The Occurrence and Risk Assessment of Aflatoxin M1 in Yoghurt Samples from Hamadan, Iran
Ali Heshmati1, Amir Sasan Mozaffari Mozaffari Nejad2, *, Tayebeh Ghyasvand1
Identifiers and Pagination:Year: 2020
First Page: 512
Last Page: 517
Publisher Id: TOPHJ-13-512
Article History:Received Date: 27/5/2020
Revision Received Date: 19/7/2020
Acceptance Date: 27/7/2020
Electronic publication date: 22/09/2020
Collection year: 2020
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Aflatoxin M1 (AFM1) is a hepatocarcinogenic and hydroxylated metabolite of aflatoxin B1, detected in milk and milk products.
The aim of our research was to determine the incidence and risk assessment of AFM1 through the consumption of yoghurt in Hamadan province of Iran.
Fifty yoghurt samples were gathered from various areas of Hamadan province, Iran and tested for AFM1 by ELISA technique. The estimated daily intake (EDI) and the liver cancer incidence of aflatoxin M1 were determined.
AFM1 was detected in 43 (86%) samples, (mean: 28.56 ng/kg; range: <5-98.65 ng/kg). The level of AFM1 in 9 (18%) samples was above the maximum tolerance limit (50 ng/kg). The AFM1 intake through yoghurt consumption in various population groups ranged from 0.016 to 0.032 ng/kg bw/day in mean consumers and 0.019 to 0.046 ng/kg bw/day in high consumers.
The AFM1 intake through yoghurt contributed a slight part from the overall incidence of liver cancer in the Iranian population. From the findings of the current study, it can be derived that although the high percentage of yoghurt samples in Iran proved to be contaminated with AFM1 contents, did not show a public health concern considering the European Commission (EC) and the Institute of Standards and Industrial Research of Iran (ISIRI) maximum limits.
Yoghurt is one of the most important and popular products among fermented dairy products, which has been used as food consumed by the world population for thousands of years. Yoghurt contains nearly all nutrients necessary to sustain life [1, 2]. Also, it is a rich source of dietary minerals including vitamins (B6 and B12), calcium (Ca), magnesium (Mg), zinc (Zn), phosphorus (P), and potassium (K), and so many others . It is also a great source of essential amino acids of great biological modality, generally including premier protein levels toward milk. Nevertheless, on the useful effects of yoghurt consumption, a substantial number of studies have presented the presence of heavy metals and aflatoxin M1 in foodstuff, which is a subject of serious concern, which has been increasing over the last few years .
Aflatoxin M1 (AFM1) is one of the groups of mycotoxin produced by Aspergillus species, especially A. nomius, A. flavus and A. parasiticus and found in contaminated milk (breast and animal), and dairy products. Researchers have demonstrated that the concentration of AFM1 is a dangerous toxic and carcinogenic for humans and animals [4-8].
Many countries have implemented regulations to control the amount of mycotoxins groups among food and agricultural products, especially for AFM1 in milk products. These regulations differ among several countries with respect to their economic considerations [4, 7, 9-12]. Hence, the European Commission (EC) and the Institute of Standards and Industrial Research of Iran (ISIRI) have set a limit of 50 ng/kg for AFM1 in yoghurt varieties.
The immediate detection of contamination is one of the beneficial methods to control aflatoxin M1 . The current detection methods such as enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC) are generally performed for the AFM1 analysis [4-8, 11-14]. In Iran, the ELISA technique is the most usual and popular among researchers because it is an ordinary, rapid, and low-cost technique for the survey of AFM1 [4-8, 15].
The aim of the current survey was to evaluate the occurrence and risk assessment of exposure of AFM1 through the consumption of yoghurt in the Hamadan province of Iran.
2. MATERIALS AND METHODS
2.1. Sample Collection
The current research is a cross-sectional study. For this purpose, 50 yoghurt samples were randomly purchased from supermarkets located in popular markets in different parts of Hamadan province, Iran, from October 2017 to August 2018. Eventually, all samples were carried to the lab and kept in the refrigerator at 4 oC. All samples were analyzed for AFM1 before the expiration date of the samples. All procedures of study were approved by the Ethics Committee of Hamadan University of Medical Sciences with No. IR.UMSHA.REC.1396.617.
The quantitative measurement of AFM1 in samples was distinguished by competitive ELISA using AFM1 test kit (RIDASCREEN® AFM1 Art. No.: R1121, R-Biopharm, Darmstadt, Germany). The preparation of the yoghurt samples and AFM1 measurement were performed according to the method described by the kit manufacturer. The mean lower limit of detection (LOD) for AFM1 in yoghurt was 5 ng/kg.
2.3. Risk Assessment for Exposure to AFM1 Through Yoghurt
AFM1 intake (ng/kg bw/day) = the concentration of AFM1 in yoghurt (ng/kg) × 95th percentile (for high consumers) or mean (for mean consumers) of per capita yoghurt consumption (kg)/ body weight (kg) Equation 1.
95th percentile (for high consumers) or mean (for mean consumers) of per capital yoghurt consumption was obtained by food frequency questionnaires (FFQ), Before the study, FFQ was prepared and given to people with different age-sex groups to complete it during a month. The people were randomly selected. For all participants in FFQ study, written informed consent was obtained.
For risk assessment, AFM1 value in yoghurt samples in which the concentration of this mycotoxin was lower than LOD was considered as LOD .
The liver cancer incidence due to AFM1 consumption was estimated according to Equation 2 :
Liver cancer incidence (cancers/yr/108 persons) = AFM1 intake (ng/kg bw/day) × potency
Potency= 0.001 × (1-P) + 0.03 × P
In these equations, the liver cancer potency of AFM1 in individuals positive for negative hepatitis B surface antigen (HBsAgz +) and individuals negative for negative hepatitis B surface antigen (HBsAgz -) was considered as 0.03 and 0.001 cancers/year/ ng of AFM1/ kg body weight/ day in a population of 100,000 . According to previous studies, HBsAgz + prevalence rate in Iran was 1.7% .
2.4. Statistical Analysis
The concentrations of AFM1 in milk samples were analyzed by SPSS Statistics 16.0 for Windows. One-side t-test was applied to compare the mean concentration of AFM1 samples with the maximum acceptable amount of the ISIRI and European Union (50 ng/kg) regulation. Differences between values were considered significant at P ≤ 0.05.
The occurrence and levels of AFM1 in yoghurt samples consumed in Hamadan province are presented in Table 1. AFM1 was detected above an acceptable level of 86% (43/50) in the analyzed samples, ranging from <5 to 98.65 ng/kg. Levels of the AFM1 in 9 (18%) yoghurt samples exceeded the ISIRI and European union i.e. 50 ng/kg. On the other hand, considering the US FDA  limits for AFM1 in milk (500 ng/l), none of the samples had levels above the maximum tolerance limit.
The AFM1 intakes through yoghurt in different age-sex groups of Iran population are shown in Table 2. The mean AFM1 intake in various population groups ranged from 0.016 to 0.032 ng/kg bw/day in mean consumers and 0.019 to 0.046 ng/kg bw/day in high consumers. Data regarding the potential liver cancer risk of AFM1 in yoghurt in the Iranian population were estimated and presented in Table 2.
Table 1. The occurrence of AFM1 in yoghurt samples from Hamadan (Iran) market.
|Sample type||N||Positive||Mean (ng/kg)||Standard Deviation (ng/kg)||No. of Positive Samples with AFM1||Range (ng/kg)|
|<5 ng/kg||5-50 ng/kg||>50 ng/kg|
|Yoghurt||50||43 (86%)||28.56||26.39||7 (14%)||34 (68%)||9 (18%)||<5-98.65|
|Group and Sex||No. of Consumers||
|Mean Consumers||High Consumers (percentile 95)|
|Yoghurt intake (kg/day)||AFM1 intake (ng/kg bw/day)||Estimation of cancer risk (cancers/yr/108 persons)||Yoghurt intake (kg/day)||AFM1 intake (ng/kg bw/day)||Estimation of cancer risk (cancers/yr/108 persons)|
|Infant (4-9 years)|
|Teenagers (10-19 years)|
|Adults (20-65 years)|
|Elderly (>65 years)|
|Location||No. of Samples||No. Positive Samples (%)||Detection Method||Mean (ng/kg)||Range (ng/kg)||Exceeded Regulation, n (%)||Reference|
|Iran||61||30 (49.2)||TLC||26||15 – 102||10 (16.4)||Fallah et al. |
|Turkey||80||70 (87.5)||ELISA||66.1||10 – 475||16 (20)||Atasever et al. |
|Iran||60||59 (98.33)||ELISA||51.66||6.2 – 87||38 (63.33)||Issazadeh et al. |
|Turkey||26||26 (100)||ELISA||238||125 – 269||26 (100)||Tosun et al. |
|Pakistan||96||59 (61)||HPLC||90.4||4 – 615.8||28 (47)||Iqbal et al. |
|Pakistan||96||32 (33.33 )||HPLC||90.4||LOD – 880||21 (21.87)||Iqbal et al. |
|Turkey||50||50 (100)||ELISA||55.28||40.62 – 72.04||5 (10)||Temamogullari & Kanici |
|Iran||60||48 (80)||ELISA||130.5||19.7 – 319.4||3 (5)||Rahimi |
|Iran (Traditional)||40||40 (100)||ELISA||33.6||6 – 91||1 (2.5)||Mason et al. |
|Iran||42||10 (23.8)||ELISA||15.1||6.3 – 21.3||0||Bahrami et al. |
|South Korea||55||15 (27.27)||HPLC||51*||20 – 150||NR||Kim-Soo et al. |
|Malaysia||5||2 (40)||ELISA||16.45*||7.5 – 31||0||Nadira et al. |
|Pakistan||66||26 (39.39)||HPLC||56||LOD – 196.3||8 (12.12)||Iqbal et al. |
|Qatar||21||16 (76.1)||ELISA||31.32||4.16-38.21||0||Hassan et al. |
|Kenya||38||NR||ELISA||117||17 – 1100||25 (65.78 )||Lindahl et al. |
|China||27||15 (55.5)||ELISA||17.2||4 – 47||0||Guo et al. |
Yoghurt is a favorite fermented dairy product, which is used as part of the popular diet in Iran because yoghurt is useful to affect human’s health and has nutritional value. Various types of fermented dairy products have been made and consumed in individual households in Iran, Turkey, Qatar, Lebanon, and other Middle Eastern countries for centuries.
Considering the present findings, we detected a high level of AFM1 contamination in yoghurt samples from Iran. In a prior survey, Cano-Sancho et al.  reported the lower level of AFM1 at a detectable level in yoghurt samples; but in a recent study, Altun et al.  detected AFM1 in 100% of yoghurt samples. Various studies by other researchers from different countries have previously been conducted on high or low contamination levels of AFM1 in yoghurt. Table 3 shows the compilation of data level of AFM1 contamination in yoghurt samples from previous studies from several countries measured by different techniques including HPLC and ELISA.
Hassan and Kassaift  from Lebanon using ELISA method reported that 49 (72%) of 68 samples of yoghurt were detected with AFM1 and in 9 (14%) samples, the amount of AFM1 was higher than the EU regulations (50 ng/kg), this result is approximately similar to our research results. The other survey from Iran by Tavakoli et al  that were done with ELISA technique on 50 samples of yoghurt, 35 (70%) of samples were contaminated with AFM1. Also, 6 (17.4%) samples of yoghurt had greater AFM1 content than the limit allowed in European Union (EU) 50 (ng/kg).
The other obtained results reported by Tabari et al.  determined AFM1 levels in 120 yoghurt samples from Guilan province in Iran using the ELISA method. They have reported that 100% of the samples found aflatoxin M1 by a mean concentration of 28.2 ng/kg. Also, 16 (13.3%) samples were above the permissible quantity according to the EC (50 ng/kg). However, this result is in contrast to our findings, that showed 86% (43/50) were contaminated with AFM1.The other conducted results were revealed in Serbia by Tomašević et al. , which were observed with ELISA, from 56 samples of yoghurt, all samples (100%) were contaminated with AFM1. Also, 22 (39.2%) samples were above the permissible level according to the EC (50 ng/kg). But, our results reported that approximately some of the yoghurt samples were contaminated with AFM1. This result from Serbia is similar to another study from Iran by Nikbakht et al. . They detected that all yoghurt samples (100%) were contaminated with AFM1; and also, 20 (22.22%) of the samples were above the permissible level according to EU (50 ng/kg).
In a previous study performed in Iran, Mason et al.  revealed that in 37 (92.5%)out of 40 industrial yoghurt samples, aflatoxin M1 was detected in concentration between <5 and 71 ng/kg; and in 3 (7.5%) samples, the contamination level exceeded the maximum permissible limit (50 ng/kg). In agreement with our research, these reports proved a widespread incidence of aflatoxin M1 in yoghurt samples ready and consumed in Iran. Compared to some reports from several countries, our results showed higher contamination. In Turkey, for example, aflatoxin M1 was detected in 2 (3.3%) out of 60 yoghurt samples with a range of 24 to 28 ng/kg. Also, none of the samples had aflatoxin M1 above the maximum tolerance limit (50 ng/kg) set by the EU . In another survey from China using the ELISA method , it was observed that in 8 (4.49%), out of 178 samples, aflatoxin M1 was present. The contamination level was detected in 8 (4.49%) of the samples which were above 50 ng/kg according to the EU.
According to several studies carried out in different neighboring countries of Iran, Turkey Altun et al. ; Tosun & Ayyildiz ; Atasever et al.  detected a high incidence of aflatoxin M1 in yoghurt samples (100%, 100% and 87.5%, respectively). These results were detected by the ELISA method.
As seen in Table 3, the contamination levels of aflatoxin M1 in yoghurt samples vary from one study to another. This variability can be explained by different factors such as geographical region, yoghurt-making procedures, analytical method employed and seasons variability [4, 29]. On the other hand, the previous study by Iha et al.  showed that the process of fermentation of yoghurt has no effect on aflatoxin M1. Also, the other surveys revealed that the quality of raw materials in yoghurt is effective on the presence and levels of AFM1. It is also noted that little or no reduction in aflatoxin M1 levels occurs as a result of pasteurization .
The incidence of liver cancer in Iran was 3.53 cancers per year per 105 persons or 3530 cancers/yr/108 persons  and AFM1 intake through yoghurt contributed 0.023-0.048 cancers/yr/108 person for mean consumers and 0.028-0.069 cancers/yr/108 person for high consumers. Therefore, our findings indicated AFM1 in yoghurt contributed a slight part from the overall incidence of liver cancer in the Iranian population. The intake of AFM1 and liver cancer incidence due to the consumption of this mycotoxin through yoghurt and milk was reported in other countries including China, Spain, Greece and Serbia [16, 17, 33]. The range of liver cancer incidence or hepatocellular carcinoma (HCC) due to AFM1 intake through milk and yoghurt was 0.025–0.033 case or cancers/yr/108 person in China that it was similar to our study while in Serbia and Greece was 3.6–0.4.7 and 0.7–0.9 case or cancers/yr/108 person, respectively that it was higher than the current study [17, 33]. The dispenses were related to the AFM1 level and consumption value of yoghurt.
From our findings of the current study, it can be derived that although a high percentage of yoghurt samples in Iran proved to have AFM1 contents, but it does not show a public health concern considering the European Commission (EC) and the Institute of Standards and Industrial Research of Iran (ISIRI) maximum limits. However, regarding the important role of milk, especially dairy products in the human diet, there is a huge concern about the presence of AFM1 in milk and dairy products. Hence, it is important to use fast methods in the detection of AFM1 in milk and dairy products; and also, the Iranian public health authorities have to monitor ceaselessly to detect AFM1 contamination.
Ali Heshmati, and Amir Sasan Mozaffari Nejad conceived, designed, analyzed, and interpreted the data; Tayebeh Ghyasvand and Amir Sasan Mozaffari Nejad performed data collection. Ali Heshmati and Amir Sasan Mozaffari Nejad wrote the first draft and finalized it. All authors read and approved the final manuscript.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
All procedures of the study were approved by the Ethics Committee of Hamadan University of Medical Sciences, Iran with No. IR.UMSHA.REC.1396.617.
HUMAN AND ANIMAL RIGHTS
CONSENT FOR PUBLICATION
AVAILABILITY OF DATA AND MATERIALS
The data that support the findings of this study are with the corresponding author, [ASMN], and can be made available on reasonable request.
The authors appreciate the Vice-Chancellor of Research and Technology of Hamadan University of Medical Sciences for financial support (Project No. 9609286051).
CONFLICT OF INTEREST
The authors declare no conflicts of interest, financial or otherwise.