Occurrence of Aflatoxin M₁ in yogurt samples found in markets in Kosovo during spring 2023

A. Camaj Ibrahimi, B. Berisha, A. Haziri, A. Camaj Isa, H. Dibrani Sopjani, S. Muriqi, N. Shala and I. Hoxha*

https://doi.org/10.46419/vs.55.3.6

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Abstract

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Aflatoxin M₁ (AFM₁), a toxic byproduct of aflatoxin B₁ (AFB₁) produced by Aspergillus fungi, is a carcinogenic mycotoxin that can contaminate various agricultural commodities. It can be transferred from AFB₁-contaminated feed to milk and dairy products, including yogurt, posing a potential health risk to consumers. In spring 2023, a total of 74 yogurt samples were collected from the largest food suppliers in Kosovo for analysis, including samples produced in Kosovo and seven other countries: Albania, North Macedonia, Bosnia and Herzegovina, Slovenia, Greece, Italy, and Germany. A rapid and sensitive analytical method, Enzyme-linked immunosorbent assay (ELISA), was used for the analysis. The results of the study highlight discernible differences in the maximum tolerable levels of AFM₁ between the countries. More specifically, yogurt samples from Slovenia and Germany had lower levels than those from other countries. Additionally, the median levels of AFM₁ in samples from Slovenia and Germany were significantly lower. The mean concentrations of AFM₁ in yogurt samples from Kosovo and other countries were 0.071 µg/kg and 0.080 µg/kg, respectively. Out of all samples, 66 (89%) exceeded the maximum tolerable limit of 0.05 µg/kg. Among the exporting countries, Albania had the highest median AFM₁ level of 0.085 µg/kg and the highest maximum level of 0.195 µg/kg. Slovenia had the lowest median AFM₁ level, while Germany had the lowest maximum AFM₁ level. All samples from Albania, Greece, and Bosnia and Herzegovina exceeded the maximum tolerable limit. High prevalence was also observed in samples from Kosovo, North Macedonia, and Slovenia. Considering the average daily consumption of about 250 grams of yogurt, and the total median value of Aflatoxin M₁ concentration (0.071 µg/kg), the estimated daily intake was calculated to be 0.017 µg. These findings highlight the importance of monitoring and enforcing regulatory limits to ensure yogurt safety and to protect public health. Efforts should be focused on mitigating AFM₁ contamination and implementing measures to minimise its presence in dairy products, especially in regions where levels exceed the established limits.

Key words: yogurt; ELISA; aflatoxin M₁; food; contamination

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Abbreviations

AFM₁ = Aflatoxin M₁
AFB₁ = Aflatoxin B₁
ELISA = Enzyme-linked immunosorbent assay

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Introduction

Aflatoxin M₁ (AFM₁) is a toxic metabolite of aflatoxin B₁ (AFB₁), a carcinogenic mycotoxin produced by Aspergillus fungi that can contaminate various agricultural commodities (IARC, 2002). AFM₁ can be transferred from AFB₁-contaminated feed to milk and dairy products, including yogurt, posing a potential health risk for humans who consume them (Prandini et al., 2009). Approximately 0.3–6.2% of AFB₁ is converted into metabolised AFM₁ and excreted in milk, depending on various factors (Iqbal et al., 2015). The International Agency for Research on Cancer (IARC) has classified AFM₁ with AFB₁ as a Group 1 carcinogen (IARC, 2002). Studies have shown that the presence of AFM₁ in milk and milk products, including yogurt, is a health issue due to their regular consumption by all age groups (Iqbal et al., 2015). The main harm of AFM₁ is its carcinogenicity and mutagenicity, which can destroy human and animal liver tissue, resulting in hepatocarcinoma and even death. AFM₁ alone can also cause damage to DNA by covalently binding to it, which may enhance the genotoxicity already caused by AFB₁ (Saha Turna et al., 2021).
The presence of AFM₁ in milk and dairy products has been known for several decades and is an important problem worldwide, especially for developing countries (Iqbal et al., 2015).

Aflatoxin-producing fungi are found in areas with hot, humid climates and aflatoxins in food are a result of both pre- and post-harvest fungal contamination (EFSA et al., 2020). Climate change is anticipated to impact the presence of aflatoxins in food in Europe. Aflatoxin M₁ has stable physical and chemical properties and is not destroyed by pasteurisation. According to Iha et al. (2013), processing and storage have little effect on AFM₁ content in milk and milk products, with total AFM₁ mass in milk being reduced by 3.2% in cheese and 6% in yogurt (pH 4.4).

The European Commission has specified that AFM₁ in milk should not exceed 0.05 μg/kg (European Commission, 2006). Kosovo has adopted several EU regulations and standards for food safety and quality, including those related to dairy products such as yogurt. Dairy products are an important part of Kosovo’s diet and economy, with significant annual per capita consumption of milk and cheese (Kosovo Agency of Statistics, 2018). In Kosovo, only a few studies have been conducted on AFM₁ in milk in recent years, and no research has been published on the presence of AFM₁ in yogurt.
According to a 2016 study in Kosovo by Camaj et al. (2018), a high percentage of milk samples were found to be non-compliant with maximum AFM₁ levels. This suggests that efforts are needed to reduce contamination levels of aflatoxin B₁ in cow feed in Kosovo. In the Balkan region, several studies have been conducted over the past ten years on the incidence of aflatoxin M₁ in milk and dairy products (Camaj et al., 2018; Ilievska et al., 2022; Topi et al., 2022), indicating further efforts are needed to reduce the contamination levels of AFB₁ in cow feed, and regular monitoring of the milk is needed.

The objective of this research was to assess the occurrence of AFM₁ in yogurt samples found in markets and major food suppliers in Kosovo during spring 2023.
These samples were produced in Kosovo and in countries exporting to Kosovo.

This study will contribute to safeguarding consumer health and promoting the production and consumption of safe and high-quality yogurt.

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Materials and methods

Sample Collection

Out of this total, the 74 samples of yogurt were collected from markets (largest food suppliers) in Kosovo during spring 2023, for analysis of the quantity of AFM₁ toxin. Of the 74 samples, 40 samples were produced in Kosovo and 34 in other countries: North Macedonia (5), Albania (7), Bosnia and Hercegovina (6), Slovenia (6), Greece (6), Italy (2), and Germany (2).
The samples were tested immediately after collection.

Laboratory analyses

The quantitative analysis of AFM₁ in the yogurt samples was done using enzyme-linked immunosorbent assay (ELISA) method, one of several methods used for the detection of AFM₁ in dairy products. It offers many advantages including a shorter analysis time, simultaneous analysis of many samples, limited use of organic solvents, absence of complicated sample preparation steps, simple analytical procedure compared to long-lasting and expensive chromatographic techniques (Jukić et al., 2020). To test AFM₁, we used the MEIZHENG BioTech Co. Aflatoxin M₁ ELISA Test Kit, following the manufacturer`s instructions, and summarised as follows. Each testing reagent kit offered a certificate with the validation results (Table 1). While the specific ELISA protocol was not validated in our laboratory for this determination, it is important to note that this method is well-established and routinely used in our lab for aflatoxin M₁ testing, and the reagent kit offered a validation certificate.

Our lab has extensive experience with this methodology, ensuring reliability and reproducibility of results.

All samples were tested at room temperature (20-25°C) immediately after collection. The dilution factor was 1. For testing, 1 g of each sample was measured and diluted with 4 mL of sample diluting buffer I, and vortexed for 2 minutes. Then samples were centrifuged for 5 minutes at 4000 rpm. For the assay, 50 μL of the sample dilution was measured.

The principle of the Aflatoxin M₁ ELISA Test Kit is an indirect competitive enzyme-labelled immunoassay. The aflatoxin M₁ antigen is precoated on the wells. The aflatoxin M₁ in the sample competes with the aflatoxin M₁ antibody with aflatoxin M₁ antigen on the well, while the AFM₁ antibody combines with the enzyme conjugate. Then the substrate solution is pipetted to the wells to convert the colour. The colour of the unknown samples is compared to the colour of the standards and the aflatoxin M₁ concentration is derived. The absorbance values were obtained using a plate reader set at 450 nm, and the level of AFM₁ was calculated using a logarithmic standard curve, and the average of duplicates was used as the result. According to the manufacturer’s certificate, sensitivity was 0.015 μg/kg (the same as the LOD of this kit for raw milk determination), which seems to suggest it should be capable of detecting aflatoxin M₁ at levels lower than the LOD.
Sensitivity is usually the concentration of the second standard (Table 1). Recovery was 100%±30%, and the precision for intra-lab assay was CV% < 10%.

In the current study, it is essential to note that the Limit of Detection (LOD) of the employed kit for fermented milk and milk beverage is 0.075 µg/kg, which is higher than the maximum tolerable limit of aflatoxin M₁ in yogurt as set by the regulatory guidelines (0.05 µg/kg). Interestingly, we observed results that were lower than the specified Limit of Detection (LOD) of the kit (0.075 µg/kg). These results indicate that some of the yogurt samples have very low or even undetectable levels of aflatoxin M₁.

Aflatoxin M₁ ELISA Test Kit standard summary

Table 1 provides the absorbance values A₄₅₀, B/B0 ratios, and CV% values, for different concentrations of Aflatoxin M₁ standards used in the ELISA test Kit validation, as provided on the certificate of analysis by the manufacturer. These values serve as reference points for comparing the results obtained from samples tested with the Aflatoxin M₁ ELISA Test Kit.

CV% (Coefficient of Variation) is calculated as CV= (SD/μ) x 100%, where SD is the standard deviation of the replicates (two replicates for each concentration of the standard), and μ is the average of replicates.

Statistical analysis

Statistical analysis was performed using SPSS. The results were grouped into two categories by country of production (Kosovo vs. other countries). The results obtained for the AFM₁ content were expressed as mean values with standard deviation (SD), as median, and as maximum concentration of AFM₁.

We used statistical tests to determine if there were any significant differences between the groups. For two groups, we used student’s t-test and for three or more groups, we used One-Way Analysis of Variance (ANOVA) with a significance level of P < 0.05 for both tests. To confirm the outcomes of the independent t-test, we used effect size as an additional variable (Dankel et al., 2017). Effect size is a measure of the disparity between two group means (Lakens, 2013). Both substantive importance (effect size) and statistical importance (P value) must be presented to interpret the findings (Sullivan and Feinn, 2012).

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Results and discussion

Table 2 presents the analysis of yogurt samples for the presence of AFM₁ (in μg/kg). The samples are categorised into two groups by country of production, i.e., Kosovo and imported from other countries.

Out of these samples, 40 samples of yogurt produced in Kosovo were tested and had a median AFM₁ level of 0.070 µg/kg, with a maximum level of 0.110 µg/kg. Of these, 16 samples (40%) exceeded the LOD (0.075 µg/kg) and 37 (92%) exceeded the maximum level of 0.05 µg/kg.

In comparison, 34 samples of yogurt produced in other countries were tested and had a median AFM₁ level of 0.072 µg/kg, with a maximum level of 0.195 µg/kg.
Of these, 11 samples (32%) exceeded the LOD (0.075 µg/kg) and 29 (85%) exceeded the maximum level of 0.05 µg/kg.

In total, out of the 74 samples tested, the median AFM₁ level was 0.071 µg/kg and the maximum level was 0.195 µg/kg.
Of all samples, 27 (36%) exceeded the LOD and 66 (89%) exceeded the maximum level of 0.05 µg/kg.

Table 3 shows the corresponding values of AFM₁ for the samples produced and imported from seven other countries.

Table 3 shows that yogurt produced in Albania had the highest median AFM₁ level of 0.085 µg/kg and the highest maximum level of 0.195 µg/kg among all countries. All seven samples (100%) exceeded the maximum level of 0.05 µg/kg. In comparison, yogurt produced in North Macedonia had a lower median AFM₁ level of 0.070 µg/kg and a lower maximum level of 0.075 µg/kg. None of these samples exceeded the LOD and four (80%) exceeded the maximum level of 0.05 µg/kg. Yogurt produced in Bosnia and Herzegovina had a median AFM₁ level of 0.090 µg/kg, which is higher than that of North Macedonia but lower than that of Albania. The maximum level was 0.150 µg/kg, which was also higher than that of North Macedonia but lower than that of Albania. All six samples (100%) exceeded the maximum level of 0.05 µg/kg.

Yogurt produced in Greece had a median AFM₁ level of 0.072 µg/kg and a maximum level of 0.100 µg/kg, both lower than those of Albania and Bosnia and Herzegovina but higher than those of North Macedonia. All six samples (100%) exceeded the maximum level of 0.05 µg/kg. Yogurt produced in Slovenia had the lowest median AFM₁ level among all countries at 0.057 µg/kg and a low maximum level of 0.065 µg/kg. None of these samples exceeded the LOD and four (67%) exceeded the maximum level of 0.05 µg/kg. Yogurt produced in Italy had a median AFM₁ level of 0.077 µg/kg and a maximum level of 0.110 µg/kg, both higher than those of Slovenia but lower than those of Albania, Bosnia and Herzegovina, and Greece. Yogurt produced in Germany had the lowest maximum AFM₁ level among all countries at 0.055 µg/kg and a low median AFM₁ level of 0.042 µg/kg, which is lower than the median of all other countries.

In total, of the 34 samples tested from these countries, the median AFM₁ level was 0.070 µg/kg and the maximum level was 0.195 µg/kg.

According to the data calculated in Table 4, the findings of this study revealed that no statistically significant disparities (x̄ _Kosovo = 0.071 µg/kg, x̄ _{other countries} = 0.080 µg/kg, t=-0.1.307, P=0.198) were observed between Kosovo and other countries with respect to AFM₁ concentrations. The mean concentrations of Aflatoxin M₁ (AFM₁) in the analysed yogurt samples were 0.071 ± 0.018 µg/kg and 0.080± 0.037 µg/kg for yogurt from Kosovo and other countries, respectively.

The current study aimed also to investigate the variations in average levels of Aflatoxin M₁ (AFM₁) in yogurt across seven distinct countries that import yogurt into Kosovo (Table 5).

The findings from this study highlight discernible differences in the maximum tolerable levels of AFM₁ among countries. More specifically, yogurt samples from Slovenia and Germany had lower levels than those from other countries. Additionally, the median levels of AFM₁ in samples from Slovenia and Germany were significantly lower. The mean AFM₁ levels were as follows: Albania (0.11 ± 0.05 µg/kg), North Macedonia (0.08 ± 0.04 µg/kg), Bosnia and Hercegovina (0.08 ± 0.17 µg/kg), Greece (0.07 ± 0.01 µg/kg), Slovenia (0.05 ± 0.00 µg/kg), Italy (0.07 ± 0.04 µg/kg), and Germany (0.04 ± 0.001 µg/kg).

Means that share the same letter are significant.

A two-year survey of aflatoxin M₁ in milk marketed in Albania, by Topi et al. (2022) found that AFM₁ was detected in 62 out of 119 milk samples (52.10%) and that AFM₁ contamination in pasteurised milk was higher than that in UHT milk samples (59.68% and 43.86%, respectively). Seven milk samples (5.88%) contained AFM₁ at levels exceeding the European Union maximum residue permitted level. A study published in 2022 (Ilievska et al.) revealed a high incidence of AFM₁ in UHT milk and dairy products marketed in North Macedonia. According to this study, the highest incidence of AFM₁ was found in yogurt samples (93.8%), with 178 yogurt samples tested.

Calculating Risk Assessment for aflatoxin M₁ in yogurt

To estimate the exposure to aflatoxin M₁ from yogurt, we need to calculate the estimated daily intake based on consumption patterns and the concentration of aflatoxin M₁ detected in yogurt. The formula to calculate the estimated daily intake is as follows:

Estimated Daily Intake (in micrograms) = average yogurt consumption (in grams) x aflatoxin M₁ concentration (in micrograms per kilogram) / per body weight

The Dietary Guidelines for Americans 2020–2025 recommends 3 cups of dairy per day for anyone over the age of 9 years. Each cup of yogurt counts as 1 cup of dairy, or about 250 grams daily. Based on this recommendation, if we consider that the average yogurt consumption per day is one cup, or about 250 grams, and if we take the total median value (0.071 µg/kg) as per aflatoxin M₁ concentration, then the estimated daily intake would be as follows:

Estimated Daily Intake (in micrograms) = 0.25 kg x 0.071 µg/kg = 0.017 µg

According to this assumption, if a grown person consumes one cup of yogurt per day, or about 250 grams, the estimated daily intake of aflatoxin M₁ from yogurt is 0.017 micrograms per body weight of the individual (in kg). Because no regulatory agency has set a tolerable daily intake (TDI) for AFM₁, it is not possible to compare exposure estimates to a TDI to determine at-risk populations (Saha Turna et al., 2021). According to Mollayusefian et al. (2021), the aflatoxin level in food commodities should be reduced to the lowest possible level.

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Conclusions

In conclusion, the findings from this study highlight discernible differences in the maximum tolerable levels of AFM₁ among countries. More specifically, yogurt samples from Slovenia and Germany had lower levels than those from other countries. Additionally, the median levels of AFM₁ in samples from Slovenia and Germany were significantly lower.

The mean concentrations of AFM₁ in yogurt samples from Kosovo and other countries were 0.071 µg/kg and 0.080 µg/kg, respectively. Out of the total number of samples, 66 (89%) exceeded the maximal tolerable limit of 0.05 µg/kg. Among the countries that export to Kosovo, yogurt produced in Albania had the highest median AFM₁ level of 0.085 µg/kg and the highest maximum level of 0.195 µg/kg among all countries. Yogurt produced in Germany had the lowest maximum AFM₁ level among all countries at 0.055 µg/kg and the lowest median AFM₁ level of 0.042 µg/kg. All the samples from Albania, Greece, and Bosnia and Hercegovina exceeded the maximum tolerable limit for AFM₁. A high prevalence was shown in samples from Kosovo, North Macedonia, and Slovenia.

Considering the average yogurt consumption of one cup or about 250 grams per day and considering the total median value of aflatoxin M₁ concentration (0.071 µg/kg), the estimated daily intake was calculated to be 0.017 µg. The risk assessment for aflatoxin M₁ in yogurt was conducted to estimate the exposure to this toxin based on consumption patterns and its concentration in yogurt samples.

It is important to note that no regulatory agency has established a tolerable daily intake (TDI) for AFM₁. Therefore, comparing exposure estimates to a TDI to identify at-risk populations is not possible. However, existing research suggests that aflatoxin levels in food commodities should be minimised to the lowest possible level to ensure food safety.

These findings emphasise the importance of monitoring and enforcing regulatory limits to ensure the safety of yogurt and to protect public health. Efforts should be made to mitigate AFM₁ contamination and implement measures to minimise its presence in dairy products, particularly in regions where levels exceed the set limits. These measures will contribute to safeguarding consumer health and promoting the production and consumption of safe and high-quality yogurt.

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Acknowledgements

The authors acknowledge the University “Haxhi Zeka” in Peja, Kosovo, for funding the research, and the Food and Veterinary Agency of Kosovo for hosting the laboratory work.

References [… show]

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Pojavnost aflatoksina M₁ u uzorcima jogurta pronađenima na tržištu u Kosovu tijekom proljeća 2023.

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Arieta CAMAJ IBRAHIMI, PhD, Assistant Professor, Faculty of Agribusiness, University “Haxhi Zeka”, Peja, Kosovo; Bajram BERISHA, PhD, habil., Full Professor, Faculty of Agriculture and Veterinary, University of Prishtina, Kosovo; Arben HAZIRI, PhD, Full Professor, Faculty of Math and Science, University of Prishtina, Kosovo; Aferdita CAMAJ ISA, MSc., Kosova Medicines Agency, Prishtina, Kosovo; Hata DIBRANI SOPJANI, MSc., Food and Veterinary Agency, Prishtina, Kosovo; Shyhrete MURIQI, PhD, Assistant Professor, Faculty of Agribusiness, University “Haxhi Zeka”, Peja, Kosovo; Nexhdet SHALA, PhD., Full Professor, Faculty of Agribusiness, University “Haxhi Zeka”, Peja, Kosovo; Ibrahim HOXHA, PhD, Associate Professor, Faculty of Agribusiness, University “Haxhi Zeka”, Peja, Kosovo

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Aflatoksin M₁ (AFM₁), toksični nusproizvod aflatoksina B₁ (AFB₁) kojeg proizvode gljivice Aspergillus kancerogeni je mikotoksin koji može kontaminirati različite poljoprivredne proizvode; može se prenijeti iz hrane za životinje kontaminirane s AFB₁ na mlijeko i mliječne proizvode, uključujući i jogurt, predstavljajući potencijalni rizik za zdravlje potrošača. U proljeće 2023. godine prikupljena su ukupno 74 uzoraka jogurta od najvećih dobavljača hrane na Kosovu za analizu, uključujući uzorke proizvedene u Kosovu i u sedam drugih zemalja: Albaniji, Sjevernoj Makedoniji, Bosni i Hercegovini, Sloveniji, Grčkoj, Italiji i Njemačkoj. Za analizu je rabljena brza i osjetljiva analitička metoda, enzimski povezani imunosorbentni test (ELISA). Rezultati studije ukazuju da nalazi ove studije naglašavaju velike razlike u maksimalno dopuštenim razinama AFM₁ između različitih zemalja. Točnije, u uzorcima jogurta iz Slovenije i Njemačke uočene su niže razine od onih iz drugih zemalja. Uz to, srednje razine AFM₁ u uzorcima iz Slovenije i Njemačke bile su značajno niže. Srednje koncentracije AFM₁ u uzorcima jogurta s Kosova i iz drugih zemalja bile su 0,071 µg/kg, odnosno 0,080 µg/kg. Od svih uzoraka, 66 (89 %) bilo je više od dopuštenog ograničenja od 0,05 µg/kg. Od zemalja uvoznica, Albanija je imala najveću srednju razinu AFM₁ od 0,085 µg/kg i najveću maksimalnu razinu od 0,195 µg/kg. Slovenija je imala najnižu srednju razinu AFM₁, a Njemačka najnižu maksimalnu razinu AFM₁. Svi uzorci iz Albanije, Grčke i Bosne i Hercegovine bili su veći od maksimalno dopuštene razine. Visoka prevalencija zamijećena je i u uzorcima s Kosova, iz Sjeverne Makedonije i Slovenije. Razmatrajući prosječnu dnevnu konzumaciju od jedne čašice od oko 250 g jogurta i ukupnu srednju vrijednost koncentracije aflatoksina M₁ (0,071 µg/kg), izračunat je procijenjeni dnevni unos od 17,75 µg. Ovi nalazi naglašavaju važnost nadziranja i provođenja regulatornih ograničenja kako bi se osigurala sigurnost jogurta i zaštitilo zdravlje ljudi. Potrebno je uložiti napore za smanjenje kontaminacije AFM₁ i provedbu mjera za smanjenje njegove prisutnosti u mliječnim proizvodima, posebice u regijama u kojima razine prekoračuju utvrđena ograničenja.

Ključne riječi: jogurt, ELISA, aflatoksin M₁, hrana, kontaminacija