Mon, Aug 25, 2025
[Archive]
Volume 10, Issue 3 (Aug 2025)
JNFS 2025, 10(3): 453-466 |
Back to browse issues page
Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Nemati M, Khajeali E, Rashidi mehr A. Evaluation of pH, Microbial Contamination, and Aflatoxin M1 Levels in Raw Milk: A Health Risk Assessment. JNFS 2025; 10 (3) :453-466
URL: http://jnfs.ssu.ac.ir/article-1-1196-en.html
URL: http://jnfs.ssu.ac.ir/article-1-1196-en.html
Department of Microbiology, Faculty of Veterinary Medicine, Ilam University, Ilam, Iran
Full-Text [PDF 765 kb]
(105 Downloads)
| Abstract (HTML) (485 Views)
1 Department of Microbiology, Faculty of Veterinary Medicine, Ilam University, Ilam, Iran; 2 Department of Food Hygiene, Faculty of Veterinary Medicine, Ilam University, Ilam, Iran; 3 Department of Microbiology and Food Hygiene, Faculty of Veterinary Medicine, Lorestan University, Khorramabad, Lorestan, Iran.
Introduction
.PNG)
.PNG)
.PNG)
Evaluation of AFM1
This study investigated the level of AFM1 in 84 raw milk samples collected from cattle farms and collection centers in Ilam province. According to the findings (Table 2, Figure 4), 65 samples showed AFM1 contamination levels below 20 ng/l (the detection limit of the device). A total of 17 samples exhibited contamination levels ranging from 20-90 ng/l, while two samples surpassed 100 ng/l, exceeding Iran's permissible limit. According to the European :union:, the concentration of AFM1 exceeded the permissible limit of 50 ng/l in seven samples.
AF mean levels in two cohorts of dairy cattle farms and collection centers were compared. As indicated in Figure 3, the farm (25.96 ng/l) exhibited a higher AFM1 level than the centers (15.14 ng/l). Nevertheless, both mean levels remained below the permissible limit.
.PNG)
Full-Text: (55 Views)
Evaluation of pH, Microbial Contamination, and Aflatoxin M1 Levels in Raw Milk: A Health Risk Assessment
Mostafa Nemati; PhD*1, Elahe Khajehali; PhD2 & Azadeh Rashidimehr; PhD3
Mostafa Nemati; PhD*1, Elahe Khajehali; PhD2 & Azadeh Rashidimehr; PhD3
1 Department of Microbiology, Faculty of Veterinary Medicine, Ilam University, Ilam, Iran; 2 Department of Food Hygiene, Faculty of Veterinary Medicine, Ilam University, Ilam, Iran; 3 Department of Microbiology and Food Hygiene, Faculty of Veterinary Medicine, Lorestan University, Khorramabad, Lorestan, Iran.
| ARTICLE INFO | ABSTRACT | |
| ORIGINAL ARTICLE | Background: This research aimed to assess pH, microbial load, and aflatoxin (AF) M1 levels in milk from cattle dairy farms and milk distribution and collection centers in Ilam province. Methods: In this study, a total of 84 raw milk samples from Ilam province, collected between April and June, were analyzed for total bacterial count (TBC), pH, and AFM1 using high-performance liquid chromatography. Daily AFM1 intake was calculated to assess health risks based on milk consumption, using different risk assessment indices. Results: The highest microbial load was found in farms Location 5 (7.84 log CFU/ml) and the lowest average microbial load was recorded in farms Location 4 (5.92 log CFU/ml), while collection centers showed an average microbial count of 5.48 log CFU/ml, also linked to Location 4. AFM1 was found in 84 samples: 65 had levels below 20 ng/g, 17 ranged from 20 to 90 ng/g, and 2 exceeded 150 ng/g, surpassing safety standards. The analysis indicates that young children consuming milk may face health risks from AFM1, notably liver cancer. The increased incidence of elevated liver cancer risk (LCR) and associated hazard index (HI) values in children under the age of seven necessitate the implementation of more stringent regulations regarding AFM1 contamination in dairy products, specifically targeting this demographic. Conclusion: Although the risk of liver cancer declines with age, it is imperative to maintain vigilant monitoring of cumulative exposure to AFM1. This is particularly important for older adults, who may represent a vulnerable population with heightened susceptibility to AFM1-related health complications. Ensuring rigorous surveillance and enforcement of safety standards for aflatoxins levels in milk products is essential for protecting these at-risk groups from potential adverse health outcomes. | |
| Article history: Received: 27 Nov 2024 Revised: 17 Mar 2025 Accepted: 21 Mar 2025 |
||
| *Corresponding author m.nemati@ilam.ac.ir Department of Microbiology, Faculty of Veterinary Medicine, Ilam University, Ilam, Iran. Postal code: 69391-77111 Tel: +98 9181413378 |
||
| Keywords Aflatoxins; Food quality; HPLC; Dairy products; Mycotoxins. Aflatoxins; Milk |
Introduction
Food security is crucial for ensuring that the population has access to a sufficient quantity and quality of food (Augustin et al., 2016). In Iran, milk is provided to school students to combat malnutrition among children. Milk is a key food for human nutrition, especially for children, as it contains essential macronutrients and micronutrients that meet the nutritional needs of all age groups. It is also a significant source of high-quality proteins that supply essential amino acids (Hashemi, 2016). Although milk offers various health benefits, it is susceptible to contamination by mycotoxins, rendering it unsafe for consumption and potentially leading to health complications (Kortei et al., 2022). The Food and Agricultural Organization (FAO) report states that the mean value of per capita milk consumption globally is approximately 100 kg/year, but this can vary significantly between countries, ranging from 10 to 300 kg/year (Rahmani et al., 2018).
Approximately 25% of food products are contaminated yearly with mycotoxins, known as secondary toxic byproducts of fungi. Aflatoxins (AFs) comprise about 90% of around 300 different mycotoxins (Smith et al., 2020). AF is a type of mycotoxin produced by Aspergillus fungi, including A. nomius, A. parasiticus, and A. flavus (Mahmoudi and Norian, 2015). AFB1 can contaminate human food such as cereals, corn, rice, oilseeds, tree nuts, and spices (Assaf et al., 2020, Marchese et al., 2018), as well as animal feed such as peanut meal, maize, and cottonseed meal (Kamkar et al., 2011). When consumed by dairy animals, it is absorbed into their gastrointestinal tracts and metabolized in their livers to produce AFM1, which is then secreted into the milk of lactating mammals, thus contaminating milk and dairy products (Marchese et al., 2018).
The presence of this contamination may lead to adverse effects including liver toxicity, mutagenicity, genotoxicity, teratogenicity, carcinogenicity, neurotoxicity, cytotoxicity, immunosuppression, and neoplastic effects (De Roma et al., 2017, Fakhri et al., 2019, Hashemi, 2016, Pour et al., 2020). As a result, in 2002, the International Agency for Research on Cancer (IARC) classified AFM1 as a Group 1 compound, which means it is carcinogenic to humans (World Health Organization and International Agency for Research on Cancer, 2002). AFM1 is found worldwide, particularly in tropical and subtropical climates with varying temperatures, rainfall, and humidity (Fakhri et al., 2019). Contamination can occur during the processing and storage of milk and its products. Moreover, AFM1 is resistant to heat, pasteurization, autoclaving, and other food processing methods. Therefore, to ensure high-quality milk, feeds must be free from AFB1 contamination (Omar, 2016, Sadeghi et al., 2009). Due to the high toxicity and heat stability of AFM1, coupled with the significant consumption of milk and dairy products, various countries have established maximum levels (ML) of AFM1 in milk, which vary widely based on their economic status and development (Kortei et al., 2022, Mollayusefian et al., 2021). The European :union: (E.U.) and the U.S. Food and Drug Administration (FDA) have set the ML for AFM1 in liquid milk at 50 and 500 ng/L, respectively (Hassan et al., 2018). Hence, it is crucial to recognize that AFM1 contamination in milk of dairy cattle presents a significant risk and should not be overlooked. Ongoing global research is being conducted on the presence of AFM1 in raw milk, highlighting the seriousness of this issue (Ketney et al., 2017). It is essential to continue investigating the presence of AFM1 in raw milk to gather data on human exposure and to assess the potential long-term health risks associated with consuming low levels of AFM1 in milk (Ketney et al., 2017).
One of the most effective ways to determine the likelihood and severity of liver cancer risk (LCR) is quantitative risk assessment for milk and dairy products contaminated with AFM1 (Tsakiris et al., 2013). This research aimed to assess pH, microbial load, and AFM1 levels in milk from cattle dairy farms and distribution and collection centers in Ilam province, Iran. Furthermore, the health risks of AFM1 across various age groups were evaluated by calculating cancer risk and considering factors such as estimated daily intake (EDI), hazard index (HI), margin of exposure (MoE), and LCR.
Materials and Methods
Description of the study site
Ilam province, situated in the western region of Iran, is inhabited by approximately 557,599 residents and encompasses a total area of 20133 square kilometers. Its geographical coordinates range between 45°24' and 48°10' longitude, and 31°58' and 34°15' latitude. The province was geographically partitioned into six distinct regions, delineated based on the density of cattle farms and the location of milk collection and distribution centers, as depicted in Figure 1
Approximately 25% of food products are contaminated yearly with mycotoxins, known as secondary toxic byproducts of fungi. Aflatoxins (AFs) comprise about 90% of around 300 different mycotoxins (Smith et al., 2020). AF is a type of mycotoxin produced by Aspergillus fungi, including A. nomius, A. parasiticus, and A. flavus (Mahmoudi and Norian, 2015). AFB1 can contaminate human food such as cereals, corn, rice, oilseeds, tree nuts, and spices (Assaf et al., 2020, Marchese et al., 2018), as well as animal feed such as peanut meal, maize, and cottonseed meal (Kamkar et al., 2011). When consumed by dairy animals, it is absorbed into their gastrointestinal tracts and metabolized in their livers to produce AFM1, which is then secreted into the milk of lactating mammals, thus contaminating milk and dairy products (Marchese et al., 2018).
The presence of this contamination may lead to adverse effects including liver toxicity, mutagenicity, genotoxicity, teratogenicity, carcinogenicity, neurotoxicity, cytotoxicity, immunosuppression, and neoplastic effects (De Roma et al., 2017, Fakhri et al., 2019, Hashemi, 2016, Pour et al., 2020). As a result, in 2002, the International Agency for Research on Cancer (IARC) classified AFM1 as a Group 1 compound, which means it is carcinogenic to humans (World Health Organization and International Agency for Research on Cancer, 2002). AFM1 is found worldwide, particularly in tropical and subtropical climates with varying temperatures, rainfall, and humidity (Fakhri et al., 2019). Contamination can occur during the processing and storage of milk and its products. Moreover, AFM1 is resistant to heat, pasteurization, autoclaving, and other food processing methods. Therefore, to ensure high-quality milk, feeds must be free from AFB1 contamination (Omar, 2016, Sadeghi et al., 2009). Due to the high toxicity and heat stability of AFM1, coupled with the significant consumption of milk and dairy products, various countries have established maximum levels (ML) of AFM1 in milk, which vary widely based on their economic status and development (Kortei et al., 2022, Mollayusefian et al., 2021). The European :union: (E.U.) and the U.S. Food and Drug Administration (FDA) have set the ML for AFM1 in liquid milk at 50 and 500 ng/L, respectively (Hassan et al., 2018). Hence, it is crucial to recognize that AFM1 contamination in milk of dairy cattle presents a significant risk and should not be overlooked. Ongoing global research is being conducted on the presence of AFM1 in raw milk, highlighting the seriousness of this issue (Ketney et al., 2017). It is essential to continue investigating the presence of AFM1 in raw milk to gather data on human exposure and to assess the potential long-term health risks associated with consuming low levels of AFM1 in milk (Ketney et al., 2017).
One of the most effective ways to determine the likelihood and severity of liver cancer risk (LCR) is quantitative risk assessment for milk and dairy products contaminated with AFM1 (Tsakiris et al., 2013). This research aimed to assess pH, microbial load, and AFM1 levels in milk from cattle dairy farms and distribution and collection centers in Ilam province, Iran. Furthermore, the health risks of AFM1 across various age groups were evaluated by calculating cancer risk and considering factors such as estimated daily intake (EDI), hazard index (HI), margin of exposure (MoE), and LCR.
Materials and Methods
Description of the study site
Ilam province, situated in the western region of Iran, is inhabited by approximately 557,599 residents and encompasses a total area of 20133 square kilometers. Its geographical coordinates range between 45°24' and 48°10' longitude, and 31°58' and 34°15' latitude. The province was geographically partitioned into six distinct regions, delineated based on the density of cattle farms and the location of milk collection and distribution centers, as depicted in Figure 1
Data collection
This study analyzed 84 raw milk samples, comprising 47 from dairy farms and 37 from collection and distribution centers in the province. Sampling was conducted randomly from April to June 2019, with each center selected based on specific criteria. Before sampling, milk temperature was measured, and 100 milliliters were collected into two separate falcon tubes—one for microbiological tests and one for chemical tests. The samples were promptly transported to the laboratory under cold conditions (4±1 0C) for further examination.
Determination of the microbiological load
Total bacterial count (TBC) was performed to assess the microbial load in raw milk samples, following the guidelines outlined by the Iranian National Standardization Organization (INSO, n: 5272-2) (Iranian National Standardization Organization, 2008).
Determination of pH
The pH meter was calibrated using pH 4 and pH 7 buffer solutions prior to measurements. A small amount of milk was placed into a 30- to 50-milliliter flask and stirred using an electric mixer until it was completely smooth (Gemechu and Amene, 2016).
AFM1 analysis
Raw milk samples were collected from all dairy farms and dairies in Ilam province. The samples were stored in 100 ml Falcon tubes and frozen at -18 0C until AFM1 determination test was conducted. To measure AFM1 levels in milk, the samples were processed according to the guidelines outlined in reference method a-5925 of the ISIRI (Iranian National Standardization Organization, 2020). Initially, 60 ml of milk was heated to 37 °C, after which the fat was separated from milk through centrifugation, and the upper layer was discarded. Subsequently, an immunoaffinity column was used to purify milk samples. It should be noted that homogenization prevents fat separation during centrifugation, rendering this statement inaccurate.
The milk was centrifuged at 2000 ×g to separate fat, and the upper layer was discarded. Subsequently, 10 ml of phosphate-buffered saline solution was added to immunoaffinity column. After passing through two stages of filter paper, 20 ml of skim milk was passed through the column. The sample container was washed twice with grade 3 water, and wash water was passed through the column. The column was then washed with 10 ml of grade 3 water. Then, 2500 µl of acetonitrile was passed through the column at a flow rate of 2-3 ml/min, collected in a vial, and dried in a water bath at a temperature of 50-40 0C. Subsequently, 1 ml of mobile phase was added to the vial and mixed for the second time with a vortex mixer (Labnet, Tehran, Iran). The column was washed with 20 ml of phosphate saline buffer, and finally, 200 μl of this solution was injected into the HPLC device. The resulting peaks from the inhibition test were compared with standard peaks to identify and quantify the contamination, as determined by the calibration curve (Figure 2).
This study analyzed 84 raw milk samples, comprising 47 from dairy farms and 37 from collection and distribution centers in the province. Sampling was conducted randomly from April to June 2019, with each center selected based on specific criteria. Before sampling, milk temperature was measured, and 100 milliliters were collected into two separate falcon tubes—one for microbiological tests and one for chemical tests. The samples were promptly transported to the laboratory under cold conditions (4±1 0C) for further examination.
Determination of the microbiological load
Total bacterial count (TBC) was performed to assess the microbial load in raw milk samples, following the guidelines outlined by the Iranian National Standardization Organization (INSO, n: 5272-2) (Iranian National Standardization Organization, 2008).
Determination of pH
The pH meter was calibrated using pH 4 and pH 7 buffer solutions prior to measurements. A small amount of milk was placed into a 30- to 50-milliliter flask and stirred using an electric mixer until it was completely smooth (Gemechu and Amene, 2016).
AFM1 analysis
Raw milk samples were collected from all dairy farms and dairies in Ilam province. The samples were stored in 100 ml Falcon tubes and frozen at -18 0C until AFM1 determination test was conducted. To measure AFM1 levels in milk, the samples were processed according to the guidelines outlined in reference method a-5925 of the ISIRI (Iranian National Standardization Organization, 2020). Initially, 60 ml of milk was heated to 37 °C, after which the fat was separated from milk through centrifugation, and the upper layer was discarded. Subsequently, an immunoaffinity column was used to purify milk samples. It should be noted that homogenization prevents fat separation during centrifugation, rendering this statement inaccurate.
The milk was centrifuged at 2000 ×g to separate fat, and the upper layer was discarded. Subsequently, 10 ml of phosphate-buffered saline solution was added to immunoaffinity column. After passing through two stages of filter paper, 20 ml of skim milk was passed through the column. The sample container was washed twice with grade 3 water, and wash water was passed through the column. The column was then washed with 10 ml of grade 3 water. Then, 2500 µl of acetonitrile was passed through the column at a flow rate of 2-3 ml/min, collected in a vial, and dried in a water bath at a temperature of 50-40 0C. Subsequently, 1 ml of mobile phase was added to the vial and mixed for the second time with a vortex mixer (Labnet, Tehran, Iran). The column was washed with 20 ml of phosphate saline buffer, and finally, 200 μl of this solution was injected into the HPLC device. The resulting peaks from the inhibition test were compared with standard peaks to identify and quantify the contamination, as determined by the calibration curve (Figure 2).
Risk assessment of exposure to AFM1
Estimation of exposure: To assess the potential risks of daily milk consumption and exposure to AFM1, the first step was to estimate the daily intake of AFM1 in ng/day.kg of body weight. This calculation was performed using the following equation (Hooshfar et al., 2020, Sharma et al., 2020):
EDI=C × I × E B . W
In the provided equation, C represents the concentration of the contaminant (AFM1) in the milk (ng/l), I stands for daily milk intake (L/d), E denotes the exposure duration per day, and B.W represents the average weight of individuals in the test population (kilograms). According to the report from the Agriculture Ministry of Iran, the average daily milk intake for the general population was approximately 0.25 L (Moghaddam et al., 2019, Nejad et al., 2020, Sharma et al., 2020). BW represents the mean body weight, which was assumed to be 15, 45.2, 78.7, and 74.5 kg for males under 7, aged 7–16, aged 16–65, and over 65 years old, respectively. For females, these values were assumed to be 15, 43.9, 65.5, and 66.3 kg, respectively (United States Environmental Protection Agency, 2011).
MoE characterization for AFM1: Genotoxic and carcinogenic substances like AF are appropriately evaluated for risk using the MoE method. These three indicators were determined based on the average daily intake of AFM1 and the average milk consumption within the community. The assessment of the risk of AFM-induced liver cancer involved multiplying the probability of cancer by the average AFM1 exposure (Benford et al., 2010).
When calculating the MoE for average exposures to AFM, the Benchmark Dose (BMD) was utilized as the dose showing the least measurable response (570 ng/d.kg bw, representing AFM potency for hepatocellular carcinoma (HCC) based on a 2-year study in male Fischer rats) (Serraino et al., 2019, Udovicki et al., 2019). This value was determined by dividing the reference value by the EDI in consumers (European Food Safety Authority, 2005, Udovicki et al., 2019). Any value above 10,000 was regarded as a low-level public health concern.
= 570 ng kg bw / day EDI ng kg bw / day
Health index (HI) of AFM1: The HI was determined using equation (3) to ensure the safety of milk about AFM1. In this equation, the EDI represents the average daily intake of AFM1 through milk consumption, and RFD is the reference dose derived from the TD50 of AFM1 (10.38 μg/kg bw/day). The TD50 is the amount of AFM1 that causes tumors in half of the laboratory animals. The specified TD50 value was divided by an uncertainty factor of 50,000 to obtain the RFD for AFM1 in humans, resulting in a risk of 1:100,000. Considering this process, the value of RFD was established at 0.2 ng/kg bw/day. If the HI value is equal to or less than 1, it will be considered an acceptable risk. Conversely, if the HI value exceeds 1, it will be regarded as an unacceptable risk, indicating a potential increase in LCR (Bahrami et al., 2016, Hooshfar et al., 2020).
HI = EDI ng kg bw / day RFD ng kg bw / day
Estimated LCR: People infected with hepatitis B virus (HBV) seem to be at a greater risk of developing HCC as a result of exposure to AFs. This study examined the combined impact of exposure to AFM1 and HBV on the development of HCC. It used a specific formula to calculate the estimated risk of HCC (cancer cases per year per 100,000 people) due to AFM1 exposure in the community.
The Joint FAO/WHO Expert Committee on Food Additives (JECFA) provided the LCR assessment method related to AFM1 (FAO/WHO, 2017). JECFA indicated a potential LCR exposure of 1 ng AFB1/kg bw/day in 100,000 individuals. Consequently, the estimated potential LCR attributed to AFM1 was 0.049 (95% upper bound) additional cancer cases per 100,000 for HBsAg-negative individuals and 0.562 (95% upper bound) for HBsAg-positive individuals.
AFM1 has a lower carcinogenic potency compared to AFB1, even in sensitive species such as rainbow trout and Fischer rats, with a potency that is one-tenth of AFB1. The carcinogenic potency of AFB1 for HBsAg- and HBsAg+ has been documented as 0.0049 and 0.0562, respectively (FAO/WHO, 2017, Hooshfar et al., 2020). The prevalence of HBsAg+ in Iran, according to the report of Iran’s Center for Disease Control and Prevention, is 1.5%. Therefore, the percentage of the population (Pop) related to HBsAg+ and HBsAg- is 0.015 and 0.985, respectively (Hooshfar et al., 2020, Moghaddam et al., 2019). Equations (4) and (5) were used, based on the information mentioned above, to calculate the cancer potency (CP) and cancer risk (CR) generated by AFM1. As per the US Environmental Protection Agency guidelines, a carcinogenic risk lower than 10-4 is deemed acceptable, while a risk higher than 10-4 is considered to be carcinogenic (Environmental Protection Agency, 2001).
CP= (PHBAg+ × %PopHBsAg+) + (PHBsAg− × %PopHBsAg−) Equation 4
LCR=CP × EDI Equation 5
Data analysis
The statistical analysis was conducted using IBM SPSS version 20 software (SPSS Inc., Chicago, IL, USA). The one-way analysis of variance (ANOVA) test was used to compare mean scores between cities, distribution centers, and dairy farms. The average microbial load and pH were also compared using an independent t-test. Both tests were performed at a significance level of α = 0.05 and a confidence level of 95% CI.
Results
TBC
The examination of TBC in raw milk from dairy farms in Ilam province revealed that L5 had the highest microbial load (log CFU/ml 7.84), while the lowest was observed at L4 (log CFU/ml 5.91) (Figure 3. A). A statistically significant difference in microbial load was found between these locations (P<0.05).
Similarly, when investigating the TBC in raw milk at the collection and distribution centers of Ilam province, it was found that the lowest TBC was associated with L1 (5.25 log CFU/ml) (Figure 3. B), and this difference was also statistically significant (P<0.05). The mean TBCs in samples collected from L1, L4, and L5 were 5.25, 8.08, and 8.57 log10 CFU/ml, respectively. A high TBC was detected in raw cattle milk sample collected at L4, while low counts were detected in milk sample collected at L1. Except for raw milk collection centers in L1, the other areas were not up to standard.
Estimation of exposure: To assess the potential risks of daily milk consumption and exposure to AFM1, the first step was to estimate the daily intake of AFM1 in ng/day.kg of body weight. This calculation was performed using the following equation (Hooshfar et al., 2020, Sharma et al., 2020):
EDI=
In the provided equation, C represents the concentration of the contaminant (AFM1) in the milk (ng/l), I stands for daily milk intake (L/d), E denotes the exposure duration per day, and B.W represents the average weight of individuals in the test population (kilograms). According to the report from the Agriculture Ministry of Iran, the average daily milk intake for the general population was approximately 0.25 L (Moghaddam et al., 2019, Nejad et al., 2020, Sharma et al., 2020). BW represents the mean body weight, which was assumed to be 15, 45.2, 78.7, and 74.5 kg for males under 7, aged 7–16, aged 16–65, and over 65 years old, respectively. For females, these values were assumed to be 15, 43.9, 65.5, and 66.3 kg, respectively (United States Environmental Protection Agency, 2011).
MoE characterization for AFM1: Genotoxic and carcinogenic substances like AF are appropriately evaluated for risk using the MoE method. These three indicators were determined based on the average daily intake of AFM1 and the average milk consumption within the community. The assessment of the risk of AFM-induced liver cancer involved multiplying the probability of cancer by the average AFM1 exposure (Benford et al., 2010).
When calculating the MoE for average exposures to AFM, the Benchmark Dose (BMD) was utilized as the dose showing the least measurable response (570 ng/d.kg bw, representing AFM potency for hepatocellular carcinoma (HCC) based on a 2-year study in male Fischer rats) (Serraino et al., 2019, Udovicki et al., 2019). This value was determined by dividing the reference value by the EDI in consumers (European Food Safety Authority, 2005, Udovicki et al., 2019). Any value above 10,000 was regarded as a low-level public health concern.
Health index (HI) of AFM1: The HI was determined using equation (3) to ensure the safety of milk about AFM1. In this equation, the EDI represents the average daily intake of AFM1 through milk consumption, and RFD is the reference dose derived from the TD50 of AFM1 (10.38 μg/kg bw/day). The TD50 is the amount of AFM1 that causes tumors in half of the laboratory animals. The specified TD50 value was divided by an uncertainty factor of 50,000 to obtain the RFD for AFM1 in humans, resulting in a risk of 1:100,000. Considering this process, the value of RFD was established at 0.2 ng/kg bw/day. If the HI value is equal to or less than 1, it will be considered an acceptable risk. Conversely, if the HI value exceeds 1, it will be regarded as an unacceptable risk, indicating a potential increase in LCR (Bahrami et al., 2016, Hooshfar et al., 2020).
Estimated LCR: People infected with hepatitis B virus (HBV) seem to be at a greater risk of developing HCC as a result of exposure to AFs. This study examined the combined impact of exposure to AFM1 and HBV on the development of HCC. It used a specific formula to calculate the estimated risk of HCC (cancer cases per year per 100,000 people) due to AFM1 exposure in the community.
The Joint FAO/WHO Expert Committee on Food Additives (JECFA) provided the LCR assessment method related to AFM1 (FAO/WHO, 2017). JECFA indicated a potential LCR exposure of 1 ng AFB1/kg bw/day in 100,000 individuals. Consequently, the estimated potential LCR attributed to AFM1 was 0.049 (95% upper bound) additional cancer cases per 100,000 for HBsAg-negative individuals and 0.562 (95% upper bound) for HBsAg-positive individuals.
AFM1 has a lower carcinogenic potency compared to AFB1, even in sensitive species such as rainbow trout and Fischer rats, with a potency that is one-tenth of AFB1. The carcinogenic potency of AFB1 for HBsAg- and HBsAg+ has been documented as 0.0049 and 0.0562, respectively (FAO/WHO, 2017, Hooshfar et al., 2020). The prevalence of HBsAg+ in Iran, according to the report of Iran’s Center for Disease Control and Prevention, is 1.5%. Therefore, the percentage of the population (Pop) related to HBsAg+ and HBsAg- is 0.015 and 0.985, respectively (Hooshfar et al., 2020, Moghaddam et al., 2019). Equations (4) and (5) were used, based on the information mentioned above, to calculate the cancer potency (CP) and cancer risk (CR) generated by AFM1. As per the US Environmental Protection Agency guidelines, a carcinogenic risk lower than 10-4 is deemed acceptable, while a risk higher than 10-4 is considered to be carcinogenic (Environmental Protection Agency, 2001).
CP= (PHBAg+ × %PopHBsAg+) + (PHBsAg− × %PopHBsAg−) Equation 4
LCR=CP × EDI Equation 5
Data analysis
The statistical analysis was conducted using IBM SPSS version 20 software (SPSS Inc., Chicago, IL, USA). The one-way analysis of variance (ANOVA) test was used to compare mean scores between cities, distribution centers, and dairy farms. The average microbial load and pH were also compared using an independent t-test. Both tests were performed at a significance level of α = 0.05 and a confidence level of 95% CI.
Results
TBC
The examination of TBC in raw milk from dairy farms in Ilam province revealed that L5 had the highest microbial load (log CFU/ml 7.84), while the lowest was observed at L4 (log CFU/ml 5.91) (Figure 3. A). A statistically significant difference in microbial load was found between these locations (P<0.05).
Similarly, when investigating the TBC in raw milk at the collection and distribution centers of Ilam province, it was found that the lowest TBC was associated with L1 (5.25 log CFU/ml) (Figure 3. B), and this difference was also statistically significant (P<0.05). The mean TBCs in samples collected from L1, L4, and L5 were 5.25, 8.08, and 8.57 log10 CFU/ml, respectively. A high TBC was detected in raw cattle milk sample collected at L4, while low counts were detected in milk sample collected at L1. Except for raw milk collection centers in L1, the other areas were not up to standard.
pH
The average pH levels in cattle dairy farms and collection and distribution centers in Ilam province are provided in Table 1. The results indicated that the lowest pH value in raw milk collected from dairy farms in Ilam province was associated with raw milk from cattle farms in L5 (P<0.05). It is important to emphasize that only three milk collection centers were located in areas 1, 4, and 5, from which samples were obtained. The highest quantity was associated with L4.
The average pH levels in cattle dairy farms and collection and distribution centers in Ilam province are provided in Table 1. The results indicated that the lowest pH value in raw milk collected from dairy farms in Ilam province was associated with raw milk from cattle farms in L5 (P<0.05). It is important to emphasize that only three milk collection centers were located in areas 1, 4, and 5, from which samples were obtained. The highest quantity was associated with L4.
| Table 1. Mean pH in dairy farms and collection and distribution centers in Ilam province (mean ±SD). | ||
| Sampling Location | Dairy farm | Collection Center |
| 1 | 6.64±0.10 | 6.28±0.33 |
| 2 | 6.67±0.21 | - |
| 3 | 6.70±0.10 | - |
| 4 | 6.5±0.014 | 6.32±0.035 |
| 5 | 6.41±0.169 | 6.305±0.09 |
| 6 | 6.53±0.014 | - |
Evaluation of AFM1
This study investigated the level of AFM1 in 84 raw milk samples collected from cattle farms and collection centers in Ilam province. According to the findings (Table 2, Figure 4), 65 samples showed AFM1 contamination levels below 20 ng/l (the detection limit of the device). A total of 17 samples exhibited contamination levels ranging from 20-90 ng/l, while two samples surpassed 100 ng/l, exceeding Iran's permissible limit. According to the European :union:, the concentration of AFM1 exceeded the permissible limit of 50 ng/l in seven samples.
AF mean levels in two cohorts of dairy cattle farms and collection centers were compared. As indicated in Figure 3, the farm (25.96 ng/l) exhibited a higher AFM1 level than the centers (15.14 ng/l). Nevertheless, both mean levels remained below the permissible limit.
| Table 2. Occurrence of AFM1 in raw cattle milk in Ilam province, Iran. | ||||||
| Distribution of samples n (ng/l) | Sample(n) | Cohort | ||||
| >500 | 100-200 | 50-100 | 20-50 | <20 | ||
| 0 | 1 | 3 | 5 | 38 | 47 | Dairy farm |
| 1 | 0 | 2 | 7 | 27 | 37 | Collection and distribution centers |
| 1 | 1 | 5 | 12 | 65 | 84 | Total (N) |
The Iran National Standards Organization (INSO) has set a maximum allowable level of 100 ng/l for AFM1 in milk. The European :union: has established a limit of 50 ng/l for AFM1 in milk, while the Food and Drug Administration has set the maximum permissible level for AFM1 in milk at 500 ng/l.
Health risk assessment of AFM1
The EDI of AFM1 through milk consumption presented in Table 3 is supported by health hazard indices such as HI, MoE, and LCR for different ages and sexes. EDI of total AFM1 in raw cow milk samples from Ilam province were 0.36, 0.12, 0.07, 0.07, and 0.06 ng/kg bw/day for male age groups including <7, 7-16, 16-6, and >65 years, respectively, and for this, female age groups had respective daily intakes of 0.36, 0.12, 0.08, and 0.08 ng/kg bw/day. The highest EDI for males and females in age groups below 7 years was 0.36 ng/kg bw/d, since younger age groups consume more milk per body weight compared to older age groups. The data on EDI showed a remarkable decrease with age, and values for age groups 16-65 and >65 years were quite low, ranging from 0.07 to 0.08 ng/kg bw/day.
The latest findings of the study revealed that among males, the HI rates for milk consumption were 1.79, 0.59, 0.34, and 0.36 in the age groups <7, 7–16, 16–65, and >65 years, respectively. For females in the corresponding age groups, the HI rates were 1.79, 0.61, 0.41, and 0.4, respectively. The study showed that the potential non-carcinogenic effects of AFM1 from milk consumption in the age group >7 exceeded the allowable limit (Table 3). In the younger age group (<7 years), the HI was determined to be 1.79 for both males and females, raising concern for significant risk due to AFM1 exposure through milk during this developmental stage. On the contrary, the HI decreased with age, and the lowest values of 0.34-0.41 were recorded for the 16-65-and >65-year age groups, indicating a low non-carcinogenic risk in adults.
The MoE level serves as a pivotal parameter for characterizing risk, with a value below 10,000 indicating a potential health hazard. The current study findings demonstrated that the MoE value for AFM1 in all types of milk sourced from diverse sampling locations and across all age groups of both genders was consistently recorded below 10,000 (Table 3). This suggests a tangible health risk associated with the consumption of AFM1-contaminated milk. Moreover, MoE highlighted the health risks associated with the presence of AFM1 in milk. For both males and females under 7 years, the MoE was the lowest at 1595.98, indicating a higher risk compared to older age groups. In contrast, values of MoE were considerably higher for older children (7-16 years), with up to 4808.96 in males and 4670.65 in females. For adults, 16-65 years old , it increased further, indicating lower carcinogenic risk with age. However, a slight decrease was observed in MoE values for the >65-year age group, representing marginally increased vulnerability among the elderly.
In the current study, the results of LCR calculation indicated that for all types of milk collected from various sampling locations and for all age groups of males and females, the LCR level was higher than 4-10 (Table 3). However, the LCR level for children under 7 years old (2.025×10-3) was below 10-4, indicating its carcinogenicity. The highest estimated LCR level was in males and females under 7 years at 2.025E-3, indicating a higher risk of liver cancer in this age group. The LCR decreased with age, indicating reduced exposure and risk in the 16-65 age group. However, in the >65 years group, the LCR was still lower compared to other age groups, but considerably indicated the long-term effects of chronic exposure to AFM1.
The EDI of AFM1 through milk consumption presented in Table 3 is supported by health hazard indices such as HI, MoE, and LCR for different ages and sexes. EDI of total AFM1 in raw cow milk samples from Ilam province were 0.36, 0.12, 0.07, 0.07, and 0.06 ng/kg bw/day for male age groups including <7, 7-16, 16-6, and >65 years, respectively, and for this, female age groups had respective daily intakes of 0.36, 0.12, 0.08, and 0.08 ng/kg bw/day. The highest EDI for males and females in age groups below 7 years was 0.36 ng/kg bw/d, since younger age groups consume more milk per body weight compared to older age groups. The data on EDI showed a remarkable decrease with age, and values for age groups 16-65 and >65 years were quite low, ranging from 0.07 to 0.08 ng/kg bw/day.
The latest findings of the study revealed that among males, the HI rates for milk consumption were 1.79, 0.59, 0.34, and 0.36 in the age groups <7, 7–16, 16–65, and >65 years, respectively. For females in the corresponding age groups, the HI rates were 1.79, 0.61, 0.41, and 0.4, respectively. The study showed that the potential non-carcinogenic effects of AFM1 from milk consumption in the age group >7 exceeded the allowable limit (Table 3). In the younger age group (<7 years), the HI was determined to be 1.79 for both males and females, raising concern for significant risk due to AFM1 exposure through milk during this developmental stage. On the contrary, the HI decreased with age, and the lowest values of 0.34-0.41 were recorded for the 16-65-and >65-year age groups, indicating a low non-carcinogenic risk in adults.
The MoE level serves as a pivotal parameter for characterizing risk, with a value below 10,000 indicating a potential health hazard. The current study findings demonstrated that the MoE value for AFM1 in all types of milk sourced from diverse sampling locations and across all age groups of both genders was consistently recorded below 10,000 (Table 3). This suggests a tangible health risk associated with the consumption of AFM1-contaminated milk. Moreover, MoE highlighted the health risks associated with the presence of AFM1 in milk. For both males and females under 7 years, the MoE was the lowest at 1595.98, indicating a higher risk compared to older age groups. In contrast, values of MoE were considerably higher for older children (7-16 years), with up to 4808.96 in males and 4670.65 in females. For adults, 16-65 years old , it increased further, indicating lower carcinogenic risk with age. However, a slight decrease was observed in MoE values for the >65-year age group, representing marginally increased vulnerability among the elderly.
In the current study, the results of LCR calculation indicated that for all types of milk collected from various sampling locations and for all age groups of males and females, the LCR level was higher than 4-10 (Table 3). However, the LCR level for children under 7 years old (2.025×10-3) was below 10-4, indicating its carcinogenicity. The highest estimated LCR level was in males and females under 7 years at 2.025E-3, indicating a higher risk of liver cancer in this age group. The LCR decreased with age, indicating reduced exposure and risk in the 16-65 age group. However, in the >65 years group, the LCR was still lower compared to other age groups, but considerably indicated the long-term effects of chronic exposure to AFM1.
| Table 3. The EDI and HI, MoE, and LCR of AFM1 via milk consumption based on various age groups for males and females | |||||
| LCR | HI | MoE | EDI (ng/kg bw/day) | Sex and age groups(year) | |
2.025E-3 |
1.79 |
1595.98 |
0.36 |
Male <7 |
|
| 6.72E-4 | 0.59 | 4808.96 | 0.12 | 7-16 | |
| 3.86E-4 | 0.34 | 8373.12 | 0.07 | 16-65 | |
| 4.07E-4 | 0.36 | 7947.55 | 0.07 | 65< | |
2.025E-3 |
1.79 |
1595.98 |
0.36 |
Female <7 |
|
| 6.91E-4 | 0.61 | 4670.65 | 0.12 | 7-16 | |
| 4.64E-4 | 0.41 | 1393.75 | 0.08 | 16-65 | |
| 4.58E-4 | 0.40 | 7053.85 | 0.08 | 65< | |
| EDI: Estimated daily intake; MoE: Margin of exposure; HI: Health index; LCR: Liver cancer risk | |||||
Discussion
Evaluation of TBC, pH, and AFM1
Iran Veterinary Organization (IVO) has established standard limits for TBC in raw milk, which have been categorized into four groups: excellent (≤4.48), first-grade (4.48–5), second-grade (5–5.70), and third-grade (5.70–6) log10 CFU/ml. The results showed that TBC levels in milk from all regions exceeded the standard. High TBCs can result from insufficient milk cooling, inadequate udder preparation methods, and unclean milking equipment.
The findings in L3 and L4 showed higher TBC levels compared to those reported by Arefeh et al., who recorded a TBC of 5.25±0.13 in Kerman, Iran (Arefeh et al., 2021). Its mean value (7.30) was mostly based on values reported by Kazeminia et al., who found 6.41±1.22 in milk sampled from milk collection centers in Qazvin, Iran (Kazeminia et al., 2023). Elevated TBC levels may be indicative of a diseased udder, improper milk handling practices, or unfavorable storage temperatures (Nasir and Gemede, 2024). The variation in the collected samples could be attributed to unhygienic handling of milking apparatus. This suggests that equipment management during milking process could significantly affect the variability in the samples.
TBC levels observed in this study exceeded the acceptable thresholds established by both national and international regulatory standards. These limits are set to ensure food safety and public health, as elevated TBC levels indicate potential microbial contamination, which can compromise product quality and pose significant health risks to consumers. Compliance with these standards is crucial for minimizing the likelihood of foodborne illnesses and ensuring the microbiological safety of consumable products.
Analysis of the samples revealed significant differences in pH levels and TBC growth across various sampling locations (P<0.05). In L5, where the pH was lower than in other areas, the number of microorganisms was higher. This is in agreement with the previous research that obtained an average pH value of 6.30±0.41 (Kazeminia et al., 2023). Furthermore, Moosavi et al. in Tabriz, Iran, reported that the pH of most samples ranged from 5.00 to 7.00, with an average of 6.75 (Moosavi et al., 2018). This increase can be attributed to the growth of psychotropic microorganisms at lower temperatures and the production of acidic metabolites.
A review study analyzed 11,370 samples of AFM1 from various types of milk in Iran. The distribution of samples was as follows: 21.7% from dairy factories and milk collection centers (location 1), 48.8% from livestock farms (location 2), 26.8% from distribution centers and the milk market (location 3), and 2.7% from unspecified locations (location 4). The average AFM1 level was 77±159 ng/kg (ND-1137 ng/kg), surpassing the EU limit of 50 ng/kg, but remaining under the INSO threshold of 100 ng/kg (Massahi et al., 2023). Furthermore, the study revealed that livestock farms had the highest AFM1 levels (90±209 ng/kg) among milk sampling locations, with raw milk showing a significant correlation (84±84 ng/kg) (Massahi et al., 2023), consistent with the current findings.
Hasninia et al. also found that the average total AFM1 of raw milk was 31.2±1.7 ng/l (Hasninia et al., 2022). The variation in AFM1 levels was primarily affected by type of milk, timing of sampling, and presence of AFB1 in the feed given to lactating animals. Furthermore, differences in AFB1 levels can arise from various factors, including conditions under which animal feed is stored, such as ventilation, temperature, humidity, storage duration, and type of feed itself; therefore, each of these factors can significantly affect the growth of Aspergillus fungi that produce AFB1. AF-producing fungi thrive in conducive environments characterized by humidity levels exceeding 15%, minimum temperatures of 25 °C, sufficient oxygen, and particularly warm and humid climates affecting cereals and animal feed (Berghofer et al., 2003, Ghaneian et al., 2016, Ranjbar et al., 2010, Sales and Yoshizawa, 2005). Iran’s diverse geography leads to a variety of weather conditions, contributing to different environments that can promote the growth of AF-producing fungi across different regions of the country.
Health risk assessment of AFM1
AFM1 is a metabolite of AFB1 that contains carcinogenic properties; therefore, its presence in milk is dangerous for the health of consumers, especially in liver cancer. Health risk indicators were analyzed across various populations, offering insights into age- and sex-related changes. The results of similar studies align with the present study findings on EDI. Massahi et al. reported EDI values of 0.94, 0.42, 0.24, and 0.25 ng/kg bw/day for age groups <7, 7-16, 16-65, and >65 years, respectively, but for females, the corresponding values were 1.26, 0.43, 0.29, and 0.28 ng/kg bw/day, respectively (Massahi et al., 2023). Hasninia et al. also found that EDI values from milk consumption in summer for males were 0.943, 0.542, and 0 for the age groups 7-16, 16-65, and over 65 years, respectively, while for females in the same age groups, the values were 0.971, 0.651, and 0.643 ng/kg bw/day, respectively (Hasninia et al., 2022). Based on the findings of the study, during winter, the estimated exposure range for males in the specified age groups was 0–0.314, 0–0.181, and 0–0.191 ng/kg bw/day, respectively. For females in the same age groups, the exposure range was 0–0.324, 0–0.217, and 0–0.214 ng/kg bw/day, respectively (Hasninia et al., 2022).
Pardakhti et al. reported that the EDI of AFM1 from milk consumption in various cities was as follows: Tehran (0.057 ng/kg bw/day), Mashhad (0.063 ng/kg bw/day), Ahwaz (0.118 ng/kg bw/day), Babol (0.289 ng/kg bw/day), Esfahan (0.069 ng/kg bw/day), Kermanshah (3.319 ng/kg bw/day), Miandoab (0.007 ng/kg bw/day), Hamedan (0.036 ng/kg bw/day), and Urmia (0.210 ng/kg bw/day) (Pardakhti and Maleki, 2019). Variations in the EDI of AFM1 between studies can be attributed to multiple factors, such as the concentration of AFM1, daily milk intake (DMI), consumer age group, and body weight (BW), as well as the type, quality, and storage conditions of dairy cattle feed (Massahi et al., 2023).
In a related study, HI values for males in Iran consuming milk, across the age groups of under 7 years, 7 to 16 years, 16 to 65 years, and over 65 years were reported to be 4.7, 2.1, 1.2, and 1.3, respectively, while for females in the same age groups, HI values were 6.3, 2.1, 1.4, and 1.4, respectively (Massahi et al., 2023), which was higher than the values observed in the present study. The variation in HI across different age groups is attributed to differences in EDI, the possible causes of which have been discussed earlier. According to the findings, the potential HI for non-carcinogenic effects associated with AFM1 exposure due to milk consumption in Iran exceeded the permissible limit, particularly for children and adolescents. Since an HI value greater than 1 indicates an increased risk of liver cancer, immediate action is required to mitigate AFB1 contamination in dairy cattle feed, especially during the summer season. Key interventions should focus on disrupting the growth conditions of Aspergillus species found in animal feed storage areas (Berghofer et al., 2003, Ghaneian et al., 2016, Ranjbar et al., 2010, Sales and Yoshizawa, 2005). A review by Rahmani et al. reported that HI values for AFM1 exposure through raw milk consumption in adults from Iran, Turkey, Syria, Palestine, Lebanon, and Egypt were 0.26, 0.47, 0.52, 0.34, 0.23, and 0.18, respectively (Rahmani et al., 2018). As mentioned earlier, variations in parameters affecting the EDI of AFM1, such as BW, DMI, and AFM1 concentration in milk, likely explain the differences between the findings of this study and previous research.
According to EFSA Scientific Committee guidelines, MoE based on a BMD Lower Confidence Limit (BMDL) of 10,000 or more from animal studies is not considered a public health concern and is deemed a low priority for risk management (European Food Safety Authority, 2005). However, the present study results for raw milk showed that MoE values for AFM1 exposure among consumers were below 10,000, indicating a health concern related to AFM1 exposure. A study by Udoveicki et al. in Serbia and Greece assessed AFM intake in milk and yogurt, reporting MoE values ranging from 213.2 to 460.4 for Serbia and 1142.3 to 1628.6 for Greece (Udovicki et al., 2019). These findings, along with those of Hasninia and Massahi show MoE levels below 10,000 (Hasninia et al., 2022, Massahi et al., 2023) and Mokhtarib et al. with an MoE range of 1892.9 to 3921.6 (Mokhtari et al., 2022), align with the current study results.
Similar studies have reported the LCR associated with AFM1 exposure through milk consumption in urban areas, with calculated risk values ranging from 0.0008 to 0.0017, which corresponds to the second sampling series (traditional milk) and the first sampling series (pasteurized milk), respectively (Mokhtari et al., 2022). These results represent the estimated probability of additional liver cancer cases per year per million individuals. Based on these findings, the overall cancer risk from AFM1 exposure to milk appears to be very low, and there is no significant public health concern in this regard. In a study conducted by Hooshfar et al. in Iran (Hooshfar et al., 2020), the risk assessment of primary LCR due to AFM1 in milk revealed that cancer risk, based on a more realistic scenario accounting for average AFM1 exposure and milk consumption, was calculated at 0.0001 additional cancer cases per 100,000 person-years. This finding aligns with the present study, which ranged from 0.0007 to 0.0016 additional cases per million person-years (Hooshfar et al., 2020).
This study aimed to assess the levels of microbial contamination and the presence of aflatoxins in livestock farms throughout the province. We measured AFM1 levels and microbial contamination in cattle milk, ensuring that all active livestock farms were included in our research. Future studies should focus on investigating the causes and sources of bacterial contamination, as well as isolating and identifying the microorganisms and bacterial strains present in milk. Moreover, it will be important to examine the levels of AFM1 across different seasons and how seasonal variations may impact these levels.
Conclusion
This study evaluated the presence and concentration of TBC, pH, and AFM1 in raw cattle milk in Ilam province, Iran. The analysis of TBC in raw milk samples collected from dairy farms showed that L5 exhibited the highest microbial load, with a log CFU/ml value of 7.84. In contrast, L4 had the lowest microbial load, with a recorded log CFU/ml value of 5.91. Furthermore, the lowest pH of raw milk from cattle farms was in L5, while the highest was in L3. Based on the findings, the mean AFM1 level in raw milk cattle produced in Ilam province was dairy farms (25.96 ng/l) and collection centers (15.14 ng/l), which was below EU regulation (50 ng/l) and INSO regulations (100 ng/l). The results showed a statistically significant variation in mean levels of pH, TBC, and AFM1 between dairy farms, collection and distribution centers, and various sampling locations (p<0.05). These discrepancies probably result from individual lactating animals ingesting AFB1 at different levels, since AFB1 is metabolized into AFM1 and excreted into milk. The present analysis points out that in health risk assessment of AFM1, in the case of young children, the intake of AFM1 through milk may pose overall health risks, with a particular incidence of liver cancer. High LCR and HI values among children under 7 years suggest stricter enforcement of the regulations regarding contamination by AFM1 in milk and its products, with special emphasis on product consumption within this age group. Although the risk decreases with age, it supports the continued vigilance in the long-term impact of cumulative exposure, particularly for the elderly, to protect vulnerable populations from AFM1-related health risks.
Acknowledgments
The authors are very grateful to the Ilam Veterinary Organization for financial support of this study.
Authors' contributions
Nemati M, Khajehali E, and Rashidimehr A conducted the experiment, analyzed data, interpreted statistical analysis, and wrote the main draft of the manuscript. All authors reviewed and approved the manuscript.
Conflicts of interest
The authors declare no conflicts of interest.
Funding
The research reported in this publication was supported by Ilam Veterinary Organization and Ilam University, Ilam, Iran.
References
Arefeh AJ, Ebrahimnejad H & Aghamiri SM 2021. A study on dairy cow management and the related bulk tank milk bacteria in Kerman County during cold and hot seasons. Iranian Veterinary Journal. 17(1): 24-32.
Assaf JC, et al. 2020. Assorted Methods for Decontamination of Aflatoxin. Toxins. 11(304): 1-23.
Augustin MA, et al. 2016. Role of food processing in food and nutrition security. Trends in Food Science & Technology. 56: 115-125.
Bahrami R, Shahbazi Y & Nikousefat Z 2016. Aflatoxin M1 in milk and traditional dairy products from west part of Iran: Occurrence and seasonal variation with an emphasis on risk assessment of human exposure. Food Control. 62: 250–256.
Benford D, et al. 2010. Application of the Margin of Exposure (MOE) approach to substances in food that are genotoxic and carcinogenic. Food and chemical toxicology. 48: S2-S24.
Berghofer L, Hocking A, Miskelly D & Jansson E 2003. Microbiology of wheat and flour milling in Australia. International journal of food microbiology. 85 (1-2): 137-149.
De Roma A, Rossini C, Ritieni A, Gallo P & Esposito M 2017. A survey on the Aflatoxin M1 occurrence and seasonal variation in buffalo and cow milk from Southern Italy. Food control. 81: 30–33.
Environmental Protection Agency 2001. Risk assessment guidance for superfund: Volume III part A, process for conducting probabilistic risk assessment. . Washington, DC: US
European Food Safety Authority 2005. Oinion of the scientifc panel on genetically modifed organisms on a request from the commission related to the notifcation (Reference C/GB/02/M3/3) for the placing on the market of glyphosate-tolerant and insect-resistant genetically modifed maize NK603× MON810, for import and processing, under part C of directive 2001/18/EC from Monsanto. EFSA Journal. 3 (12): 308.
Fakhri Y, et al. 2019. Concentration and prevalence of aflatoxin M1 in human breast milk in Iran: Systematic review, meta-analysis, and carcinogenic risk assessment: A review. Journal of food protection. 82: 785-795.
FAO/WHO 2017. Evaluation of certain contaminants in food: Eighty-third report of the Joint FAO/WHO Expert committee on food Additives. https://apps.who.int/ iris/handle/ 10665/254893.
Gemechu T & Amene T 2016. Physicochemical properties and microbial quality of raw cow milk produced by smallholders in Bench Maji-Zone, Southwestern Ethiopia. Food science and quality management. 54: 47–54.
Ghaneian M, et al. 2016. Survey the frequency and type of fungal contaminants in animal feed of Yazd dairy cattles. Iranian journal of animal science research. 7 (4): 422-427.
Hashemi M 2016. A survey of aflatoxin M1 in cow milk in Southern Iran. . Journal of food and drug analysis. 24 (4): 888-893.
Hasninia D, et al. 2022. Human health risk assessment of aflatoxin M1 in raw and pasteurized milk from the Kermanshah province, Iran. Journal of food composition and analysis. 110: 104568.
Hassan Z, et al. 2018. Evidence of low levels of aflatoxin M1 in milk and dairy products marketed in Qatar. Food control. 92: 25-29.
Hooshfar S, et al. 2020. Health risk assessment of aflatoxin M1 in infant formula milk in IR Iran. Food and chemical toxicology. 142: 111455.
Iranian National Standardization Organization 2008. Microbiology of the food chain -Horizontal method for the enumeration of microorganisms-Part 2 :Colony count at 30 °C by the surface plating technique. no 5272-2.
Iranian National Standardization Organization 2020. Food and feed- Maximum tolerated level of mycotoxins. no. 5925, 1st Revision.
Kamkar A, Jahed K & Alavi S 2011. Occurrence of aflatoxin M1 in raw milk produced in Ardebil of Iran. Iranian journal of environmental health science and engineering. 8 (2): 123-128.
Kazeminia M, Mahmoudi R, Mousavi S & Mehrabi A 2023. Raw cow milk quality: Physicochemical, microbiological, and seasonal variation. Journal of microbiology, biotechnology & food sciences. 13 (3): e10078.
Ketney O, Santini A & Oancea S 2017. Recent aflatoxin survey data in milk and milk products: A review. International journal of dairy technology. 70 (3): 320-331.
Kortei N, et al. 2022. Exposure assessment and cancer risk characterization of aflatoxin M1 (AFM1) through ingestion of raw cow milk in southern Ghana. Toxicology reports. 9: 1189-1197.
Mahmoudi R & Norian R 2015. Aflatoxin B1 and M1 contamination in cow feeds and milk from Iran. Food and agricultural immunology. 26 (1): 131-137.
Marchese S, et al. 2018. Aflatoxin B1 and M1: Biological properties and their involvement in cancer development. Toxins. 10 (6): 1–19.
Massahi T, et al. 2023. A systematic literature review for aflatoxin M1 of various milk types in Iran: Human health risk assessment, uncertainty, and sensitivity analysis. Food control. 150: 109733.
Moghaddam A, et al. 2019. Risk associated with the intake of AFM from milk in Iran. World mycotoxin journal. 12 (2): 191–200.
Mokhtari SA, et al. 2022. Aflatoxin M1 in distributed milks in northwestern Iran: occurrence, seasonal variation, and risk assessment. Environmental science and pollution research. 29 (27): 41429-41438.
Mollayusefian I, et al. 2021. The concentration of aflatoxin M1 in raw and pasteurized milk: A worldwide systematic review and meta-analysis. Trends in food science & technology. 115: 22-30.
Moosavi M, Mahmoudi R, Ghorbanpour E & Khatibi S 2018. Evaluation of microbial and physicochemical characteristics of raw cow milk delivered to pasteurized milk plants in Tabriz city, Iran. Journal of food research. 28 (1): 183-196.
Nasir L & Gemede HF 2024. Aflatoxin M1 concentrations, adulterants, microbial loads, and physicochemical properties of raw milk collected from Nekemte City, Ethiopia. Scientific world journal. 2024 (1): 3796985.
Nejad A, Heshmati A & Ghiasvand T 2020. The occurrence and risk assessment of aflatoxin M1 in Cheeses samples from Hamadan, Iran. Iranian journal of pharmaceutical research. 19 (4): 44.
Omar S 2016. Aflatoxin M1 levels in raw milk, pasteurised milk and infant formula. Italian journal of food safety. 5 (3): 5788.
Pardakhti A & Maleki S 2019. Risk assessment of Aflatoxin M1 contamination of milk in Iran. International journal of environmental research. 13: 265-271.
Pour S, et al. 2020. Aflatoxin M1 contamination level in Iranian milk and dairy products: A systematic review and meta-analysis. World mycotoxin journal. 13: 67–82.
Rahmani J, et al. 2018. The prevalence of aflatoxin M1 in milk of Middle East region: A systematic review, metaanalysis and probabilistic health risk assessment. Food and chemical toxicology. 118: 653–666.
Ranjbar S, Noori M & Nazari R 2010. Study of milk aflatoxin M1 and its relationship with feed fungi flora in Markazi Province. Journal cell tissue. 1 (1): 9–18.
Sadeghi N, et al. 2009. Incidence of aflatoxin M1 in human breast milk in Tehran, Iran. Food control. 20 (1): 75-78.
Sales A & Yoshizawa T 2005. Updated profile of aflatoxin and Aspergillus section Flavi contamination in rice and its byproducts from the Philippines. Food additives & contaminants. 22 (5): 429–436.
Serraino A, et al. 2019. Occurrence of aflatoxin M1 in raw milk marketed in Italy: Exposure assessment and risk characterization. Frontiers in microbiology. 10: 2516.
Sharma H, Jadhav V & Garg S 2020. AFM in milk in Hisar city, Haryana, India and risk assessment. Food additives & contaminants: Part B. 13 (1): 59–63.
Tsakiris I, et al. 2013. Risk assessment scenarios of children’s exposure to afatoxin M1 residues in diferent milk types from the Greek market. Food and chemical toxicology. 56: 261–265.
Udovicki B, Djekic I, Kalogianni E & Rajkovic A 2019. Exposure assessment and risk characterization of aflatoxin M1 intake through consumption of milk and yoghurt by student population in Serbia and Greece. Toxins. 11 (4): 205.
United States Environmental Protection Agency 2011. Exposure Factors Handbook: 2011 Edition. Office of Research and Development: Washington, DC.
World Health Organization & International Agency for Research on Cancer 2002. Working group on the evaluation of carcinogenic risks to humans. Some traditional herbal medicines, some mycotoxins, naphthalene and styrene. IARCpress: Lyon, France.
Evaluation of TBC, pH, and AFM1
Iran Veterinary Organization (IVO) has established standard limits for TBC in raw milk, which have been categorized into four groups: excellent (≤4.48), first-grade (4.48–5), second-grade (5–5.70), and third-grade (5.70–6) log10 CFU/ml. The results showed that TBC levels in milk from all regions exceeded the standard. High TBCs can result from insufficient milk cooling, inadequate udder preparation methods, and unclean milking equipment.
The findings in L3 and L4 showed higher TBC levels compared to those reported by Arefeh et al., who recorded a TBC of 5.25±0.13 in Kerman, Iran (Arefeh et al., 2021). Its mean value (7.30) was mostly based on values reported by Kazeminia et al., who found 6.41±1.22 in milk sampled from milk collection centers in Qazvin, Iran (Kazeminia et al., 2023). Elevated TBC levels may be indicative of a diseased udder, improper milk handling practices, or unfavorable storage temperatures (Nasir and Gemede, 2024). The variation in the collected samples could be attributed to unhygienic handling of milking apparatus. This suggests that equipment management during milking process could significantly affect the variability in the samples.
TBC levels observed in this study exceeded the acceptable thresholds established by both national and international regulatory standards. These limits are set to ensure food safety and public health, as elevated TBC levels indicate potential microbial contamination, which can compromise product quality and pose significant health risks to consumers. Compliance with these standards is crucial for minimizing the likelihood of foodborne illnesses and ensuring the microbiological safety of consumable products.
Analysis of the samples revealed significant differences in pH levels and TBC growth across various sampling locations (P<0.05). In L5, where the pH was lower than in other areas, the number of microorganisms was higher. This is in agreement with the previous research that obtained an average pH value of 6.30±0.41 (Kazeminia et al., 2023). Furthermore, Moosavi et al. in Tabriz, Iran, reported that the pH of most samples ranged from 5.00 to 7.00, with an average of 6.75 (Moosavi et al., 2018). This increase can be attributed to the growth of psychotropic microorganisms at lower temperatures and the production of acidic metabolites.
A review study analyzed 11,370 samples of AFM1 from various types of milk in Iran. The distribution of samples was as follows: 21.7% from dairy factories and milk collection centers (location 1), 48.8% from livestock farms (location 2), 26.8% from distribution centers and the milk market (location 3), and 2.7% from unspecified locations (location 4). The average AFM1 level was 77±159 ng/kg (ND-1137 ng/kg), surpassing the EU limit of 50 ng/kg, but remaining under the INSO threshold of 100 ng/kg (Massahi et al., 2023). Furthermore, the study revealed that livestock farms had the highest AFM1 levels (90±209 ng/kg) among milk sampling locations, with raw milk showing a significant correlation (84±84 ng/kg) (Massahi et al., 2023), consistent with the current findings.
Hasninia et al. also found that the average total AFM1 of raw milk was 31.2±1.7 ng/l (Hasninia et al., 2022). The variation in AFM1 levels was primarily affected by type of milk, timing of sampling, and presence of AFB1 in the feed given to lactating animals. Furthermore, differences in AFB1 levels can arise from various factors, including conditions under which animal feed is stored, such as ventilation, temperature, humidity, storage duration, and type of feed itself; therefore, each of these factors can significantly affect the growth of Aspergillus fungi that produce AFB1. AF-producing fungi thrive in conducive environments characterized by humidity levels exceeding 15%, minimum temperatures of 25 °C, sufficient oxygen, and particularly warm and humid climates affecting cereals and animal feed (Berghofer et al., 2003, Ghaneian et al., 2016, Ranjbar et al., 2010, Sales and Yoshizawa, 2005). Iran’s diverse geography leads to a variety of weather conditions, contributing to different environments that can promote the growth of AF-producing fungi across different regions of the country.
Health risk assessment of AFM1
AFM1 is a metabolite of AFB1 that contains carcinogenic properties; therefore, its presence in milk is dangerous for the health of consumers, especially in liver cancer. Health risk indicators were analyzed across various populations, offering insights into age- and sex-related changes. The results of similar studies align with the present study findings on EDI. Massahi et al. reported EDI values of 0.94, 0.42, 0.24, and 0.25 ng/kg bw/day for age groups <7, 7-16, 16-65, and >65 years, respectively, but for females, the corresponding values were 1.26, 0.43, 0.29, and 0.28 ng/kg bw/day, respectively (Massahi et al., 2023). Hasninia et al. also found that EDI values from milk consumption in summer for males were 0.943, 0.542, and 0 for the age groups 7-16, 16-65, and over 65 years, respectively, while for females in the same age groups, the values were 0.971, 0.651, and 0.643 ng/kg bw/day, respectively (Hasninia et al., 2022). Based on the findings of the study, during winter, the estimated exposure range for males in the specified age groups was 0–0.314, 0–0.181, and 0–0.191 ng/kg bw/day, respectively. For females in the same age groups, the exposure range was 0–0.324, 0–0.217, and 0–0.214 ng/kg bw/day, respectively (Hasninia et al., 2022).
Pardakhti et al. reported that the EDI of AFM1 from milk consumption in various cities was as follows: Tehran (0.057 ng/kg bw/day), Mashhad (0.063 ng/kg bw/day), Ahwaz (0.118 ng/kg bw/day), Babol (0.289 ng/kg bw/day), Esfahan (0.069 ng/kg bw/day), Kermanshah (3.319 ng/kg bw/day), Miandoab (0.007 ng/kg bw/day), Hamedan (0.036 ng/kg bw/day), and Urmia (0.210 ng/kg bw/day) (Pardakhti and Maleki, 2019). Variations in the EDI of AFM1 between studies can be attributed to multiple factors, such as the concentration of AFM1, daily milk intake (DMI), consumer age group, and body weight (BW), as well as the type, quality, and storage conditions of dairy cattle feed (Massahi et al., 2023).
In a related study, HI values for males in Iran consuming milk, across the age groups of under 7 years, 7 to 16 years, 16 to 65 years, and over 65 years were reported to be 4.7, 2.1, 1.2, and 1.3, respectively, while for females in the same age groups, HI values were 6.3, 2.1, 1.4, and 1.4, respectively (Massahi et al., 2023), which was higher than the values observed in the present study. The variation in HI across different age groups is attributed to differences in EDI, the possible causes of which have been discussed earlier. According to the findings, the potential HI for non-carcinogenic effects associated with AFM1 exposure due to milk consumption in Iran exceeded the permissible limit, particularly for children and adolescents. Since an HI value greater than 1 indicates an increased risk of liver cancer, immediate action is required to mitigate AFB1 contamination in dairy cattle feed, especially during the summer season. Key interventions should focus on disrupting the growth conditions of Aspergillus species found in animal feed storage areas (Berghofer et al., 2003, Ghaneian et al., 2016, Ranjbar et al., 2010, Sales and Yoshizawa, 2005). A review by Rahmani et al. reported that HI values for AFM1 exposure through raw milk consumption in adults from Iran, Turkey, Syria, Palestine, Lebanon, and Egypt were 0.26, 0.47, 0.52, 0.34, 0.23, and 0.18, respectively (Rahmani et al., 2018). As mentioned earlier, variations in parameters affecting the EDI of AFM1, such as BW, DMI, and AFM1 concentration in milk, likely explain the differences between the findings of this study and previous research.
According to EFSA Scientific Committee guidelines, MoE based on a BMD Lower Confidence Limit (BMDL) of 10,000 or more from animal studies is not considered a public health concern and is deemed a low priority for risk management (European Food Safety Authority, 2005). However, the present study results for raw milk showed that MoE values for AFM1 exposure among consumers were below 10,000, indicating a health concern related to AFM1 exposure. A study by Udoveicki et al. in Serbia and Greece assessed AFM intake in milk and yogurt, reporting MoE values ranging from 213.2 to 460.4 for Serbia and 1142.3 to 1628.6 for Greece (Udovicki et al., 2019). These findings, along with those of Hasninia and Massahi show MoE levels below 10,000 (Hasninia et al., 2022, Massahi et al., 2023) and Mokhtarib et al. with an MoE range of 1892.9 to 3921.6 (Mokhtari et al., 2022), align with the current study results.
Similar studies have reported the LCR associated with AFM1 exposure through milk consumption in urban areas, with calculated risk values ranging from 0.0008 to 0.0017, which corresponds to the second sampling series (traditional milk) and the first sampling series (pasteurized milk), respectively (Mokhtari et al., 2022). These results represent the estimated probability of additional liver cancer cases per year per million individuals. Based on these findings, the overall cancer risk from AFM1 exposure to milk appears to be very low, and there is no significant public health concern in this regard. In a study conducted by Hooshfar et al. in Iran (Hooshfar et al., 2020), the risk assessment of primary LCR due to AFM1 in milk revealed that cancer risk, based on a more realistic scenario accounting for average AFM1 exposure and milk consumption, was calculated at 0.0001 additional cancer cases per 100,000 person-years. This finding aligns with the present study, which ranged from 0.0007 to 0.0016 additional cases per million person-years (Hooshfar et al., 2020).
This study aimed to assess the levels of microbial contamination and the presence of aflatoxins in livestock farms throughout the province. We measured AFM1 levels and microbial contamination in cattle milk, ensuring that all active livestock farms were included in our research. Future studies should focus on investigating the causes and sources of bacterial contamination, as well as isolating and identifying the microorganisms and bacterial strains present in milk. Moreover, it will be important to examine the levels of AFM1 across different seasons and how seasonal variations may impact these levels.
Conclusion
This study evaluated the presence and concentration of TBC, pH, and AFM1 in raw cattle milk in Ilam province, Iran. The analysis of TBC in raw milk samples collected from dairy farms showed that L5 exhibited the highest microbial load, with a log CFU/ml value of 7.84. In contrast, L4 had the lowest microbial load, with a recorded log CFU/ml value of 5.91. Furthermore, the lowest pH of raw milk from cattle farms was in L5, while the highest was in L3. Based on the findings, the mean AFM1 level in raw milk cattle produced in Ilam province was dairy farms (25.96 ng/l) and collection centers (15.14 ng/l), which was below EU regulation (50 ng/l) and INSO regulations (100 ng/l). The results showed a statistically significant variation in mean levels of pH, TBC, and AFM1 between dairy farms, collection and distribution centers, and various sampling locations (p<0.05). These discrepancies probably result from individual lactating animals ingesting AFB1 at different levels, since AFB1 is metabolized into AFM1 and excreted into milk. The present analysis points out that in health risk assessment of AFM1, in the case of young children, the intake of AFM1 through milk may pose overall health risks, with a particular incidence of liver cancer. High LCR and HI values among children under 7 years suggest stricter enforcement of the regulations regarding contamination by AFM1 in milk and its products, with special emphasis on product consumption within this age group. Although the risk decreases with age, it supports the continued vigilance in the long-term impact of cumulative exposure, particularly for the elderly, to protect vulnerable populations from AFM1-related health risks.
Acknowledgments
The authors are very grateful to the Ilam Veterinary Organization for financial support of this study.
Authors' contributions
Nemati M, Khajehali E, and Rashidimehr A conducted the experiment, analyzed data, interpreted statistical analysis, and wrote the main draft of the manuscript. All authors reviewed and approved the manuscript.
Conflicts of interest
The authors declare no conflicts of interest.
Funding
The research reported in this publication was supported by Ilam Veterinary Organization and Ilam University, Ilam, Iran.
References
Arefeh AJ, Ebrahimnejad H & Aghamiri SM 2021. A study on dairy cow management and the related bulk tank milk bacteria in Kerman County during cold and hot seasons. Iranian Veterinary Journal. 17(1): 24-32.
Assaf JC, et al. 2020. Assorted Methods for Decontamination of Aflatoxin. Toxins. 11(304): 1-23.
Augustin MA, et al. 2016. Role of food processing in food and nutrition security. Trends in Food Science & Technology. 56: 115-125.
Bahrami R, Shahbazi Y & Nikousefat Z 2016. Aflatoxin M1 in milk and traditional dairy products from west part of Iran: Occurrence and seasonal variation with an emphasis on risk assessment of human exposure. Food Control. 62: 250–256.
Benford D, et al. 2010. Application of the Margin of Exposure (MOE) approach to substances in food that are genotoxic and carcinogenic. Food and chemical toxicology. 48: S2-S24.
Berghofer L, Hocking A, Miskelly D & Jansson E 2003. Microbiology of wheat and flour milling in Australia. International journal of food microbiology. 85 (1-2): 137-149.
De Roma A, Rossini C, Ritieni A, Gallo P & Esposito M 2017. A survey on the Aflatoxin M1 occurrence and seasonal variation in buffalo and cow milk from Southern Italy. Food control. 81: 30–33.
Environmental Protection Agency 2001. Risk assessment guidance for superfund: Volume III part A, process for conducting probabilistic risk assessment. . Washington, DC: US
European Food Safety Authority 2005. Oinion of the scientifc panel on genetically modifed organisms on a request from the commission related to the notifcation (Reference C/GB/02/M3/3) for the placing on the market of glyphosate-tolerant and insect-resistant genetically modifed maize NK603× MON810, for import and processing, under part C of directive 2001/18/EC from Monsanto. EFSA Journal. 3 (12): 308.
Fakhri Y, et al. 2019. Concentration and prevalence of aflatoxin M1 in human breast milk in Iran: Systematic review, meta-analysis, and carcinogenic risk assessment: A review. Journal of food protection. 82: 785-795.
FAO/WHO 2017. Evaluation of certain contaminants in food: Eighty-third report of the Joint FAO/WHO Expert committee on food Additives. https://apps.who.int/ iris/handle/ 10665/254893.
Gemechu T & Amene T 2016. Physicochemical properties and microbial quality of raw cow milk produced by smallholders in Bench Maji-Zone, Southwestern Ethiopia. Food science and quality management. 54: 47–54.
Ghaneian M, et al. 2016. Survey the frequency and type of fungal contaminants in animal feed of Yazd dairy cattles. Iranian journal of animal science research. 7 (4): 422-427.
Hashemi M 2016. A survey of aflatoxin M1 in cow milk in Southern Iran. . Journal of food and drug analysis. 24 (4): 888-893.
Hasninia D, et al. 2022. Human health risk assessment of aflatoxin M1 in raw and pasteurized milk from the Kermanshah province, Iran. Journal of food composition and analysis. 110: 104568.
Hassan Z, et al. 2018. Evidence of low levels of aflatoxin M1 in milk and dairy products marketed in Qatar. Food control. 92: 25-29.
Hooshfar S, et al. 2020. Health risk assessment of aflatoxin M1 in infant formula milk in IR Iran. Food and chemical toxicology. 142: 111455.
Iranian National Standardization Organization 2008. Microbiology of the food chain -Horizontal method for the enumeration of microorganisms-Part 2 :Colony count at 30 °C by the surface plating technique. no 5272-2.
Iranian National Standardization Organization 2020. Food and feed- Maximum tolerated level of mycotoxins. no. 5925, 1st Revision.
Kamkar A, Jahed K & Alavi S 2011. Occurrence of aflatoxin M1 in raw milk produced in Ardebil of Iran. Iranian journal of environmental health science and engineering. 8 (2): 123-128.
Kazeminia M, Mahmoudi R, Mousavi S & Mehrabi A 2023. Raw cow milk quality: Physicochemical, microbiological, and seasonal variation. Journal of microbiology, biotechnology & food sciences. 13 (3): e10078.
Ketney O, Santini A & Oancea S 2017. Recent aflatoxin survey data in milk and milk products: A review. International journal of dairy technology. 70 (3): 320-331.
Kortei N, et al. 2022. Exposure assessment and cancer risk characterization of aflatoxin M1 (AFM1) through ingestion of raw cow milk in southern Ghana. Toxicology reports. 9: 1189-1197.
Mahmoudi R & Norian R 2015. Aflatoxin B1 and M1 contamination in cow feeds and milk from Iran. Food and agricultural immunology. 26 (1): 131-137.
Marchese S, et al. 2018. Aflatoxin B1 and M1: Biological properties and their involvement in cancer development. Toxins. 10 (6): 1–19.
Massahi T, et al. 2023. A systematic literature review for aflatoxin M1 of various milk types in Iran: Human health risk assessment, uncertainty, and sensitivity analysis. Food control. 150: 109733.
Moghaddam A, et al. 2019. Risk associated with the intake of AFM from milk in Iran. World mycotoxin journal. 12 (2): 191–200.
Mokhtari SA, et al. 2022. Aflatoxin M1 in distributed milks in northwestern Iran: occurrence, seasonal variation, and risk assessment. Environmental science and pollution research. 29 (27): 41429-41438.
Mollayusefian I, et al. 2021. The concentration of aflatoxin M1 in raw and pasteurized milk: A worldwide systematic review and meta-analysis. Trends in food science & technology. 115: 22-30.
Moosavi M, Mahmoudi R, Ghorbanpour E & Khatibi S 2018. Evaluation of microbial and physicochemical characteristics of raw cow milk delivered to pasteurized milk plants in Tabriz city, Iran. Journal of food research. 28 (1): 183-196.
Nasir L & Gemede HF 2024. Aflatoxin M1 concentrations, adulterants, microbial loads, and physicochemical properties of raw milk collected from Nekemte City, Ethiopia. Scientific world journal. 2024 (1): 3796985.
Nejad A, Heshmati A & Ghiasvand T 2020. The occurrence and risk assessment of aflatoxin M1 in Cheeses samples from Hamadan, Iran. Iranian journal of pharmaceutical research. 19 (4): 44.
Omar S 2016. Aflatoxin M1 levels in raw milk, pasteurised milk and infant formula. Italian journal of food safety. 5 (3): 5788.
Pardakhti A & Maleki S 2019. Risk assessment of Aflatoxin M1 contamination of milk in Iran. International journal of environmental research. 13: 265-271.
Pour S, et al. 2020. Aflatoxin M1 contamination level in Iranian milk and dairy products: A systematic review and meta-analysis. World mycotoxin journal. 13: 67–82.
Rahmani J, et al. 2018. The prevalence of aflatoxin M1 in milk of Middle East region: A systematic review, metaanalysis and probabilistic health risk assessment. Food and chemical toxicology. 118: 653–666.
Ranjbar S, Noori M & Nazari R 2010. Study of milk aflatoxin M1 and its relationship with feed fungi flora in Markazi Province. Journal cell tissue. 1 (1): 9–18.
Sadeghi N, et al. 2009. Incidence of aflatoxin M1 in human breast milk in Tehran, Iran. Food control. 20 (1): 75-78.
Sales A & Yoshizawa T 2005. Updated profile of aflatoxin and Aspergillus section Flavi contamination in rice and its byproducts from the Philippines. Food additives & contaminants. 22 (5): 429–436.
Serraino A, et al. 2019. Occurrence of aflatoxin M1 in raw milk marketed in Italy: Exposure assessment and risk characterization. Frontiers in microbiology. 10: 2516.
Sharma H, Jadhav V & Garg S 2020. AFM in milk in Hisar city, Haryana, India and risk assessment. Food additives & contaminants: Part B. 13 (1): 59–63.
Tsakiris I, et al. 2013. Risk assessment scenarios of children’s exposure to afatoxin M1 residues in diferent milk types from the Greek market. Food and chemical toxicology. 56: 261–265.
Udovicki B, Djekic I, Kalogianni E & Rajkovic A 2019. Exposure assessment and risk characterization of aflatoxin M1 intake through consumption of milk and yoghurt by student population in Serbia and Greece. Toxins. 11 (4): 205.
United States Environmental Protection Agency 2011. Exposure Factors Handbook: 2011 Edition. Office of Research and Development: Washington, DC.
World Health Organization & International Agency for Research on Cancer 2002. Working group on the evaluation of carcinogenic risks to humans. Some traditional herbal medicines, some mycotoxins, naphthalene and styrene. IARCpress: Lyon, France.
Type of article: orginal article |
Subject:
public specific
Received: 2024/11/27 | Published: 2025/07/6 | ePublished: 2025/07/6
Received: 2024/11/27 | Published: 2025/07/6 | ePublished: 2025/07/6
Send email to the article author
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
