Sample collection and preparation

A total of 246 representative samples of various food samples and different categories (local (154), imported (73), and exported (19) were obtained from the Egyptian market in 2022 according to AOAC 977.16 (http://www.aoacofficialmethod.org/index.php?main_page=product_info&products_id=2065 (accessed on 20 August 2022) (Omar et al. 2020). The collected samples were tested for the presence of the four major MTs, including Aflatoxins (AFs; 196 samples), Ochratoxin A (OTA; 139), Zearalenone (ZEN; 70), and Deoxynivbyalenol (DON; 100) using the ELISA followed by immunoaffinity fluorometric analysis as previously reported by EOSQC standard guidelines (2010) (https://www.eos.org.eg/en/standard/12561, (accessed on 8 august 2023).

For lots weighing more than 50 tons, we took 100 incremental samples from the sub-lots to get an aggregate sample weighing 10 kg; but, for lots weighing less than 50 tons, we only took 3 to 10 incremental samples, depending on the lot weight, to produce an aggregate sample weighing 1 to 10 kg (Rahmani et al. 2009). To establish homogeneity, samples were transferred to the laboratory and then crushed (by means of sanitized food cutters) and mixed carefully by a horizontal shaker (Benchmark Scientific, Orbi Shaker, Edison, USA) to be prepared for subsequent analysis (Elkenany and Awad 2020).

Screening of certain mycotoxins (AFs, OTA, ZEN and DON) using ELISAChemicals and reagents

Screening for the four different MTs was done using an ELISA Test kit (RIDASCREEN®, Manufacturer R- Bio pharm AG, Darmstadt, Germany). For AFs (Art. Nr. R4701), OTA (Art. No. R1312), ZEN (Art. No.: R1401), DON (Art. No.: R5906). All reagents required for the enzyme immunoassay, were included in the test kits. Ready to use standards were included in the test kits with concentrations 0 µg/L, 0.05 µg/L, 0.15 µg/L, 0.45 µg/L, 1.35 µg/L, 4.05 µg/L (1.3 mL each) for AFs; 0 µg/L, 0.03 µg/L, 0.1 µg/L, 0.3 µg/L, 1 µg/L, 3 µg/L (1.3 mL each) for OTA; 0 ng/L, 50 ng/L, 150 ng/L, 450 ng/L, 1350 ng/L, 4050 ng/L (1.3 mL each) for ZEN and 0 µg/L, 3.7 µg/L, 11.1 µg/L, 33.3 µg/L, 100 µg/L (1.3 mL each) for DON. diluted ECO extractor (10x concentrate) was included in the test kit of OTA to be used in extraction (dilution was done 1:10 with distilled or deionized water at 2 to 8 ℃). In the test kit of ZEN, Buffer 1 (50 mL) was included. A microtiter plate spectrophotometer was required for semi-quantification (screening). Special software, RIDASOFT® Win.NET (Art. No. Z9996FF) was used in the screening process. Filter paper: Whatman No. 1 or its equivalent was purchased from VICAM (https://www.vicam.com/category/aflatoxin-testing-solutions (accessed on 12 August 2023). Methanol (HPLC grade, purity ≥ 99.9%) was purchased from Sigma Aldrich (Merck, Kga, Darmstadt, Germany). Ultra-pure water was purchased and products by the Milli-Q purification system (Milli-Q from Millipore, USA).

Method of analysis for mycotoxinsAflatoxins (AFs)

Ground samples (5 g) and 25 mL of 70% methanol were mixed for 10 min at room temperature by vortexing and filtered through a Whatman No. 1 filter or centrifuged (10 min / 3500 g / room temperature). Then, 100 µL of the filtrate/supernatant was diluted with 600 µL distilled water. The wells were then filled with 50 µL of standard or sample in duplicate together with 50 µL of the conjugate. Then, 50 µL of the antibody was added to each well, and the plate was gently shaken for mixing and then allowed to sit at room temperature (20 to 25 ℃) in the dark for 30 min. After incubation, the well contents were discarded and the microwell holder was tapped upside down strongly (three times) on absorbent paper. Then wells were washed with 250 µL wash buffer 3 times after which 100 µL of substrate/chromogen was added to each well, gently mixed by hand shaking the plate, and incubated for 15 min at room temperature (between 20 and 25 ℃) in the dark. The stop solution (100 µL) was then pipetted into each well followed by manual shaking of the plate. After 30 min, the extinction was determined at 450 nm.

Ochratoxin A (OTA)

An aliquot of 10 gm ground sample was weighed and 50 mL of diluted ECO extractor was added followed by vortexing (10 s). The sample was then shaken violently for 5 min (either manually or with a shaker set to 420 rounds per minute) followed by centrifugation for 5 min at room temperature (20 to 25 ℃, 3500 g). Subsequently, 1 mL of the supernatant was diluted with 1 mL of wash buffer. An aliquot of 50 µL of standard or sample was then used to fill the wells in duplicate. Each well then received 50 µL of the conjugate which was gently combined by manually shaking the plate for 30 min at room temperature (20 to 25 ℃) in the dark. After that, the well contents were discarded, and the assay was continued as mentioned previously for AFs.

Zearalenone (ZEN)

Aliquots of 5 g of ground samples were weighed into a suitable container and 25 mL of methanol (70%) were added. Then, vigorous shaking (either manually or using a shaker) was carried out for three min. The extracts were centrifuged (10 min/3500 g, room temperature) or filtered. Following that, sample dilution buffer (buffer 1) was used to dilute the filtrates or supernatants 1:7 (100 µL supernatant or filtrate + 600 µL buffer 1). Then 50 µL of standard or sample were used to fill the wells in duplicate. The ZEN enzyme conjugate (diluted 1:11 in buffer) was added in 50 µL portions to each well. After that, the plate was gently stirred by handshaking and incubated for 2 h at room temperature (20 to 25  ℃) in the dark. After that, the well contents were discarded, and the assay was continued as mentioned previously for AFs.

Deoxynivalenol (DON)

Aliquots of 5 g of ground samples were weighed into a suitable container and 25 mL of distilled water was added and shaken for three min. Whatman No. 1 filter was used to filter the extract. Then 50 µL of standard or sample were added to the wells in duplicate. Then each well received 50 µL of the conjugate. Subsequently, each well received 50 µL of the anti-DONantibody, which was carefully mixed by hand shaking the plate and incubated for 30 min at room temperature (20 to 25 ℃) in the dark. After that, the well contents were discarded, and the assay was continued as mentioned previously for AFs.

Quantitative determination of certain mycotoxins (AFs, OTA and ZEN) using VICAMAflatest, Ochratest, zeralatest immunoaffinity column (IAC) followed by fluorometric methodChemicals and reagents

AflaTest Columns (25 per box), OchraTest Columns (25 per box), zearalaTest Columns (25 per box), Microfiber Filters, filter paper Whatman no.1, 1.0 m, 9 cm (100), Tween-20 (50 mL), 10X Concentrate of 0.01% Tween/PBS (150 mL), 5X Concentrate of 2% Tween/PBS (300 mL), 10X Concentrate of 0.1% Tween/PBS (150 mL), 10x concentrate of PBS (Phosphate Buffered Saline), AflaTest Developer (50 mL), OchraTest Eluting Solution (50 mL)and Zearalatest developer were purchased from VICAM A., WATERS, USA. Methanol (HPLC Grade, Acetonitrile HPLC Grade), ACS Grade Salt (100 g) (nonionized salt, NaCl) Zinc acetate powder, and AlCl3 powder were purchased from Sigma Aldrich. A commercial Blender with a Stainless Steel Container (Robot coupé, Inc, Ridgeland, USA) was used. Using a Milli-Q filtration system (Milli-Q from Millipore, USA), ultra-pure water was created. AFs standard product (product number CRM46304 Lot no. XA26847V with concentration of total AFs 2.6 ng/µL), OTA standard product (product number CRM46912 Lot no. LRAD1407 with concentration approximately 50 ng/µL in benzene: acetic acid (99:1), ampoule of 1 mL) and ZEN standard product ( product number CRM46916 Lot no.XA20006V with concentration 50 ng/µL in Acetonitrile, ampoule of 1 mL) were purchased from Supelco (Merck, Darmstadt, Germany).

Aflatoxins (afs)

Methanol/water solutions (80%, 70%, 60%, 20%) were prepared to extract AFs out of the samples. AflaTest Developer solution was prepared by mixing 45 mL of filtered water and 5 mL of AflaTest Developer concentrate. 10% Tween-20, 10X concentrate PBS, 10X concentrate 0.01% Tween-20 and 10X concentrate 0.1% Tween-20 solutions were prepared by adding 10 mL from each to 90 mL distilled water. 5 X concentrate 2% Tween-20 solution was prepared by adding 20 mL to 80 mL of distilled water. ZnCl2/AL(C2H3O2) 3 was prepared by adding 25 g of zinc acetate to 6.25 g AlCl3 dissolved in 125 mL deionized water.

The assay was done according to the VICAM international standard guidelines (VICAM manual; https://www.vicam.com/category/aflatoxin-testing-solutions (accessed on 12 August 2023).). Briefly, a 25 g ground sample was weighed with 5 g salt (NaCl) for paprika, chili, spices, oily seeds, nuts, Gramineae, cereals, chocolate, and cocoa and placed in a blender jar. For green coffee, a 50 g ground sample was weighed with 5 g salt (NaCl). For dried fruits, dried figs, and dates, 25 g ground samples were weighed with no NaCl added. Then 100 mL methanol: water (80%) for chili, paprika, spices, Gramineae, cereals, and green coffee, 125 mL methanol: water (60%) for oily seed and nuts, 100 mL methanol: water (70%) for dried fruits, dried figs and dates and 100 mL of absolute methanol for chocolate and cocoa were added to the jar. The blender jar was then covered and blended for one minute at a high speed. 5 mL of filtered extract was then diluted with 20 mL purified water in case of paprika, chili, Gramineae, cereals, or green coffee or diluted with 20 mL 10% tween 20 solution in case of spices and mixed well.

For oily seeds and nuts, 20 mL of filtered extract was diluted with 20 mL of purified water. For dried fruits, dried figs, and dates, 5 mL of filtered extract was diluted with 20 mL 0.01% Tween/PBS solution and mixed well. For chocolate and cocoa, 5 mL of filtered extract was mixed with 20 mL of ZnCl2/Al(C2H3O2)3 solution. Then, using marks on the barrel to quantify 4 mL, the diluted extract was filtered through a 1.5 m microfiber filter into a clean vessel or straight into a glass syringe barrel. At a rate of approximately 1 drop/second, 4 mL of filtered diluted extract was completely passed through an AflaTest column (4 mL = 0.2 g sample equivalent) for chili and paprika, but for the other types of samples, 10 mL were passed through the column. Then,10 mL of methanol: water (20%) was passed through the column at a rate of about 1–2 drops/second in the case of paprika, chili, chocolate, and cocoa or 10 mL of distilled water was passed in case of spices, oily seeds, nuts, Gramineae and cereals, green coffee. The previous step was repeated once more until air went through the column. For dried fruits, dried figs, and dates, 10 mL of 0.01% Tween/PBS solution was passed through the column at a rate of 1–2 drops/second then 10 mL of purified water was passed through the column. After that, a glass cuvette was placed under the column and 1 mL HPLC grade methanol was added into a glass syringe barrel. By allowing methanol to run through the AflaTest column and collecting all the samples eluate in a glass cuvette, the column was eluted at a rate of 1 drop/second or slower. 1 mL of AflaTest Developer solution was added to eluate in the cuvette and thoroughly mixed after that. Then the cuvette was immediately placed in a calibrated fluorometer Series 4EX Fluorometer 110 V, U.S.A. (Part Number/N G8001) and 220 V, International (P/N G8002). Total AF concentration was read after 60 s.

Ochratoxin A (OTA)

Methanol/water solutions (80%, 60%) were prepared to extract OTA out of the samples. Methanol: 1% Sodium bicarbonate solution was also prepared as previously reported. This test was performed using the VICAM international standard guidelines (https://www.vicam.com/category/ochratoxin-testing-solutions (accessed on 12 August 2023). The remaining solutions were prepared as mentioned previously for AFs. The assay was done according to the manufacturer’s manual (VICAM manual for OTA) as follows: 50 g ground samples were weighed with 5 g salt (NaCl) for paprika, chili, and spices and placed in the blender jar. But for Gramineae and cereals, 50 g ground samples were weighed with no NaCl added. For green coffee, roasted coffee, and Nescafe, 25 g ground samples were weighed with no NaCl added. Aliquots of 100 mL methanol: water (80%) for paprika, chili, spices, Gramineae, and cereals, 50 mL methanol:1% sodium bicarbonate (70%) for green and roasted coffee were added to the jar. The blender jar was covered and blended for 1 min at high speed, then 5 mL of filtered extract was diluted with 20 mL purified 10% tween 20 in case of paprika and spices and mix well. For Gramineae and cereals, 5 mL of filtered extract was diluted with 20 mL of purified 10% PBS solution. For green roasted coffee and Nescafe, 5 mL of the filtered extract was diluted with 20 mL 2% Tween-20/PBS. After that, the diluted extract was filtered through a 1.5 m microfiber filter into a clean vessel or straight into a glass syringe barrel using markings to measure 10 mL on the barrel. Then, 10 mL of filtered diluted extract were entirely run through the Ochratest column at a rate of around 1 drop/second until air went through the column (10 mL = 1.0 g sample equivalent).

The column was then cycled through with 10 mL of 10% PBS at a rate of roughly 1–2 drops per second in the case of paprika, chili, and spices. The previous step was then repeated once more until air went through the column. For Gramineae and cereals, 10 mL of 10% PBS was passed through the column at a rate of 1–2 drops/second then 10 mL of purified water was passed until air went through the column. For green coffee, 10 mL of 2% Tween-20/PBS was passed through the column at a rate of about 1–2 drops/second then 10 mL of purified water was passed until air came through the column. For roasted coffee and Nescafé, 10 mL of 2% Tween‐20/PBS were passed through the column at a rate of about 1–2 drops/second then 5 mL of 20%methanol\water were passed through the column at a rate of about 1–2 drops/second then 5 mL of 20% methanol\water were passed until air came through the column. Then a glass cuvette (VICAM part # 34,000) was placed under the OchraTest column and the glass syringe barrel was filled with the OchraTest Elution Solution. The column was eluted at a rate of one drop per second and all the sample eluate (1.5 mL) was collected in the glass cuvette. The cuvette was mixed and then placed into the previously mentioned fluorometer. OTA concentration was read after 60 s.

Zearalenone (ZEN)

This test was performed using the VICAM international standard guidelines https://www.vicam.com/category/zearalenone-testing-solutions (accessed on 12 August 2023). Methanol/water solution (80%) and acetonitrile/water solution (90%) were prepared to extract ZEN from the sample. To prepare Dilute ZearalaTest Developer Solution, aluminum chloride hexahydrate was dissolved in 50 mL of HPLC Grade-methanol prior to use. The dissolved zearalatest developer was stored at room temperature for up to one month. The remaining solutions were prepared as mentioned previously for AFs and OTA. For Gramineae, 20 g ground samples were weighed with 2 g salt (NaCl) and put in a blender jar. Then 50 mL of either acetonitrile: water (90%) or methanol: water (80:20%) were added to the jar. The blender jar was then covered and blended for 2 min at high speed. 5 mL of filtered extract were then diluted with 20 mL 0.1% tween PBS buffer and then mixed well. Following that, the diluted extract was filtered through a 1.5 m microfiber filter into a clean container or straight into a glass syringe barrel utilizing markings on the barrel to measure 10 mL. These 10 mL of filtered diluted extract were entirely run through a zearalatest column at a rate of around 1 drop/second until air passed through the column (10 mL = 0.8 g sample equivalent). The column was then cycled through with 10 mL of 0.1% tween PBS buffer at a rate of around 1–2 drops/second until air flowed through the column then, 10 mL of deionized, or distilled water was run through it at a rate of around 2 drops per second. Finally, 1.0 mL of HPLC-grade methanol was injected into a glass syringe barrel while a glass cuvette (VICAM part # 34,000) was positioned beneath the ZearalaTest column. All the sample eluate (1 mL) from the column was collected in a glass cuvette after it was eluted at a rate of 1 drop/second. To eluate in the cuvette, 1.0 mL of ZearalaTest Developer solution was added. After thoroughly mixing the cuvette, it was immediately placed into the previously mentioned fluorometer, and the ZEN concentration was measured after 300 s.

Standard preparation and spiking

Using certified Iso17034 and traceable to NIST (National Institute of Standards and Technology) standards, the AFs mix standard solution was prepared at 10 µg/kg while OTA standard was prepared at 20 µg/kg. ZEN standard solution was prepared at 50 µg/kg. In addition, blank (samples known to be zero MTs) samples were spiked with those prepared standard solutions at each run to be employed as quality control to guarantee accurate assessment of the data quality for all targeted MTs in regular sample analysis.

Statistical analysis

Data was analyzed using Excel 365, Minitab 20 and SPSS 26. Count and percentage were calculated for qualitative variables while for quantitative variables, mean, SD, SE, median, first quartile, third quartile, and inter quartile range were determined as descriptive statistics. Before performing any statistical analyses, the data was cleaned. Missing information and typographical issues have been examined. Inferential statistics has been used to find correlations and differences among different food and beverage categories or type and source (export, import or local). All parametric assumptions for quantitative variables have been examined, and when necessary, the best approach was used to apply the Box-Cox transformation for non-normal dependent variables. The various models matched the data well, and following data transformation, all analyses had linear attitudes in the normal residual probability plots. p-values were considered significant at α < 0.05. Qualitative variables have been tested using X2 test, The General Linear Model’s quantitative variables were evaluated using the Student’s t test and One Way ANOVA. Post hoc analysis using Tukey was done after ANOVA where groups sharing similar letters had no significant differences. Bar charts were graphed showing percentages of each level within each category using Excel 365.