Occurrence of aflatoxin M1 from rural subsistence and commercial farms from selected areas of South Africa

Food Control 39 (2014) 92e96

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Occurrence of aflatoxin M1 from rural subsistence and commercial farms from selected areas of South Africa Mwanza Mulunda a, b, *, Dutton Mike b a

Department of Animal Health, Faculty of Agriculture and Technology, Mafikeng Campus, North West University, Private Bag X2046, Mmabatho 2735, South Africa b Food, Environmental and Health Research Group, Doornfontein Campus, University of Johannesburg, P.O. Box 17011, 2028 Doornfontein, Gauteng, South Africa

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 December 2012 Received in revised form 31 October 2013 Accepted 5 November 2013

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 and its presence in milk is considered to be a potential health risk for humans. Due to the important role of milk in humans, especially in infant nutrition, this study is intended to evaluate the quality of milk consumed on daily basis in South Africa by both rural and urban population in regard to AFM1 contamination. To achieve this, samples were collected from rural subsistence (RSFs) and commercial dairy farms (CDFs) in selected areas of South Africa and samples were extracted using two clean-up procedures, C18 cartridges and immunoaffinity columns (IAC) and analysed using thin layer chromatography (TLC) or by fluorometry (VICAM) (VF) and high pressure liquid chromatography (HPLC) coupled with a fluorescence detector and a coring cell (CoBrA cell) for AFM1 derivatisation. Results obtained showed a frequency of contamination with AFM1 of RSFs milk samples at 22.8% by TLC, 93.9% by VF 86% by HPLC and in CDFs of 17.8% (TLC) 96.5% (VF) and 100% (HPLC). No significant differences were obtained between milk from rural subsistence and commercial farms with mean varying between 0.15 and 0.17 mg kg
1 Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Aflatoxin M1 Rural subsistence farm Commercial farm Coring cell Fluorometry

1. Introduction Aflatoxin M1 and M2 are the hydroxylated metabolites of aflatoxin B1 (AFB1) and B2 (AFB2) formed by cytochrome P450 1A2 in humans (Faletto et al., 1988) and may be found to be a major excretion product in the milk of lactating animals and women exposed to dietary previously contaminated with AFB1 and AFB2 (Wild, Pionneau, Montesano, Mutiro, & Chetsanga, 1987). Aflatoxin M1 can also be found in the organs, e.g., kidney, liver, and excreta of animals exposed to AFB1 (Allcroft, Rogers, Lewis, Nabney, & Best, 1966). Aflatoxin M1 (AFM1) is secreted in the milk of lactating cows at a level approximately equal to 1e3% of the dietary concentration of AFB1 (Van Egmond, 1989) with 3e6 days of constant daily ingestion of AFB1, before steady-state excretion of AFM1 in milk can be achieved and it is estimated that AFM1 appears in milk within hours of consumption of AFB1 and returns to baseline levels within two to three/four days after removal of the mycotoxin from

* Corresponding author. Department of Animal Health, Faculty of Agriculture and Technology, Mafikeng Campus, North West University, Private Bag X2046, Mmabatho 2735, South Africa. Tel.: þ27 728729471; fax: þ27 183892748. E-mail addresses: [email protected], [email protected] (M. Mulunda), [email protected] (D. Mike). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.11.011

the diet (Frobish, Bradley, Wagner, Long-Bradley, & Hairston, 1986). Aflatoxin M1 has been demonstrated to be cytotoxic in human hepatocytes in vitro and its acute toxicity in several species is similar to that of AFB1 and, as in the case of AFB1, liver cancer has been related to exposure to AFM1 (Groopman, Cain, & Kenster, 1988) and in this connection exhibits a high level of genotoxic activity and thus represent s a health risk because of its chemical combination with DNA (Gursoy-Yuzugullu, Yuzugullu, Yilmaz, & Ozturk, 2011). Consequently AFM1 has been classified since 1993 by the International Agency for Research on Cancer (IARC) as a Group 2B agent (possibly carcinogenic to humans) (International Agency for Research on Cancer, 1993, pp. 397e444). Tolerable limits worldwide, including South Africa, on aflatoxin levels vary between 5 and 20 mg kg
1 (Mariko, 2012). Studies done have demonstrated that a concentration of 20 mg kg
1 of AFB1 in the total mixed ration dry matter of lactating dairy cattle will result in AFM1 levels in milk below the FDA set up limit of 0.5 mg kg
1 (Henry et al.., 2001, pp. 1e102). The European Union and several other countries, including South Africa, have however, presently set up acceptable level of AFM1 in milk and milk products at 0.05 mg kg
1 (European Commission, 2006). The aim of this study was to evaluate the quality of milk from RSFs and CDFs in regard to aflatoxin M1 (AFM1) contamination and

M. Mulunda, D. Mike / Food Control 39 (2014) 92e96

hence determine any potential health risks to children in the populations consuming this milk. Two achieve this, two different extraction techniques were used for comparison of results, and the Coring cell (CoBrA cell) combined with a fluorescence detection was used for the first time to achieve maximum sensitivity for AFM1 determination instead of derivatization. 2. Materials and methodology 2.1. Materials All chemicals and solvents (sodium chloride, nitric acid, Methanol, ethanol) used in this study were obtained from Merck & Co.; VICAM immunoaffinity column from Microscep SA and aflatoxin M1 (1 mg/ml) standards from Sigma, SA. All reagents used in this study were of analytical grade, unless otherwise specified. 2.2. Methodology 2.2.1. Sample collection A total of 225 raw milk samples were collected for this study between April 2010 and March 2011. One hundred and twenty five (125) of these samples were obtained from rural subsistence and hundred (100) from commercial farms in Mpumalanga Province, Limpopo, Mpumalanga, and Gauteng. Milk samples were collected in 200 ml plastic containers and stored in cooler boxes during transportation and deep frozen in the Food, Environmental and Health Research Group (FEHRG) University of Johannesburg laboratories until analysis. 2.2.2. Aflatoxin M1 extraction from milk 2.2.2.1. Determination of aflatoxin M1 using immuno-affinity column. The VICAM (VF) immunoaffinity method was used to extract AFM1 according to the instruction manual as follow: 50 ml of the milk sample were added and mixed with salt (NaCl). The mixture was centrifuged at 2000 g for 10 min and the skim portion was then filtered through a micro fibre filter. Ten ml of the filtered skim milk was passed through the affinity column at a rate of 1e2 drops/ second until air came through the column. The column was washed twice with 10 ml of methanol: water (10:90) solution. Aflatoxin M1 was then eluted with 2 ml of HPLC grade methanol: water (80:20) at a speed of 1e2 drops/second. Finally, 1 ml of elute was added with the Aflatest developer and place in the VICAM fluorometer. The readings were done after 1 min. The same extract, with no developer added, were run on HPLC. The remained 1 ml from elute was dried and kept for HPLC analysis. 2.2.2.2. Aflatoxin M1 extraction using solid phase extraction (C18 cartridges). The solid phase extraction method of Manetta et al. (2005) was used as follow: 50 ml of milk was homogenized and centrifuged at 300  g for 10 min. Then 10 ml of the homogenized sample was then defatted and diluted with the same volume of deionised water. The C18 cartridges (ISOLUX, Microsep SA) were conditioned by eluting with 5 ml of acetonitrile followed by 10 ml of deionised water. The diluted milk sample (20 ml) was passed through the cartridge, followed by washing with 10 ml of water and then 20 ml of acetonitrile/water (20:80, v/v) and then 10 ml of nhexane. Aflatoxin M1 was eluted with 6 ml of dichloromethane/ acetone (95/5, v/v) and the elute extract was then evaporated under a stream of nitrogen and stored in deep fridge until further analysis. Recovery of AFM1 was done for both extraction procedures, in triplicate, by mixing control blank samples of milk with a standard (obtained from MERCK SA) to concentrations of 5, 10 and 20 ng/ml and then treated as above results obtained were of 98.8% and 67.3% respectively on Immunoaffinity and C18 column.

93

2.3. Aflatoxin M1 detection and quantification in milk samples 2.3.1. Thin layer chromatography (TLC) for aflatoxin M1 detection analysis For confirmation of the presence of AFM1, 100 mg/ml of AFM1 standard and 40 ml of the extracts (previously diluted in 200 ml of DCM) were diluted in 1 ml of dichloromethane and 20 ml of the diluted standard and extracts were dispensed on TLC plates on the three different solvents systems were used: dichloromethane lacetone-propan-2-ol (85/10/5); Diethyl ether-methanol-water (94/ 4.5/5) and dichloromethane-acetone-methanol (90/10/2) were used for the detection of AFM1 on TLC (). 2.3.2. High pressure liquid chromatography analysis The HPLC analysis of AFM1 was done according to Bakirci (2001) and Manetta et al. (2005) using a Shimadzu system (Kyoto, Japan) consisting of liquid chromatography LC 20A fitted to degasser DGU 20A3, auto sampler (injection) SIL 20A, communications bus module CBM 20A, column oven CTO 20A, photodiode array detector SPD M20A and fluorescence detector RF 10AXL, all connected to a gigabyte computer with Intel Core DUO with Microsoft XP was used. The analysis of AFM1, was done using a fluorescence detector RF 10AXL coupled to a reverse phase C18 column (Waters) and a Coring cell (CoBrA cell) (DR Weber Consulting, Germany), as electrochemical cell for the derivatisation of AFM1. The mobile phase was composed of HPLC grade methanol-acetonitrile-water (20:20:60) containing 119 mg of potassium bromide and 100 ml of nitric acid. Extracts were re-dissolved in 1 ml of HPLC grade methanol and 20 ml of the diluted solution was injected in the HPLC. The HPLC conditions excitation 362 nm and emission 440 nm with a flow rate of 1 ml/min. Standard AFM1 solutions of 0.5, 1, 2, 5, 10 and 20 ng/ml were prepared in methanol (HPLC grade) and linearity was achieved with a regression of 0.9987. The mean limit of detection was 0.01 ng/ml. 3. Statistical analysis Data obtained from this study were analysed and compared by t-test using SigmaPlot10.00. Mean values were deemed to be significantly different if the level of probability was 0.05. 4. Results The results obtained from the analysis of AFM1 in all milk samples are summarized in Table 1. The frequency of AFM1 contamination in RSF samples was 22.8% (26 samples) on TLC; in 93.9% (107) by VF and in 86.0% (98) by HPLC; in samples from CDFs, it was 17.8% (15) by TLC; 96.5% (82) by VF and a 100% (85) by HPLC coupled with the Coring cell. The mean concentrations of AFM1 in RSF samples were 2.38 mg kg
1 (range 1e19 mg kg
1) by VF and 0.15 mg kg
1(range 0.01e2.85 mg kg
1) by HPLC; and in CDF samples, 1.59 mg kg
1 (range 1.0e16.05 mg kg
1) by VF and 0.14 mg kg
1 (range 0.01e2.48 mg kg
1) by HPLC. Aflatoxin M1 concentrations ranged between 1 and 19 mg kg
1 by fluorometry and 0.01e 2.85 mg kg
1 by HPLC in samples from rural farms while in samples from commercial farms means concentrations ranged between 1.0 and 16.05 mg kg
1 by fluorometry and 0.01e2.48 mg kg
1 for HPLC analysis. The peak for aflatoxin M1 by HPLC was obtained at a retention time (RT) of 7.22 min (Figs. 1 and 2) but as expect the RT moves under the influence of experimental conditions such as mobile phase and HPLC room temperature variations. Of the three methods used for analysing AFM1 in milk samples, the results obtained by TLC had the lowest frequency of occurrence with a detection incidence of 17.8e22.8% (Table 1) followed by the HPLC 85.6e100% confirming the higher sensitivity of HPLC. The lowest

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M. Mulunda, D. Mike / Food Control 39 (2014) 92e96

Table 1 Aflatoxin M1 levels in milk samples from rural and commercial farms using VICAM fluorometer and high pressure liquid chromatography.

Rural milk (125) TLC FLUOROMETER (VICAM) HPLC Commercial milk (85) TLC FLUOROMETER (VICAM) HPLC

Positive/total (mg kg
1)

Mean (mg kg
1)

26 107

2.38a

98

0.15a

15 82

1.59a

85

0.14

a

Std. Dev.

>0.05 mg/l (%)

3.46

101 (80.8)

0.01e0.20 2.09

85 (79.0)

Median

1e19

1e14

2.63

82 (92.6)

0.03e0.19 0.28

78 (85.6)

TLC: thin layer chromatography; HPLC: high performance liquid chromatography; std. dev.; standard deviation. a The differences in the mean values and Std Dev. among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P ¼ <0.001) between results obtained from rural and commercial milk samples and between HPLC and VICAM methods.

frequency of occurrence of AFM1 obtained by TLC (Table 1) with detection incidences of 17.8/22.8% (Table 1) indicates the much lower sensitivity of this method as compare to the other two. Among positive samples, it was noted that 107 of them from RSFs (93.46%) by VF and 85 (79%) by HPLC and from CDFs 82 (92.6%) by fluorometry and 78 (85.6%) on HPLC were above the South African and international regulations, which is of 0.05 mg kg
1.

5. Discussion In this study, both extractive methods gave similar incidences but with the VF method giving a much wider range of contamination than the SPE/HPLC method (Table 1). This result was not satisfactory as a comparison of both methods using HPLC of the extracts with fluorescence and the Coring cell brought the range of the VICAM extract much closer to the of the SPE/HPLC method (Table 2). However statistical analysis of the means showed a significant difference between the two means with the VF results being nearly twice as large as the SPE/HPLC method (Table 2). This shows that the former method may give spurious false positives or the latter spurious false negatives. The reasons for these discrepancies in this study is not clear but it is imperative that a standard method is chosen for the analysis of AFM1 to these low levels, which is acceptable to the various agencies with stakes in such analysis.

Ignoring the VF result, the lower frequency of contamination by AFM1 in CDF milk samples, as compared to those from RSF samples is significant but the mean and range of concentrations are not (Table 1 and Fig 2). The means and ranges of mycotoxin concentration determined by the VF method, however, are significantly higher, with respect to those of SPE/HPLC and it seems highly likely that the VICAM/Fluorometry method is giving spurious false positives, as revealed by the results in Table 2. Any discrepancies between the results for RSF and CDF samples have to be explained on the sensitivity of the method, basis of feed or metabolic/physiological performance of the animals. In the case of the difference of frequency of AFM1 in the two areas of sampling (Table 1) could be explained on the basis of all three factors. The Coring cell improves detection of AFM1 (Table 3) and this would result in a higher number of positives at the lower concentration end. It is also possible that the style of feeding RSF animals, which is basically foraging in harvested fields, being fed any other waste food materials and poor pasture (Mwanza, 2007) results in a wider exposure to contaminated material than animals fed compound feed, silage and good pasture. It is likely that the metabolism of the RSF animals are modified due to the much poorer condition of these and their lower milk yields may also affect AFM1 concentrations. Whether this would result in less or more AFM1 in the milk is a matter of conjecture. In contrast, animals raised by intensive farming may be fed on a daily basis with feed concentrates reaching up to five kilograms per animal. This means animals will be exposed to a considerable amount of AFB1 because concentrates are liable to be contaminated with AFB1 even if it is below regulation limits, even if low levels are present in the feed and hence resulting in the higher incidence of AFM1 positive milk samples (100%). Aspergillus flavus and Aspergillus parasiticus are the main producers of the AFs (Klich, 2002, 116 pp.; Pitt & Hocking, 1997) and are considered to be storage fungi, which would suggest that the main point of production of AFs in feeds and their component commodities is during their storage and further indicates that in the case of CDFs that these materials are not stored correctly, particularly in the farm scenario (Dutton, de Kock, Khilosia, & Mwanza, 2012). Where commercial compound feeds are used often the components are contaminated, e.g., in a study on cottonseed cake imported into South Africa for the production of compound feed, nearly every sample take was positive for AFB1 and in some cases many times over the legal limit (Reiter et al., 2011). These factors would explain why milk from cattle can have levels of AFM1 well over the South African/EU legal limit as found in this study. In general the results of this study on South African milk correlate with those found in other developing countries, such as Iran (Kamkar, 2005; Rahimi, Bonyadian, Rafei, & Kazemeini, 2010); India (Siddappa, Nanjegowda, & Viswanath, 2012); Brazil (Oliveira

Fig. 1. Illustration of a chromatogram of aflatoxin M1 standard at 8 ml/ml at 40 ml injection on high performance liquid chromatography coupled to a fluorescence detector connected to the Coring cell (CoBrA cell).

M. Mulunda, D. Mike / Food Control 39 (2014) 92e96

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Fig. 2. Example of a chromatogram of aflatoxin M1 standard at 8 ml/ml and 40 ml injection on high performance liquid chromatography coupled to a fluorescence detector without the Coring cell (CoBrA cell).

& Ferraz, 2007); Turkey (Tekins¸en & Eken, 2008) and a recent South African study (Dutton et al., 2012) where milk samples in many cases did not meet the EU or South African regulations. This is in contrast to developed countries, e.g., France (Boudra, Barnouin, Dragacci, & Morgavi, 2007) with only 3 positive samples (none above the limit) out of 264 and Spain (Cano-Sancho, Marin, Ramos, Peris-Vicente, & Sanchis, 2010; Markaki & Melissari, 1997) with no samples over the current EU limit except one in latter study. This contrast is basically due to poorer control of aflatoxin levels in dairy feed in developing countries as mentioned above. The result show that AFM1 contamination in milk samples from both rural and commercial farms remain of concern as AFM1 has been recently classified as a group 1 carcinogenic mycotoxin (International Agency for Research on Cancer, 2012). It is known that aflatoxin B1 is the most important contaminant of crops in African crops and can be transformed into AFM1 in cattle (Stoloff et al., 1975). For many developing countries, the EU level may be considered impractical to achieve routinely and the one adopted by the USFDA more realistic. If this level is currently adopted then many of the levels of AFM1 found in this study would be within permitted limits. Whatever the final regulated levels are agreed to be, continuous exposure to AFM1 is a health hazard for consumers, in particular young children. Table 2 Results of immunoaffinity columns (VICAM) and solid phase extraction (C18 column) methods for aflatoxin M1 detection using high Pressure liquid chromatography coupled with fluorometer detector and the coring cell. Extraction methods n ¼ 50

Positive (%)

Mean (ppb)

Ranges (ppb)

Std dev

SEM

Immunoaffinity (IAC) Solid phase extraction

49 (98) 33 (66)

0.16a 0.16a

0.01e0.20 0.01e0.20

0.05 0.02

0.18 0.18

IAC: immunoaffinity column; std dev; standard deviation; SEM: standard error of the mean. a The differences in the mean values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P ¼ <0.001).

Table 3 Results of aflatoxin M1 leavels on high performance liquid chromatography coupled with and not coupled with the Coring cell (CoBrA cell). Analysis methods n ¼ 40

Positive (%)

Mean

Ranges

Std dev

50 ng/l (%)

HPLC-Coring cell HPLC þ coring cell

31(77.5) 33(66)

0.11a 0.16a

0.02e0.20 0.02e0.31

0.03e1.22 0.06e2.07

17 (52.5) 31 (77.5)

a There was a significant difference between High Performance Liquid Chromatography (HPLC) coupled with coring cell to High Performance Liquid Chromatography (HPLC) without Coring cell (CoBrA cell) mean concentrations (P  0.001).

6. Conclusion Aflatoxin contamination remains a concern in South African milk as revealed by the results obtained in this study. Aflatoxins are carcinogenic, teratogenic and mutagenic, and hence are able to cause or potentiate great damage to human health of rural families and ordinary consumers in urban areas. In addition the higher levels of AFM1 in milk has economic consequences in that milk and milk products, like milk powered, cannot be traded on the global market where legislation is more stringent. This study revealed the exposure level to AFM1 for both rural and urban consumers with highest incidences of aflatoxins M1 observed in commercial samples as compared to rural samples. It is clear in the former case the source of the parent AFB1 is compound feed whether produced on the farm or bought in and, therefore, control of the appearance of AFM1 in commercial milk depends upon the control of AFB1 appearing in feed Although sporadic milk contamination by AFM1 is not alarming in itself, it is assumed that a continuous exposure to this mycotoxin could have negative effects on consumer’s health. This implies potential health risk factors for rural population, especially children who consume milk daily. The novelty of this study is the use of immunoaffinity clean-up and liquid chromatography coupled with a Coring cell (CoBrA cell) which enhances AFM1 detection. The method may, however, be too expensive for routine use in developing countries such as South Africa, where there is a need of regular control by regular analysis of animal food. In regard to the above obtained results, it is therefore necessary for developing countries such as South Africa to introduce cheap and reusable techniques for mycotoxins detection such as immunoultrafiltration and solegel column in order to control these mycotoxins. Acknowledgements The authors would like to thank University of Johannesburg Research Council (URC) for the financial support of this study. References Allcroft, R., Rogers, H., Lewis, G., Nabney, J., & Best, P. E. (1966). Metabolism of aflatoxin in sheep: excretion of the “milk toxin”. Nature, 209, 154e155. Bakirci, I. (2001). A study on the occurrence of aflatoxin M1 in milk and milk products produced in Van province of Turkey. Food Control, 12, 47e51. Boudra, H., Barnouin, J., Dragacci, S., & Morgavi, D. P. (2007). Aflatoxin M1 and ochratoxin A in raw bulk milk from French dairy herds. Journal of Dairy Science, 90, 3197e3201. Cano-Sancho, G., Marin, S., Ramos, A. J., Peris-Vicente, J., & Sanchis, V. (2010). Occurrence of aflatoxin M1 and exposure assessment in Catalonia, Spain. Revista Iberoamericana de Micologíia, 27, 130e135. Dutton, M. F., de Kock, S., Khilosia, L. D., & Mwanza, M. (2012). Mycotoxins in South African foods: a case study on aflatoxin M1 in milk. Mycotoxin Research, 28, 17e23. European Commission. (2006). Commission Regulation (EC) No 1881/2006 of 19 December as amended, on setting maximum levels of certain contaminants in

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