Stoloff et al., 1991). The European Commission (EC) has established a maximum admissible level of 0.05 lg/kg for AFM1 in milk (European Commission Regulation., 2001). The maximum permitted limit under Codex Alimentarius Regulations (CAC, 2001) and under the mandatory regulations of India, The Food Safety and Standards Act 2006 (FSSAI, 2011) for milk is 0.5 lg/kg. This difference in the maximum permitted limits has a direct affect on global trade. It can be seen in Table 3, that out of 45 samples of raw milk analyzed, 48.9% would not pass the EC regulations, while this number is reduced to 13.3% if the Codex or FSSAI regulations are applied. Similarly, while 100% of the UHT milk samples were above the limits laid down by EC, 33.3% had exceeded the Codex (CAC, 2001) or FSSAI (2011) mandatory regulations. Similarly, all the 29 out of 45 UHT milk samples analyzed, did not conform to the EC regulations while 22.2% were the numbers that exceeded the Codex or FSSAI, the country’s regulations. 4. Conclusions In this study it has been observed that AFM1 contamination of processed UHT milk can range from levels that are not detectable to as high as 2.1 lg L1. What is of greatest concern is that infants are more exposed to the risk of this health hazard because milk is a major constituent of their diet. It is also a major constituent of human diet of other vulnerable sections of the society, namely, the aged, the sick and the immuno-compromised (Galvano et al., 1996). It is recognized that one of the most effective methods to ensure absence of AFM1 in milk is to monitor the AFB1 in the livestock feed. While developed countries regulate the content of AFB1 in cattle feed, thus ensuring the content of AFM1 within permissible limits in milk and milk products, excepting for China, there are no mandatory limits in India and other Asian countries for cattle feed (FAO, 1997). Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the Council of Scientific & Industrial Research, Government of India, under the network project NWP017. The authors are thankful to the Director, CFTRI and to
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Occurrence of aflatoxin M1 in some samples of UHT, raw & pasteurized milk from Indian states of Karnataka and Tamilnadu Vinutha Siddappa, Divyashree Kallenahalli Nanjegowda, Prema Viswanath ⇑ Food Safety & Analytical Quality Control Laboratory, Central Food Technological Research Institute, Mysore 570020, India
a r t i c l e
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Article history: Received 15 June 2012 Accepted 14 August 2012 Available online 24 August 2012 Keywords: Aflatoxin M1 UHT milk Raw milk HPLC India
a b s t r a c t Aflatoxin M1 (AFM1) is a toxic metabolite found in the milk of lactating animals which have consumed feedstuffs contaminated with aflatoxin B1. Ultra high temperature treated (UHT) milk is a product which is becoming popular in developing countries like India as there is a lack of proper cold storage or refrigeration facilities. In this study, 45 samples of UHT milk of popular brands prevalent in the market were analyzed for the presence of AFM1 by reversed phase HPLC using fluorescent detector after cleanup of sample with immunoaffinity columns. All samples of plain UHT milk were positive for AFM1 and 38% of these contained levels more than 0.5 lg/kg, the maximum permitted limit prescribed by the Codex Alimentarius Commission and by the mandatory regulations of the country, the FSSAI Regulations, 2011. In 62.5% of flavored UHT milk, AFM1 was below detectable levels (0.02 lg L1). However, 12.5% of these samples also contained levels exceeding the maximum permitted limits. AFM1 was present in 61.6% of the 52 raw milk samples analyzed from the two states of Karnataka and Tamilnadu with a range of 0.1–3.8 lg L1. 17.3% of these samples also exceeded the regulatory limits of the country. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction India is the world’s largest milk producing country with an output of 110 million tonnes per annum (FAO, 2010). This country has a tropical climate with conditions favorable for the growth of aflatoxigenic fungi. Aflatoxins are toxic fungal metabolites produced by Aspergillus species, mainly byAspergillus flavus andAspergillus parasiticus, but also by Aspergillus nomius, Aspergillus pseudotamarii, Aspergillus ombycis, Aspergillus ochraceoroseus, and Aspergillus australis (IARC, 2012). Aflatoxins consist of a group of approximately 20 related metabolites, of which aflatoxins B1, B2, G1 and G2 are normally found in foods (Van Egmond, 1989). Their prevalence has been reported in a wide variety of agricultural products and animal fodder (Peraica et al., 1999; Eaton and Groopman, 1994) as a result of molds contamination before or during harvest or improper storage (Sweeney and Dobson, 1998). Aflatoxin B1 (AFB1) is metabolized by the animals consuming these contaminated feeds to AFM1 mainly by the hepatic microsomal mixed-function oxidase system (Marsi et al., 1974). The amount of AFM1 that is excreted in milk as a percentage of AFB1 is an average of 1–2%. This can vary from animal to animal and from season to season. The AFM1 is detected in milk 12–24 h after the first AFB1 ingestion, reaching a high level after a few days. If there is no further intake of AFB1 through feed, the AFM1 concentration in the milk decreases to an undetectable level after 72 h (Van Egmond, 1989). ⇑ Corresponding author. Tel.: +91 0821 2514972; fax: +91 0821 2412064. E-mail addresses: [email protected], [email protected] (P. Viswanath). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.08.034
Aflatoxins are one of the major etiological factors in the development of hepatocellular carcinoma (IARC, 2012), and more recently associations between childhood aflatoxin exposure and growth stunting have been reported (Gong et al., 2004). It has been calculated that about 27% of the hepatocellular carcinoma cases reported in Southeast Asia is aflatoxin induced (Liu and Wu, 2010). AFM1 has less mutagenic properties than AFB1 (Hsieh et al., 1984). However because of the toxic and carcinogenic effects of AFM1 IARC (International Agency for Research on Cancer) of WHO reconsidered its carcinogenic categorization and changed it from Group 2B to Group 1 (IARC, 2012). The prevalence of AFM1 in raw milk and milk products has been reported in many countries (WHO, 2010). One of the milk products is ultra high temperature treated milk (UHT) which is valuable for the fact that it is shelf stable. These products can therefore be marketed in places where there are no cold storage facilities. In India, UHT milk currently accounts for less than 1% of the total liquid milk market in India. However, International Market Analysis Research and Consulting Group (IMARC), one of the world’s leading research and advisory firms, expects the Indian UHT Milk market to more than triple its current size during 2010–11 and 2016–17 (Anonymous, 2011). ‘‘UHT (ultra high temperature) treatment of milk and liquid milk products is the application of heat to a continuously flowing product using such high temperatures for such time that renders the product commercially sterile at the time of processing. When the UHT treatment is combined with aseptic packaging, it results in a commercially sterile product. UHT treatment is normally in
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V. Siddappa et al. / Food and Chemical Toxicology 50 (2012) 4158–4162 Table 1 Occurrence of Aflatoxin M1 in ultra high temperature (UHT) treated milk. Sample category
No. of samples
No. of positive samples
UHT plain milk UHT flavored milk Total
21 24 45
21 (100) 9 (37.5) 30 (66.6)
Range of aflatoxin M1 concentration (lg L1) <0.02
0.02–0.05
0.05–0.1
0.1–0.5
>0.5
– 15(62.5) 15(62.5)
– – –
3 (14.2) 2 (8.3) 5 (11.1)
10 (47.6) 4 (16.6) 14 (31.1)
8 (38.0) 3 (12.5) 11 (24.4)
Values in parentheses indicate% samples.
the range of 135–150 °C in combination with appropriate holding times necessary to achieve commercial sterility’’ (FAO & WHO, 2007). There have been varying reports on the effect of heat either through pasteurization or sterilization on the AFM1 in milk. (Purchase et al., 1972) observed that a 32% reduction in AFM1 can be achieved by pasteurization at 62 °C for 30 min. In another report, depending on the conditions employed in heating of the milk, a decrease in the AFM1 content of the milk of between 12% and 35% was observed (Kabak, 2009). Differences in the reports of reduction of AFM1 can be attributed to the wide range of temperature, different analytical methods, and employment of both naturally and artificially contaminated milk. In general it has been observed that aflatoxins are stable during heat-treatments (Van Egmond et al., 1977; Wiseman and Marth, 1983; Yousef and Marth, 1989; Govaris et al., 2001; Prandini et al., 2009). This manuscript is the first report of the occurrence of AFM1 in UHT milk of the major brands prevalent in the Indian market. Occurrence of the toxin in raw and pasteurized milk randomly received for analysis from milk producers in the states of Karnataka and Tamil Nadu is also presented. The quantitative analysis of AFM1 in the milk samples was performed by reversed phase High Performance Liquid Chromatography with fluorometric detection after sample clean-up using immunoaffinity columns (AOAC, 2005). 2. Materials and methods 2.1. Samples A total of 45 samples of shelf stable UHT milk (Plain and flavoured) of different brands before their expiry date and with date of manufacture from April to July 2011 that is sold in the local market were purchased from retail shops in the city of Mysore, Karnataka state, India. These brands are also sold all over India. 52 samples of raw and pasteurized milk were voluntarily sent by milk producers from the states of Karnataka and Tamil Nadu for determination of the presence of AFM1. These samples were transported to the laboratory inside an ice box at temperature of less than 4 °C. 2.2. Chemicals Aflatoxin M1 (AFM1) was obtained from Sigma Chemical Co (St. Louis, MO, USA). HPLC grade solvents were purchased from Merck, Riedel (Darmstadt, Germany). AFM1 immunoaffinity columns (AFLAPREPÒ M) were obtained from R-BIOPHARM RHONE LTD, Glasgow Scotland. Other chemicals namely, methanol and acetonitrile were also obtained from Merck, Germany. All water used was purified by a MilliQ Water System (Millipore-Synergy UC, France). 2.3. Sample preparation The milk sample in the sachet or tetrapak was mixed well, warmed to about 40 °C, centrifuged at 4000 rpm for 15 min at room temperature and filtered through Whatman No. 1 filter paper. A volume of 50 ml of filtrate was collected for analysis as outlined in the AOAC Official method (AOAC, 2005). 2.4. Extraction procedure The procedure was as outlined in the AOAC Official method (AOAC, 2005). The 50 ml prepared filtrate was passed through the immunoaffinity column at a flow rate of about 2–3 ml/min. Two 10 ml portions of phosphate buffered saline were then passed through the column at approximately 5 ml/min. Air was then pressed through the column to remove any residual liquid. AFM1 was eluted with three
Fig. 1. HPLC chromatograms of (A) a negative sample, (B) a naturally contaminated milk sample, (C) milk sample spiked at 0.02 lg L1. 0.5 ml portions of a mixture of methanol:acetonitrile (2:3, v/v) into a clean glass tube at approximately one drop every 2–3 seconds. Care was taken to ensure com-
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V. Siddappa et al. / Food and Chemical Toxicology 50 (2012) 4158–4162 Table 2 Occurrence of Aflatoxin M1 in pasteurized and raw milk. Sample category
No. of samples
No. of positive samples
Pasteurized milk Raw milk Total
7 45 52
3 (42.9) 29 (64.4) 32 (61.5)
Range of aflatoxin M1 concentration (lg L1) <0.02
0.02–0.05
0.05–0.1
0.1–0.5
>0.5
4 (7.7) 16 (30.8) 20 (38.4)
– 7 (13.5) 7 (13.5)
– 5 (9.6) 5 (9.6)
– 11 (21.2) 11 (21.2)
3 (5.8) 6 (11.5) 9 (17.3)
Values in parentheses indicate% samples.
Table 3 AFM1 contamination in raw, pasteurized and UHT milk samples exceeding EC, Codex and FSSA 2006, India regulations. Sample category
Raw milk Pasteurized milk UHT milk
No. of samples analysed
45 7 45
No. of positive samples
45 (100) 3 (42.9) 29 (64.4)
Exceeding EC regulation 0.05 lg/kg
Exceeding Codex and FSSA, 2006 India regulation 0.5 lg/kg
No.
Range
No.
Range
22 (48.9) 3 (100) 29 (100)
0.1–3.8 1.8–3.8 0.06–0.7
6 (13.3) 3 (100) 10 (22.2)
0.6–3.8 1.8–3.8 0.5–2.1
Values in parentheses indicate% samples.
plete removal of the bound toxin from the antibody by leaving it in contact with the solvent mixture for at least 30 seconds and by back flushing during the final elution. The solvent in the tube was evaporated to dryness under a gentle stream of nitrogen. The residue was dissolved in 1 ml of mobile phase to inject into the HPLC column.
2.5. HPLC conditions Detection and quantification was by HPLC (Waters) fitted with a fluorescence detector. The excitation and emission wavelengths were set at 355 and 435 nm respectively. The chromatographic separation was achieved on a reversed phase C18 column (Waters Spherisorb, 5 lm particle size, 250 mm 4 mm I.D.), maintained at 40 °C. The mobile phase was isocratic, acetonitrile–water (33:67) mixture. The flow rate was 1.2 ml min1 and the injection volume was 50 ll. The system is interfaced, via network chromatographic software, to a personal computer for the control of instruments, data acquisition, and processing.
2.7. Quality control The immunoaffinity column was checked by passing a standard solution of 100 ng of AFM1 through the column, washing with PBS (20 ml) and eluting with the solvent mixture methanol:acetonitrile (2:3, v/v), evaporating the solvent and suspending the residue in 1 ml mobile phase. A recovery of 94.2% was obtained which was within the required recovery of not less than 80% as per the AOAC Official method. Milk sample found to be negative for aflatoxin M1 was spiked (n = 4) with the standard at 1.00 and 0.02 lg L1. 50 ml volumes of these samples were passed through the IAC column, the eluent evaporated to dryness and finally suspended in 1 ml mobile phase. Recoveries of 84.8 ± 2.5 and 76.5 ± 2% respectively were obtained. The aflatoxin M1 peak of the 0.02 lg L1 gave a signal to noise ratio of more than 10 and was taken as the LOQ.
3. Results and discussion 2.6. Preparation of standard and calibration curve AFM1 stock solutions of approximately 5 lg ml1 were prepared in benzene– acetonitrile (9:1, v/v). The real concentration was determined by recording the UV spectrum from 200 to 400 nm against the solvent used for solution in reference cell and by noting the absorbance at wavelength of maximum absorbance close to 350 nm. Actual concentration of the aflatoxin solution was determined using the equation given below, the values of molecular weight and molar absorptivity (e) in benzene: acetonitrile (9 + 1) is 328 and 18815 respectively (AOAC, 2005).
AFM1
lg=ml ¼
Absorbance oMolecular weight oCorrelation factor Molar absorptivity ðeÞ
An aliquot of the AFM1 in benzene:acetonitrile was transferred to a 5 ml volumetric flask, solvent evaporated to dryness under gentle stream of nitrogen, dissolved in acetonitrile and volume made up to give a solution of 20 lg L1 in acetonitrile. Aliquots of this solution were transferred to tubes, solvent evaporated to dryness and residue dissolved in 1 ml mobile phase. The concentrations of these solutions were 16, 8, 4, 2 and 1 lg L1 and the amount of AFM1 when injected into the 50 ll loop of the HPLC was 0.8, 0.4, 0.2, 0.1 and 0.05 ng respectively. Calibration curve was determined using the response of these solutions. The curve exhibited linearity with correlation of 0.998. The signal to noise ratio of a 0.2 lg L1 solution was more than three. Thus theoretically, if 50 ml of milk sample was passed through the IAC and the final residue suspended in 1 ml, the LOD is 0.004 lg L1. Aflatoxin M1 mass concentration of the test sample was determined using the following formula:
Wm ¼ Wa ðVf=ViÞ ð1=VsÞ where Wm = the numerical value of aflatoxin M1 in the test sample in lg/L; Wa = the numerical value of the amount of aflatoxin M1 corresponding to area of the aflatoxin M1 peak of the test extract (ng); Vf = the numerical value of the final volume of redissolved eluate (lL); Vi = the numerical value of the volume of injected eluate (lL). Vs = the numerical value of volume of prepared test portion passing through the column (mL).
The incidence and levels of AFM1 in UHT milk of the various brands in the Indian market are summarized in Table 1. Chromatograms of a negative sample, a positive sample and a spiked sample (0.02 lg L1) are presented in the Fig. 1. The content of AFM1 in the 45 samples of plain and flavored UHT milk ranged from below detection limit to 2.1 lg L1. All samples of plain UHT milk were positive for AFM1 and 38.0% of these contained AFM1 more than 0.5 lg L1. In 62.5% of the flavored milk samples, AFM1 was below detectable limits but 12.5% these contained the toxin in levels of more than 0.5 lg L1. The prevalence of AFM1 in shelf stable UHT milk has been reported in countries like Spain (Blanco et al., 1988), Iran (Fallah, 2010), Turkey (Unusan, 2006), Kuwait (Dashti et al., 2009), Brazil (Shundo et al., 2009) and Pakistan (Raza, 2006). In this report it is observed that AFM1 is present in UHT milk in all the popular brands available in the market. Out of a total of 52 samples of raw and pasteurized milk samples received from the southern states of Karnataka and Tamilnadu, 61.6% were positive for AFM1 within a range of 0.1 to 3.8 lg L1. The content in 17.3% of these samples was more than 0.5 lg L1 (Table 2). In India, there have been reports on the prevalence of AFM1 in raw milk. Choudhary et al. (1997) have reported AFM1 contamination of 94% of raw and pasteurized milk ranging from 0.066 to 0.763 lg kg1 in the 223 samples collected from Anand town in the western part of the country in Gujarat. Rajan et al. (1995) analyzed 504 raw milk samples from Thrissur, a town in the southern state of India in Kerala and found that 17.7% were contaminated with AFM1 at a range of 0.1–3.5 lg kg1. Paul et al. (1976) analyzed 81 raw milk samples from Ludhiana, in the north of the country and
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found 6.2% positive with one sample having a high concentration of 13.3 lg L1. Thus, the prevalence of AFM1 in raw and pasteurized milk has been reported from all parts of the country. This is directly related to the aflatoxin content of the cattle feed. Dhawan and Choudhary (1995) reported a range of 3–1734 lg kg1 with an average of 103 lg kg1 in cattle feedstuffs. Maize and peanut cake form a major part of livestock feed in India. The contamination of these ingredients with AFB1 has been found to be as high as 3300 lg kg1 (Reddy et al., 2000). Veldman et al. (1992) have derived a formula for the conversion of AFB1 to AFM1 by the cow’s metabolism, which is AFM1 (ng/kg1 of milk) = 1.19 AFB1 consumed (lg per cow per day) + 1.9. They have also indicated that in order to produce milk with AFM1 less than 0.05 lg kg1, the average daily individual intake in a herd should be limited to 40 lg/day. Presence of AFM1 in milk and dairy products is of worldwide concern since these products are frequently consumed by all sections of the population (Fallah, 2010). Because of the hazardous nature of the toxin in milk and milk derivatives (Sassahara et al., 2005; Unusan, 2006; Oveisi et al., 2007) several countries have set or proposed legal regulations for AFM1 levels in milk and dairy products. These regulations vary in different countries and are often based on economic considerations (Stoloff et al., 1991). The European Commission (EC) has established a maximum admissible level of 0.05 lg/kg for AFM1 in milk (European Commission Regulation., 2001). The maximum permitted limit under Codex Alimentarius Regulations (CAC, 2001) and under the mandatory regulations of India, The Food Safety and Standards Act 2006 (FSSAI, 2011) for milk is 0.5 lg/kg. This difference in the maximum permitted limits has a direct affect on global trade. It can be seen in Table 3, that out of 45 samples of raw milk analyzed, 48.9% would not pass the EC regulations, while this number is reduced to 13.3% if the Codex or FSSAI regulations are applied. Similarly, while 100% of the UHT milk samples were above the limits laid down by EC, 33.3% had exceeded the Codex (CAC, 2001) or FSSAI (2011) mandatory regulations. Similarly, all the 29 out of 45 UHT milk samples analyzed, did not conform to the EC regulations while 22.2% were the numbers that exceeded the Codex or FSSAI, the country’s regulations. 4. Conclusions In this study it has been observed that AFM1 contamination of processed UHT milk can range from levels that are not detectable to as high as 2.1 lg L1. What is of greatest concern is that infants are more exposed to the risk of this health hazard because milk is a major constituent of their diet. It is also a major constituent of human diet of other vulnerable sections of the society, namely, the aged, the sick and the immuno-compromised (Galvano et al., 1996). It is recognized that one of the most effective methods to ensure absence of AFM1 in milk is to monitor the AFB1 in the livestock feed. While developed countries regulate the content of AFB1 in cattle feed, thus ensuring the content of AFM1 within permissible limits in milk and milk products, excepting for China, there are no mandatory limits in India and other Asian countries for cattle feed (FAO, 1997). Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the Council of Scientific & Industrial Research, Government of India, under the network project NWP017. The authors are thankful to the Director, CFTRI and to
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the Head, Food Safety & Analytical Quality Control Laboratory, CFTRI, for providing the infrastructural facilities. References Anonymous, 2011. UHT milk to triple by 2016. Customer Research Frontiers. 17th November 2011. Available at:
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