Introduction

Today, humans pay considerable attention to food safety and contaminants (Milicevic et al., 2021; Rapa et al., 2021; Xinyu et al., 2020). Mycotoxins produced by fungi are among the most important food contaminants and have a negative impact on public health, food safety, and the national economy of many countries, especially developing countries (Batrinou et al., 2020; Grumi et al., 2020). The most critical factors in food contamination with mycotoxins are moisture, intrinsic properties and nutrients, long shelf life and pH, and high-water activity (Wang et al., 2018). Aflatoxins (AFs) are produced by Aspergillus fungi, especially A. flavus and A. parasitics and rarely by A. nomius (De Souza et al., 2021; Heshmati et al., 2019). They pose carcinogenic, mutagenic, immunosuppressive, and teratogenic consequences. The most common AFs are aflatoxin B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁), and G₂ (AFG₂) (Heshmati et al., 2017). AFB₁ is the most carcinogenic type for humans and animals (Mokhtarian et al., 2020). The International Agency for Research on Cancer (IARC) classified AFB₁ as a group 1 carcinogen (Elzupir et al., 2010).

Aflatoxins production may occur during harvesting, transportation, storage, or on the farm. AFs are very stable chemical compounds resistant to heat and food processes (Cui et al., 2020). They can contaminate various foods, including cereal, dairy products, oilseeds, spices, and nuts (Javanmardi et al., 2020; Khaneghah et al., 2018; Mozaffari Nejad et al., 2020).

Due to the high toxicity of AFs, exposure to these contaminations could threaten human health. The Committee on Food Additives of Joint FAO/WHO (JECFA) recommends that the presence of mycotoxins in meals be minimized to reduce the potential risk (Di Sanzo et al., 2018). Therefore, in order to control AFs in food products, some regulations were established in many countries; the measurement and monitoring of AFs in food products are crucial (Sebaei et al., 2020).

With the scientific name of Sesamum indicum L., sesame seed belongs to the Pedaliaceae family and is one of the oldest and most momentous oilseeds globally (Lee et al., 2020). It contains 58%–44% oil, 25%–25% protein, 13.5% carbohydrates, and 5% ash (Kollia et al., 2016). It is also a significant source of dietary fiber and micronutrients such as minerals, including calcium, phosphate, iron, potassium, vitamins such as E, thiamine, and niacin, lignans tocopherols, and phytosterols (Elleuch et al., 2011; Yao et al., 2021). Sesame seeds have antioxidant, anti-inflammatory, anti-fungal, anti-viral, and natural antibacterial effects (Dravie et al., 2020). Sesame seeds are consumed in different forms in the world (Namiki, 2007). It is widely used in the Iranian food industry as an ingredient in confectionery products, bread, and pastries. Therefore, ensuring the mycotoxicological quality of sesame is very important (Asadi et al., 2011; Eghbaljoo-Gharehgheshlaghi et al., 2020; Elleuch et al., 2011; Kollia et al., 2016).

Tahini is made by grinding and roasting sesame seeds known in the Middle East (Gholami et al., 2020; Sebaei et al., 2020). Tahini halva, also known as halva, helva, halawi, halawh, is produced by mixing tahini with sugar, citric or tartaric acid, and Saponaria officinalis root extract (Öğütcü et al., 2017; Osaili et al., 2018a; Var et al., 2007). Consumption of tahini halva has increased due to its excellent nutritional value and health properties in other countries, including the United States and European countries, Iran, Turkey, Saudi Arabia, Iraq, Greece, Jordan, Bulgaria, and Bosnia and Herzegovina (Anilakumar et al., 2010). Tahini halva consists of about 45% tahini, 45%–55% sugar, 2% ash, and 2.5% moisture and is consumed with bread in breakfast and dinner (Osaili et al., 2018b).

When sesame seeds were stored improperly, they can be contaminated with mycotoxins, especially AFs (Anthony et al., 2014). Therefore, the EU sets limits for AFB₁ and TAF in sesame and its products as 2 and 4 μg/kg, respectively. In contrast, the limit of these mycotoxins, according to the Iranian Institute of Standards and Industrial Research (ISIRI), was 5 and 15 μg /kg (INSO, 2020). The prevalence of AFs in sesame seeds and their products was demonstrated by previous studies (Anthony et al., 2014; Apeh et al., 2016; Asadi et al., 2011; Esan et al., 2020; Fapohunda et al., 2018; Hosseininia et al., 2014; Kollia et al., 2016; Li et al., 2009; Reddy et al., 2011; Sabry et al., 2016; Sebaei et al., 2020; Sirhan et al., 2014; Tabata, 2007; Torlak and Akan, 2013; Var et al., 2007).

No studies have been performed to evaluate mycotoxin contamination in tahini and tahini halva samples available in Iran’s market. Therefore, the current study aimed to determine the level of AFs in sesame seed, tahini, and tahini halva, and halva consumed in western Iran. Furthermore, the exposed risk due to ingestion of AFB₁ via their consumption was estimated with the aid of the Monte Carlo simulation (MCS).

Material and Methods

Materials

Acetonitrile, HNO₃ (65%), phosphate buffer solution (PBS), methanol, Potassium bromide (KBr), were purchased from Merck (Darmstadt, Germany). The AF standards were purchased from Sigma-Aldrich (St. Louis, MO, USA)

Sample collection

Samples (n = 120; 40 fresh sesame seed, 40 tahini, and 40 tahini halva) were collected from the local market in Hamadan province, Iran, from April 2020 until August 2020. Samples were stored in a refrigerator (4°C) until analysis.

Chemical properties

Moisture content (%) of sesame seed, tahini, and tahini halva was determined by drying in an oven at 100°C. The protein and fat contents of samples were determined by the Kjeldahl method and Soxhlet extraction, respectively (Zebib et al., 2015). Extracted fat acidity was measured by titration by sodium hydroxide (0.01°N) in the presence of phenolphthalein as an indicator.

Extract and cleanup of AF

The method applied for the extraction and cleanup of AF was similar to the previous one with slight modifications (Torlak and Akan, 2013). Samples were wholly powdered and mixed. Fifty grams of ground sample was weighed and transferred into a 250 mL flask. Then, 150 mL of a mixture of water and methanol (30:70; v/v) was added and placed on a shaker and stirred for 10 min. Then, it was filtered through filter paper of Whatman No. 2. Twenty milliliters of filtered solutions were collected and transferred into a 100 mL flask, and 40 mL of distilled water was added and mixed for 5 min and filtered. For the cleanup of AFs, 15 mL of filtrate was transferred through the immunoaffinity column at a flow rate of 2–3 drops/s. Further, the column was washed three times with 10 mL distilled water at the same flow rate. The elution of AFs was performed by acetonitrile (1 mL). The eluate was gathered in a vial, and its volume was reached 2 mL with acetonitrile. Then, 100 µL of eluted AFs was injected into a high-performance liquid chromatography (HPLC) instrument.

Analysis of AFs

The quantification of AFs in sesame products was carried out by HPLC (Knauer-Germany), equipped with a Smartline Pump, fluorescence detector, reverse phased C18 (150 mm × 4.6 mm i.d and 5 μm particle size). In the fluorescence detector, an excitation wavelength of 333 nm and emission wavelength of 430 nm was applied for AFs determination. The mobile phase was water/methanol/acetonitrile (6:102:94, v/v/v) and contained 100 mg KBr and 100 μL HNO₃. Then, it is diverted into HPLC in the isocratic method with a flow rate of 0.8 mL/min. The column temperature of HPLC was maintained at 40°C.

The validation of AFs analysis method

The linearity, accuracy, repeatability, limit of detection (LOD), and limit of quantification (LOQ) were determined (Heshmati et al., 2017).

Risk assessment

For risk assessment of AFB₁ intake through sesames seed, tahini, and tahini halva, the probabilistic approach was considered and estimated daily intake (EDI). The following equations are used to calculate the margin of exposure (MOE):

EDI (ng/kg bw/day) = the concentration of AFB₁ × average daily consumption (kg)/average body weight (kg)

Per capita consumption of sesame in Iran is 3 g/day (Eghbaljoo-Gharehgheshlaghi et al., 2020). In this study, 70 kg is considered as the mean body weight for an adult in Iran.

MOE = benchmark dose lower confidence limit 10% (BMDL₁₀)/EDI

where, BMDL₁₀ is the lowest dose that is 95% certain to cause no more than 10% cancer prevalence. The EFSA Panel on Contaminants in the Food Chain (CONTAM) suggested 400 ng/kg BW/day for BMDL₁₀ reference value (Chain et al., 2020). A MOE of 10,000 or larger has little concern for public health, while a MOE of less than 10,000 shows a potential danger for consumers (Heshmati et al., 2017). EDI and MOE were estimated by MCS using Crystal ball software (version 11.1.2.3 Oracle).

Statistical analysis

For statistical analysis, the SPSS software version 20 (IBM, PASW Statistics, USA) was applied. The mean and standard deviation of AFs levels in different samples were determined. One sample T-test was used to compare the mean of AFB₁ and TAF with the allowable limit. The significant level was considered P < 0.05. One-way ANOVA and Tukey’s test were applied to determine the significant difference of moisture, fat, protein content, and extracted fat acidity levels of these samples.

Result and Discussion

The chemical properties of samples

The moisture, fat, protein, and extracted fat acidity amount of sesame seed, tahini, and tahini halva samples are shown in Table 1. Sesame seed had a higher moisture, fat, and protein value than tahini and tahini halva samples.

Method validation

The LOD of AFs for sesame seeds, tahini, and tahini Halva samples ranged from 0.04 to 0.08, 0.05 to 0.09, and 0.07 to 0.08 µg/kg, respectively while LOQ for them ranged from 0.13 to 0.25, 0.18 to 0.31, and 0.21 to 0.27 µg/kg, respectively (Table 2). Moreover, the recovery range for AFs of sesame seeds, tahini, and tahini halva samples was 82.15–96.23, 77.36–95.63, and 83.65–98.65, respectively (Table 3). The determination coefficients (R² > 0.992) of the regression equations showed acceptable linearity, and good recovery was obtained for spike samples and was similar to values reported in previous studies (Heshmati et al., 2017). The findings obtained during method validation were conformed with accepted criteria (AOAC International, 2002).

The prevalence of AFs in sesame seeds

In this study, 40 samples of sesame seeds were analyzed for AFs. Sesame seeds had the highest prevalence (55%) of total AFs among the three analyzed samples. The detection rates of AFs were higher in sesame seed samples than in the tahini and tahini halva. AFB₁, AFB₂, AFG₁, and AFG₂ were detected in 19 (47.5%), 5 (12.5%), 6 (15%), and 5 (12.5%) of 40 sesame seeds samples, respectively (Table 4). AFB₁ was the most abundant AF, and its level varied from 0.21 to 12.35 μg/kg. In addition, 10 and 6 samples contained AFB₁ more than the accepted limit according to European (2 µg/kg) and Iranian standard (5 µg/kg), respectively, and the total AFs content of samples was lower than the permitted level in Iran (15 mg/kg).

There are several reports of AF contamination in sesame seeds (Table 5). The different levels of total AFs and AFB₁ have been reported in previous similar studies. For example, Anthony et al. (2014) reported that 8 (26.67%) out of 30 sesame samples studied in Nigeria were contaminated with AFB₁ at levels above the limit of European regulations (Anthony et al., 2014). Esan et al. (2020) surveyed the contamination of total AFs in sesame seeds of Nigeria. They demonstrated that the positive samples contaminated with total AFs ranged from 0.29 to 88.5 µg/kg (Esan et al., 2020). In another study, Kollia et al. (2016) investigated 30 samples of sesame seeds from the Greek market. They observed that the amount of AFB₁ in eight samples exceeded the limit of European regulations (Kollia et al., 2016). In an investigation, among 28 samples of sesame products from Egypt by Sabry et al. (2016), a higher prevalence of AFB₁ and AFG₁ than other mycotoxins in the range of 60%–100% and 33.33%–100%, respectively, were reported. The mean range of AFB₁ and AFG₁ in different provinces was 18.63–66.79 and 14.88–51.47 g/kg, respectively (Sabry et al., 2016).

The prevalence of AFs in tahini

A lower rate of AF contamination was observed in tahini than that in sesame seed. Eighteen (45%) of Tahini samples contained total AFs. The highest prevalence of AFs in tahini halva was AFB₁ (35%), followed by AFG₂ (12.5%), AFG₁ (10%), and AFB₂ (7.5%). It was observed that the AFB₁ value of seven samples was higher than the limit of European regulations (2 µg /kg) while only three samples had AFB₁ contamination higher than the recommended standard of Iran (5 µg/kg). Also, 18 samples contained total AF with an average value of 1.10 ± 0.30 µg/kg, among which the concentration of eight samples was higher than the limit of European regulations (4 µg/kg). It seems that washing and peeling of sesame seeds before tahini preparation could reduce the amount of AFs. Before tahini preparation, sesame seeds were roasted. The previous studies indicated that roasting operation could cause AFs degradation (Emadi et al., 2021; Yazdanpanah et al., 2005).

The prevalence of AFs in tahini was reported in other studies. For example, in a study performed by Sebaei et al. (2020) in Egypt, mean AFB₁ in 16 samples of branded tahini and 101 samples of local tahini reported 0.10 ± 0.24 μg/kg and 13 ± 19.3 μg/kg, respectively (Sebaei et al., 2020). Li et al. (2009) reported that 19% and 32% of 100 of the sesame paste (Tahini) samples studied in China contained AFB₁ at levels higher than the Chinese regulation (5 μg/kg) and European regulations (Li et al., 2009). In addition, Torlak and Akan (2013) studied AFs contamination in 104 samples of tahini from the Anatolia region of Turkey. The mean of AFB₁ and total AFs of samples was 0.93 ± 0.62 μg/kg and 1.17 ± 0.55 μg, respectively, which both were lower than the Turkish standard (AFB₁: 5 μg/kg and TAF: 10 μg/kg) and our study. These authors indicated that the roasting process applied in tahini production does not eliminate AFs (Torlak and Akan, 2013).

For the traditional production of tahini, sesame is roasted for 2 h at a temperature of 100°C to 150°C (Torlak and Akan, 2013). AFs are resistant to heat and are difficult to eliminate at insufficient temperatures. In general, AFs are eliminated at 237°C to 306°C. In foods heated, important parameters determining the reduction of AF include moisture content, heating temperature, and nutrient media. It has been reported that roasting dried wheat at 150°C for 30 min, Green coffee beans at 150°C to 180°C for 10 to 15 min, and Pistachio nuts at 150°C for 30 min will reduce AF levels to 50%, 42.2%–55/9%, and 63%, respectively (Pankaj et al., 2018).

The prevalence of AFs in tahini halva

The results showed that tahini halva samples contained four types of AF, i.e., AFB₁, AFB₂, AFG₂, and AFG₂, with mean values of 0.55 ± 0.17, 0.07 ± 0.03, 0.04 ± 0.03, and 0.06 ± 0.03 μg/kg, respectively (Table 4). Except for AFB₂, the detection rate of AFB₁, AFG₂, and AFG₂ was lower in tahini halva samples than in the sesame seeds and tahini samples. The average total AFs of tahini halva samples was 0.72 ± 0.22 µg/kg. According to Iranian standards, the concentration of total AFs and AFB₁ was not higher than the allowable limit. In contrast, the total AFs level of two samples was higher than the limit of European regulations (4 μg/kg). Also, in six samples, the concentration of AFB₁ was higher than the limit of European regulations (2 µg/kg). The lower level of AFs in tahini halva could be related to the dilution effect of other products, including sugar, emulsifier, in the formulation. Few studies have been performed to evaluate AFs in tahini halva. Var et al. (2007) investigated AFB₁ contamination in 34 samples of helva in Turkey. No AFB₁ was found in helva samples. It seemed the discrepancies in AFs contamination prevalence in different studies could be due to differences in sampling geographical areas and storage conditions and the AFs measuring method.

Risk assessment of AFB1 intake

Findings of percentile 50 (as the median of the population) of EDI of AFB₁ calculated using the MCS approach showed the lowest (0.02 ng/kg bw/day) and highest (0.05 ng/kg bw/day) of EDI in sesame seed and tahini halva, respectively (Figure 1). The percentile 95 of EDI of AFB₁ through sesame seed, tahini, and tahini halva was 0.09, 0.05, and 0.03 ng/kg bw/day, respectively.

As shown in Figure 2, the percentile 95 of MOE with AFB₁ through sesame seed, tahini, and tahini halva was calculated as 25,485, 50,092, and 77,114, respectively. Because data regarding the percentile 50 and 95 of MOE was more than 10,000 (Heshmati et al., 2017), it could be concluded that AFB₁ intake through sesame seed, tahini, and tahini halva consumption has no remarkable cancer risk for adults.

Conclusions

The results of this study indicated the high prevalence of AFs in sesame seed (55%), tahini (45%), and tahini halva (32.5%) samples. In addition, the levels of TAF in 7 (17.5%), 8 (20%), and 2 (5%) samples of sesame seeds, tahini, and tahini halva exceeded the limit of European regulations (4 µg/kg), respectively. Furthermore, 10 (25%), 7 (17.5%), and 6 (15%) samples of sesame seeds, tahini, and tahini halva contained AFB₁ more than the limit of European regulations (2 µg/kg), respectively. However, risk assessment indicated that the intake of AF through the consumption of mentioned products had no remarkable cancer risk for adults.