Occurrence of Aflatoxin M₁ in Cheese and Yoghurt Marketed at El-Fayoum Province, Egypt

MATERIALS AND METHODS

Materials: This study was carried out from June, 2020 to December, 2020. One hundred skim milk soft white (kareish) cheese, fifty flavoured drinking yoghurt (25 strawberry and banana flavours for each) and fifty (pepper and Tomato) flavoured cheese samples of plant origin were randomly acquired from various retail outlets and markets in AL-Fayoum province, Egypt. The quantity of kareish and flavoured cheese samples was 250 g for each, while in drinking yoghurt samples, each bottle about (440 mL) in an excellent state by observation. The obtained samples were transferred to the lab in a careful proper condition.

Methods: The measurable examination of aflatoxin M₁ was based on Competitive Enzyme Immunoassay by RIDASCREEN^® AFM₁ 30/15 (Art. no: R1111, R-Bio-Pharm, Darmstadt, Germany) kit. The utilized chemicals were obtained by the kit industrialist. Chloroform, methanol, dichloromethane and C₇H₁₆ were provided from Merck. For achieving recovery study, AFM₁ standard was got from Sigma Company (Sigma-Aldrich, A6428). This standard was obtained from Aspergillus flavus. AFM₁ stock solution (50 mg mL^–1) was done in a methanol/chloroform mix (81:19 v/v) and kept at -20°C. Before use, it was diluted with methanol/chloroform (1:1 v/v) at required concentrations according to Lopez et al.¹⁹.

Extraction and clean up using solid-phase extraction: The AFM₁ in the investigated samples were extracted with 75 mL chloroform, 1 mL saturated NaCl solution and 5 g Celite Hyflo Supercel for 45 min with unceasing shaking. The mixture was filtered using paper 110 mm (Schleicher and Schuell, Germany). The extract of chloroform was put into a 250 mL flask, evaporated to dryness in H₂O bath (Edelstahl, UK) at 30°C. One mL methanol, 30 mL distilled H₂O and 50 mL hexane was supplemented to the residue²⁸.

By separating the funnel, the mixture was relocated, then washed two times with 10 mL distilled H₂O and shaken for fifteen seconds. The lower layer was collected and utilized in the cartridge C₁₈ (Machery and Nagel, Germany) for clean-up. The cartridge C₁₈ was primed by adding 10 mL CH OH followed by 10 mL dist. H₂O. The collected layer gently pulled through the cartridge. Ten mL of distilled H₂O followed by 10 mL of C₆H₁₄ were added as washing solution and pulled through the column. Three mL of eluting dichloromethane/acetone (3:1) was pulled through the cartridge. Finally, elute was collected in a 4 mL glass vial tube and evaporated till dry.

Determination of AFM₁ using ELISA: Standard solutions (100 μL) and extracted samples were poured into independent microtiter wells to seal the binding places and kept in the absence of light at (25°C) for 60 min. Then, the solution was squandered and wells were washed by Buffer of washing (250 μL) twice. In the following, 100 μL of the watered enzyme conjugate was supplemented to fill free binding places and kept in the absence of light at room temperature for 15 min. Again, the wells were washed twice to remove any free enzyme conjugate. Subsequently, 100 μL of substrate/ chromogenic was added and put in the absence of light at 25°C for 15 min. Bound enzyme conjugate converted the colourless chromogen to blue output. Finally, 100 μL of the stop solution (1N H₂SO₄) was supplemented into the wells and the colour became yellow. The absorbance was determined at λ equal 450 nm in the plate reader of ELISA (ELX800, Bio-Tek Instruments, USA) versus air blank within 15 min. According to the RIDASCREEN kit directions, the lower detection limit is (50 ng kg^–1)²⁹.

Recovery study: For the validation of the method, a recovery study was achieved by spiking known concentrations (50, 150, 250 and 450 ng kg^–1) of AFM₁ into extracted samples just before the examination. The sample preparation and ELISA test steps were done as described above. All experiments were achieved using four samples/each treatment. Under these conditions, the mean recovery scores in spiked samples were 99.00%, with a coefficient of variation of 8.5%. Based on the kit directions, the recovery rate in the examined samples is almost 100.00%, with a mean coefficient of variation of 1.1%.

Estimated daily intake (EDI) calculation: The estimation of dietary exposure to AFM₁ was calculated from the mean concentration of toxin in skim milk cheese (ng kg^–1). The daily intake of this cheese is 22 g and the mean adult person's bodyweight is 60 kg^26,30. Estimated AFM₁ Daily Intake was achieved by these Eq.:

Statistical analysis: The p-value for significant difference between the mean values of AFM₁ concentration was predestined by T-test through Excel of Microsoft 365 enterprise. The p (less than 0.05) was approved as a significant value and 95.00% Confidence Interval.

RESULTS AND DISCUSSION

The AFM₁ prevalence in the investigated kareish cheese depicted in Table 1 and 2. Aflatoxin M₁ was found in 80.00% of the examined skim milk cheese, ranging between 59.1-875.4 ng kg^–1 and AFM₁ was not detected (<50 ng kg^–1) in all examined drinking yoghurt and flavoured cheese samples. Aflatoxin M₁ concentration in 80.00% of the examined kareish cheese was exceeded the critical value (50 ng kg^–1) which, was legislated by EC⁶.

There was a significant difference (p<0.05) between the mean value of AFM₁ concentration of kareish cheese and flavoured drinking yoghurt samples, also between the mean value of AFM₁ concentration of kareish cheese and flavoured cheese. As reported in Table 2, there is a considerable variation in the AFM₁ concentration. Most of the examined kareish cheese samples (51.00%) contained AFM₁ at 50-250 ng kg^–1, while the 10, 8.00 and 11.00% of the examined kareish cheese samples were contaminated with AFM₁ at concentrations, 251-450, 451-650 and >650 ng kg^–1, respectively. In addition, AFM₁ was not detected (<50 ng kg^–1) in 20.00% of the examined kareish cheese samples. The Estimated Daily Intake (EDI) and Hazard Index (HI) for AFM₁ from the eating of Egyptian kareish cheese were 0.091 and 0.455 ng kg^–1, respectively.

Taken into account, the preferred compatibility of AFM₁ for milk casein portion, a high toxin amount, may happen in curd processing¹⁷. Investigations indicated that the concentration of AFM₁ is four times greater in cheese than in milk, which is utilized in its production²². So, cheese could be the highest origin of aflatoxins among milk products. An elevated AFM₁ mean value in the examined skim milk cheese was recognized since multiple surveys recorded the great AFM₁ existence in plant-made and farmers-made cheese from many areas^18,26,31.

Current results agree with those recorded in many countries. In Libya, Elgerbi et al.³² found that the AFM₁ in 75.00% of twenty cheese samples, with range 0.11 to 0.52 μg kg^–1, while the percentage of AFM₁ in the examined 20 skim milk cheese samples was 55.00%¹. In Brazil, AFM₁ was found in 74.70% of white soft cheese samples collected from Minas Gerais, the range from 0.02-6.92 μg kg Minas Gerais and 26.70% samples were greater than 0.25 μg kg^{–1 33}. In another study, the AFM₁ was found in (80.00%) of the investigated samples in Kuwait³¹. Hosny et al.³⁴ found that 33.3% of the examined skim milk cheese AFM₁ polluted with an average of 0.027±0.009, a minimum of 0.01 and a maximum of 0.04. The forgoing results indicated that the dairy milk used for processing skim milk cheese must be free from AFM₁ to avoid pollution of cheese and safe human health. The existence of AFM₁ in animal milk mainly resulted from the consumption of contaminated feed with AFB₁.

In the present study, the findings were compared with the international limit of EC authorities. In this respect, Kamkar³⁵ detected AFM₁ in 82.50% of the examined skim milk cheese samples with average levels of 0.41 μg kg^–1 and (60.5%) of these samples were incriminated AFM₁ greater than the upper allowable international limit (0.25 μg kg^–1). While Rahimi et al.³⁶ recorded that 53.4% of the examined cheese samples contaminated with AFM₁ at the range from 82-1254 ng kg^–1 and 31.80% of these samples were overrun the limit 250 ng kg^–1.

Current findings were higher than obtained by Montagna et al. ³⁷, who found AFM₁ in 16.6% of soft cheese samples with an average of 88.6 ng kg^–1. In another study, 15% of the investigated soft cheese samples were found positive for AFM₁ and its concentration was (>50 ng kg^–1) in all samples³⁸.

Moreover, the AFM₁ was found in 39 soft cheese sample²¹. They resulted that 11 (28.2%) samples were polluted with AFM₁, with a maximum concentration of 188.4 ng kg^–1. Whereas, Atanda et al.³⁹ revealed that no detectable concentrations of AFM₁ were recorded in soft cheese. Similarly, Martins et al.⁴⁰ not detected AFM₁ in soft cheese and dried milk using the HPLC method. Discipline programs for operators and the exactness of (HACCP) system are necessitous for satisfying the safe food articles^41-47.

Vaz et al.⁴⁸ reported that the hazardous compounds become concentrated in food through processing. The Enriched Factor (EF) of a product is the significant key factor for determining exposure. Although the AFM₁ levels in kareish cheese have been reported to be higher than European legislation, many of the experiments concerning EFs have been performed using artificially contaminated milk. The EFs differed based on the cheese kind plus the source of milk contamination. The enriched factor was ranged from 4.1-4.9. Therefore, cheese using contaminated milk with AFM₁ was just about three folds greater than the manufactured fresh milk^18,49.

The variations in detected AFM₁ concentrations in forgoing studies may be related to different factors included cheese preparation, ripening, changes in season's climates, storage and packing²⁹. It has been reported that the higher levels of milk AFM₁ processed during cold times⁵⁰.

Hassan and Kassaify⁵¹ recorded a higher EDI of 0.14 ng kg^–1 daily body weight in the examined cheese samples. When the HI value was <1, it means that the risk degree for hepatic cancer in Egyptian kareish cheese consumers is not common. HI obtained in this investigation was greater than the number recorded by Shahbazi et al.²⁷, who reported that the EDI is affected by weather and the technique of AFM₁ determination.

The low aflatoxin M₁ level (<50 ng kg^–1) in flavoured cheese of plant origin samples may be attributed to mixing ingredients (non-dairy fats and/or proteins) for cheese processing to conform to particular specification with low cost⁵². Regarding drinking yoghurt samples, Atasever et al.⁵³ detected that the AFM₁ levels were greater than the international limit of 50 ng kg^–1 in 13.6% of the examined drinking yoghurt samples. They revealed t hat the min of AFM₁ was 6 ng kg^–1, the max was 264 ng kg^–1 and the mean value 36.5 ng kg^–1. In the current study, the detected concentrations of AFM₁ in the examined drinking yoghurt samples were similar to the recorded findings by Heshmati et al.⁵⁴.

The low AFM₁ level (<50 ng kg^–1) in the examined flavoured drinking yoghurt may be attributed to the fermentation of drinking yoghurt, the reduced AFM₁ concentration in the used milk and also the companies which manufactured this product use the verified raw milk for AFM₁ levels⁵⁵.

CONCLUSION

The prevention of dairy animals from consuming contaminated feed with AFB₁ is the perfect factor for gaining free milk and their products from AFM₁ contamination. Besides, hygienic processing, ripening and storage shall be applied through the manufacturing and handling of milk derivatives for the prevention of aflatoxins' health risks.

Whereas the calculated EDI and HI for aflatoxin M₁ represent a moderate toxic risk for Egyptian kareish cheese consumers. Simultaneously, alertness shall be offered to periodical monitoring of these toxins in feeds and milk products. Plus, the governmental agencies should aware of the farmers, owners of dairy companies and these milk products consumers on the bad health effects of this toxin.

SIGNIFICANCE STATEMENTS

This study revealed a significant contamination level of Egyptian kareish cheese with Aflatoxin M₁ and calculated EDI and HI for aflatoxin M₁ represent a moderate toxic risk for Egyptian kareish cheese consumers. Subsequently, farmers, dairy plants and consumers should have general knowledge about the potential risk of this toxin. Protecting the dairy animals from eating contaminated feed with AFB₁ is the best way to prevent the generation of aflatoxin M₁ in milk and dairy products.