Detection of aflatoxin M1 in pasteurized and raw milk marketed in metropolitan cities using ELISA

Introduction

Mycotoxin contamination of the dairy supply chain poses a persistent food-safety challenge in sub-Saharan Africa, where hot and humid storage conditions favour fungal proliferation in animal feed, and regulatory enforcement remains inconsistent [1] . Aflatoxin B1 (AFB1), the most potent naturally occurring liver carcinogen known, is produced by Aspergillus flavus and A. parasiticus on maize, groundnut and other staple crops [2] . When lactating cows ingest AFB1-contaminated feed, they metabolise it hepatically to aflatoxin M1 (AFM1), which is then secreted into milk at a carry-over rate of approximately 1-6% of the ingested dose [3] . AFM1 is classified as a Group 1 human carcinogen by the International Agency for Research on Cancer and retains roughly 10% of the carcinogenic potency of the parent compound [4] . Unlike many mycotoxins, AFM1 is heat-stable: pasteurisation, boiling and even ultra-hightemperature processing reduce its concentration by only 10-20%, meaning that thermal treatment of milk does not eliminate the hazard [5] . Children, who consume proportionally more milk per kilogram of body weight than adults, are considered the most vulnerable population [6] . Regulatory limits for AFM1 in milk vary considerably worldwide. The European Union applies the strictest standard at 0.05 µg/L, while the Codex Alimentarius Commission and the United States set the limit at 0.5 µg/L [7] . Nigeria, at the time of this research, had not established a binding national limit specific to AFM1 in liquid milk, although the Nigerian Industrial Standard for pasteurized milk references the Codex guideline [8] . This regulatory gap means that milk with AFM1 levels between 0.05 and 0.5 µg/L non-compliant by EU standards but technically permissible under Codex circulates freely in the market. Competitive ELISA has emerged as the method of choice for large-scale AFM1 screening because it combines adequate sensitivity (detection limits as low as 0.005 µg/L), high throughput and moderate equipment cost compared with instrumental methods such as HPLC and LC-MS/MS [9] . Several ELISA-based surveys have been conducted in Nigeria, but most covered a single city or a single milk type, and few have examined seasonal effects within the same sampling framework [10,11] . This research aimed to quantify AFM1 in both raw and pasteurized milk across six Nigerian metropolitan cities during two seasons and to assess compliance with the EU and Codex limits, thereby providing a comprehensive snapshot of exposure risk for urban consumers.

Materials and Methods

Reagents and Chemicals

The Ridascreen Aflatoxin M1 competitive ELISA kit (Art. No. R1121, R-Biopharm AG, Darmstadt, Germany) was used for quantitative AFM1 determination. The kit included AFM1-coated microwell strips, AFM1 standard solutions (0, 5, 10, 20, 40 and 80 ng/L), enzyme conjugate, substrate/chromogen solution and stop solution. Phosphatebuffered saline (PBS, pH 7.4) was prepared in-house. Certified reference material (ERM-BD282, European Commission Joint Research Centre) was used for method verification. All reagents were stored at 2-8 °C and used within their stated shelf life.

Instrumentation and Analytical Equipment

Absorbance was read at 450 nm on a Thermo Scientific Multiskan FC microplate reader. Sample preparation employed a Hettich EBA 20 centrifuge operating at 4, 000 rpm. A calibrated analytical balance (Mettler Toledo ME204) was used for weighing. Pipetting was performed with calibrated single-channel and multichannel micropipettes (Eppendorf Research Plus, 10-100 µL and 100-1000 µL ranges). Temperature during incubation steps was maintained at 25±1 °C using a thermostatically controlled room.

Materials

A total of 240 milk samples were purchased from retail outlets across six Nigerian cities between June 2022 and March 2023. In each city, 20 raw milk samples and 20 pasteurized milk samples were collected, split equally between the wet season (June-September 2022) and the dry season (December 2022-March 2023). Raw milk was sourced from open markets and neighbourhood vendors, while pasteurized milk was purchased from supermarkets. Samples were transported in insulated cool boxes (4 °C) to the Lagos Foundation for Food Safety Research and stored at -20 °C until analysis [12] .

Methods

Frozen samples were thawed at room temperature, vortexed and centrifuged at 4, 000 rpm for 10 minutes to remove the fat layer. A 100-µL aliquot of the defatted fraction was dispensed into AFM1-coated microwells, followed by 50 µL of enzyme conjugate. The plate was incubated at 25 °C for 60 minutes in the dark, then washed three times with PBS-Tween. Substrate solution (100 µL) was added, and colour was allowed to develop for 15 minutes before the reaction was stopped with 50 µL of 1 N sulphuric acid. Absorbance was recorded at 450 nm [9] . AFM1 concentration was calculated from a fourparameter logistic standard curve using RIDA SOFT Win software (R-Biopharm). The limit of detection (LOD) was 0.005 µg/L and the limit of quantification (LOQ) was 0.010 µg/L.

Quality Control and Calibration

Each 96-well plate included duplicate standard solutions and a blank. Three spiked milk samples (at 0.025, 0.050 and 0.100 µg/L) were analysed alongside unknowns; mean recovery ranged from 88% to 103%. The certified reference material was run once per batch, and measured values fell within the certified range on all occasions. Intra-assay and inter-assay coefficients of variation were 4.6% and 7.8%, respectively. Data were analysed using SPSS 26.0 with independent t-tests for milk-type and seasonal comparisons, one-way ANOVA for city comparisons, and chi-square for compliance proportions. Significance was set at P < 0.05.

Results: AFM1 was detected in 210 of 240 samples (87.5%). Of the 30 non-detectable samples, 22 were pasteurized dryseason specimens and eight were raw dry-season specimens. Concentrations ranged from < 0.005 to 0.094 µg/L. The overall mean across all positive samples was 0.043±0.021 µg/L. Raw milk had a significantly higher mean (0.058±0.017 µg/L) than pasteurized milk (0.027±0.011 µg/L; P < 0.001). Wet-season means exceeded dry-season means for both milk types (P<0.01).

Comprehensive Interpretation

The data reveal a widespread, low-to-moderate AFM1 contamination pattern across all six cities, with two clear gradients: raw milk is more contaminated than pasteurized, and wet-season samples exceed dry-season samples. Lagos Mainland and Port Harcourt emerge as the cities of greatest concern, likely reflecting the larger volume of informally marketed raw milk and the proximity of dairy supply chains to humid coastal storage conditions. While the overwhelming majority of samples fell below the Codex threshold reassuring from a regulatory standpoint nearly two-thirds exceeded the stricter EU limit, which is the standard increasingly referenced in international trade and consumer-advocacy discourse.

Discussion

The overall AFM1 detection rate of 87.5% is comparable to rates reported in other West African surveys. Adetunji et al. [10] found 82% positivity in raw milk from northern Nigeria, while Ogunade et al. [11] reported 91% in a Lagos-specific survey. The consistently high detection rate across cities and seasons confirms that AFM1 exposure through milk is pervasive rather than localised. At the feed level, the explanation lies in the widespread contamination of maizethe dominant energy source in Nigerian dairy rations -with Aspergillus flavus, particularly when grain is stored in openair or poorly ventilated structures [1,2] . The seasonal effect, with wet-season means approximately 40% higher than dry-season means, aligns with findings from Kenya [13] and Egypt [14] . Higher ambient moisture during the rains raises the water activity of stored feed, accelerating fungal growth and AFB1 production [2] . Because the carryover from AFB1 to AFM1 is dose-dependent, elevated feed contamination translates directly into higher milk levels [3] . That pasteurized milk contained roughly half the AFM1 concentration of raw milk might seem to contradict the known heat stability of the toxin. But the difference is better explained by dilution: pasteurized products are often pooled from multiple farm sources, which averages out individual high-concentration contributions. Additionally, the processing steps of centrifugation and standardisation remove the fat fraction, which carries a small portion of AFM1 bound to casein micelles [5,15] . From a public health perspective, the 61.7% EU noncompliance rate signals that Nigerian consumers, particularly children, face chronic low-dose AFM1 exposure. While acute toxicity is unlikely at these levels, the genotoxic and immunosuppressive effects of long-term exposure remain a valid concern [4,6] . Strengthening on-farm aflatoxin management through improved grain drying, biological control agents such as Aflasafe, and routine feed testing offers the most effective upstream intervention [16,17] .

Conclusion

The main contribution of this research is a multi-city, dualseason, dual-milk-type survey of AFM1 in the Nigerian metropolitan milk supply, executed with a validated competitive ELISA protocol and supported by rigorous quality-control measures including certified reference material and spike-recovery verification.

The supporting evidence can be grouped under four themes. First, AFM1 was detected in nearly nine out of every ten samples, confirming that contamination is endemic across both raw and processed milk channels. Second, raw milk carried significantly higher concentrations than pasteurized milk, with the gap widest during the wet season, pointing to feed-storage conditions as the root cause. Third, geographic variation was significant: Lagos Mainland and Port Harcourt showed the highest levels, likely reflecting the humid coastal climate and the larger informal dairy sector in those cities. Fourth, while virtually all samples complied with the Codex limit of 0.5 µg/L, nearly two-thirds exceeded the EU threshold of 0.05 µg/L, underscoring the regulatory gap that Nigerian consumers face. The significance of these findings resonates at several levels. For the Nigerian dairy industry, the data make a strong case for investing in pre-harvest and post-harvest aflatoxin mitigation along the feed-to-milk chain. For regulators, the absence of an enforceable national AFM1 limit specific to liquid milk represents a gap that leaves consumers, especially children, exposed to chronic low-dose carcinogen intake. For the scientific community, the research contributes city-level resolution data that can inform risk-assessment models and exposure calculations tailored to Nigerian dietary patterns.

Several questions remain open and deserve targeted followup. The carry-over rate from feed to milk in Nigerian dairy systems has not been measured directly; such data would allow back-calculation of the feed-contamination levels required to push milk AFM1 above the EU limit. Whether the same seasonal and geographic patterns apply to fermented dairy products nono and wara, which are widely consumed in Nigeria is unknown. And the cost-effectiveness of available interventions (Aflasafe biocontrol, hermetic grain storage, activated-clay binders in feed) has not been evaluated under Nigerian smallholder conditions. Answering these questions would move the conversation from surveillance to actionable mitigation.

Acknowledgement

Acknowledgements Institutional SupportThe authors acknowledge the Lagos Foundation for Food Safety Research for providing laboratory facilities, ELISA instrumentation and cold-chain logistics for sample transport.

References

1. Akinmusire OO, El-Haleem DRA, Olaitan JO. An overview of aflatoxin contamination of foods and feeds in Africa. Journal of Food Quality and Hazards Control. 2019;6(3):104-110.
2. Klich MA. Aspergillus flavus: The major producer of aflatoxin. Molecular Plant Pathology. 2007;8(6):713-722.
3. Veldman A, Meijst JAC, Borggreve GJ, Heeres-van der Tol JJ. Carry-over of aflatoxin from cows' feed to milk. Animal Production. 1992;55(2):163-168.
4. International Agency for Research on Cancer. Aflatoxins. In: Chemical agents and related occupations. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. 2012;100F:225-248.
5. Prandini A, Tansini G, Sigolo S, Filippi L, Laporta M, Piva G. On the occurrence of aflatoxin M1 in milk and dairy products. Food and Chemical Toxicology. 2009;47(5):984-991.
6. Kunter İ, Hürer N, Gülcan HO, Öztürk B, Doğan İ, Şahin G. Assessment of aflatoxin M1 and heavy metal levels in mothers' breast milk in Famagusta. Biological Trace Element Research. 2017;175(1):42-49.
7. European Commission. Commission Regulation EC No. 1881/2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union. 2006;L364:5-24.
8. Standards Organisation of Nigeria. Nigerian industrial standard for pasteurised milk, NIS 157:2007. Lagos: SON; 2007.
9. R-Biopharm AG. Ridascreen Aflatoxin M1: Enzyme immunoassay for the quantitative analysis of aflatoxin M1 in milk and milk products. Darmstadt: R-Biopharm; 2020.
10. Adetunji MC, Atanda OO, Ezekiel CN. Risk assessment of mycotoxins in stored maize grains consumed in Nigeria. Food Control. 2014;41:98-103.
11. Ogunade IM, Martinez-Tuppia C, Queiroz OCM, Jiang Y, Jeong KC, Adesogan AT. Silage review: Mycotoxins in silage: Occurrence, effects, prevention, and mitigation. Journal of Dairy Science. 2018;101(5):4034-4059.
12. Codex Alimentarius Commission. Code of practice for the prevention and reduction of mycotoxin contamination in cereals, CAC/RCP 51-2003. Rome: FAO/WHO; 2014.
13. Kang'ethe EK, Lang'a KA. Aflatoxin B1 and M1 contamination of animal feeds and milk from urban centres in Kenya. African Health Sciences. 2009;9(4):218-226.
14. Elgerbi AM, Aidoo KE, Candlish AAG, Tester RF. Occurrence of aflatoxin M1 in randomly selected North African milk and cheese samples. Food Additives and Contaminants. 2004;21(6):592-597.
15. Unusan N. Occurrence of aflatoxin M1 in UHT milk in Turkey. Food and Chemical Toxicology. 2006;44(11):1897-1900.
16. Bandyopadhyay R, Ortega-Beltran A, Akande A, Mutegi C, Atehnkeng J, Kaptoge L, et al. Biological control of aflatoxins in Africa: Current status and potential challenges in the face of climate change. World Mycotoxin Journal. 2016;9(5):771-789.
17. Grace D, Lindahl J, Atherstone C, Kang'ethe E, Nelson F, Wesonga T, et al. Aflatoxin standards for feed. Nairobi: International Livestock Research Institute; 2015.