Occurrence and exposure assessment of Aflatoxin M₁ in milk and milk products in India

Abstract

Milk containing Aflatoxin M₁ (AFM₁) poses a serious health risk to consumers. Present study was undertaken to determine levels of AFM₁ in 146 milk and value added dairy products sold in retail markets of Chhattisgarh, India using HPLC coupled with fluorescence detector. A total of 52 samples (35.6%) were found to contain AFM₁ with overall concentrations ranging from nd − 2.608 µg/L. The contamination levels were higher in non-fermented milk products than fermented milk products samples, although this difference was statistically non-significant (p > 0.05). AFM₁ concentrations above maximum permissible limits established by the European Commission were found in 94.2% of positive samples. Health risk assessments ascertained that the estimated daily intakes for AFM₁ is higher than the established tolerable daily intakes for both adults and children (Hazard Index > 1), there by implying a potentially high risk to consumer’s health. Current investigation provides valuable information regarding contamination of raw as well as value added milk products sold in Indian markets. Therefore, to protect consumer’s health and promote dairy trade; there is an urgent need to increase farmer’s knowledge on good storage practices of feed and fodder. Further, stringent enforcement of food safety regulations is imperative to safeguard and promote human health.

Introduction

Milk and milk products finds an important place in human diet. With rising food safety and quality concerns, the demand for packaged milk is increasing among health conscious consumers. In India, the packaged milk is mostly marketed as pasteurized and ultra-high temperature (UHT) milk with numerous varieties depending on the fat content and flavors. The consumption of value added dairy products is also experiencing exponential growth due to rising income, urbanization, and other socio-demographic shifts. Therefore, milk is not only consumed as liquid milk, but now it is also being utilized for the preparation of processed milk and other dairy products.

Despite the fact that milk and milk products have high nutritional value and contains significant amount of macro and micronutrients, their contamination with environmental pollutants is very alarming. Among various contaminants arising from unapproved animal husbandry and farming practices, aflatoxins, antibiotics, pesticides etc. are the most important ones. These contaminants not only affects the quality of raw and processed milk products but they also pose potential human health risks, especially to new born, children, elderly and ill people (Kumar et al. 2021).

India is a tropical country and the hot/humid weather conditions of Central India favors the growth and multiplication of aflatoxigenic fungi like Aspergillus flavus, Aspergillus parasiticus etc. The important aflatoxins like B_1, B₂, G₁, and G₂ that predominate in various agricultural commodities and food are toxic fungal metabolites produced by different species of Genus Aspergillus (IARC 2012). Ingestion of Aflatoxin B₁ (AFB₁) contaminated feed and fodder by the dairy animals’ results in excretion of Aflatoxin M₁ (AFM₁) in their milk (Prandini et al. 2009). Both, AFB₁ and AFM₁ have been classified as class 1 human carcinogenic compound by International Agency for Research on cancer (IARC), though the carcinogenicity of AFM₁ is approximately 2–10% to that of AFB₁ (Creppy 2002). But, the acute toxicities exhibited by both AFB1 and AFM1 are rather similar. Studies conducted so far have elucidated that AFM₁ cause hepatocellular carcinoma in humans and may further increase the incidence rate of carcinoma by 3.3 times among people already suffering with Hepatitis B Virus (Sun et al. 1999). The occurrence of AFM1 in dairy products has drawn attention owing to its heat stability i.e. routine milk processing techniques and storage are ineffective in reducing its levels (Campagnollo et al. 2016). Trace levels of AFM1 may therefore, impose significant health risks to vulnerable population i.e. children, old and ill people. Therefore, contamination of milk with AFM₁ is a serious health concern for the milk consumers.

In order to reduce the toxic and economic impact of mycotoxins and safeguard consumer’s health, several countries have established regulatory limits for occurrence of AFM₁ in milk and milk products. However there is lack of homogeneity among various international regulations. The European Commission (EC) and Food Standards Australia New Zealand (FSANZ) has set the maximum permissible limit (MPL) for AFM₁ in milk at 0.05 µg/L (EC 2006), while the MPL up to 0.5 µg/L has been established for milk and milk products by United States (USFDA) and The Food Safety and Standards Authority of India (FSSAI 2011).

The screening and monitoring of milk products for AFM₁ at very low concentrations requires analytical methods that combine simplicity, reliability with sensitivity and a high analytical throughput. High performance liquid chromatography coupled with fluorescence detector (HPLC-FLD) technique is the most widely used for such analyses owing to its high diagnostic sensitivity and specificity (Kumar et al. 2020; Patyal et al. 2020; Pandey et al. 2021).

It has been reported that raw milk in many parts of India is contaminated with AFM₁ (Nile et al. 2016; Patyal et al. 2020; Sharma et al. 2019; Siddappa et al. 2012). However, information on contamination of milk products with AFM₁ is meagre. Hence, it becomes imperative to determine AFM₁ levels in raw milk as well as in dairy products using a highly accurate, sensitive and specific diagnostic test in order to protect consumers of various age groups from potential health hazards.

Keeping aforementioned facts in mind, the objective of this study was to estimate the levels of AFM₁ in raw and heat treated milk and other dairy products sold in urban and rural retail markets of Chhattisgarh state, India using HPLC-FLD and assess potential human health risks.

Materials and methods

Milk sampling

Assuming prevalence of 5.7% as reported in FSSAI nationwide survey (2018), the study would have required a sample size of 87 for estimating the expected proportion with 5% absolute precision and 95% confidence. After considering multistage cluster sampling with simple random sample collection at each stage and using a design effect of 1.7, a sample size of 146 was required (Dhand and Khatkar 2014). Therefore, a total of 146 samples of milk and milk products were collected from retail and wholesale markets of Chhattisgarh, India during 2019–2020.

The samples of raw milk (n = 46) were collected from milk vendors, milk retailers and dairy outlets. Other milk products of different brands comprising of pasteurized milk (n = 15), UHT plain milk (n = 12), UHT flavored milk (n = 40), milk powder (n = 10), yoghurt (n = 10), and butter milk (n = 13) were procured from retail shops. All milk and milk products samples were collected in sterile, acid washed polypropylene specimen containers, properly labelled and transported to the laboratory in ice jacketed thermo-cooled boxes and maintained at − 80 °C till analysis. All the samples were subjected to thawing prior to sample preparation, and each sample underwent only one freeze–thaw cycle.

Chemicals

AFM₁ standard with > 98% chromatographic purity and celite were procured from Supelco, Sigma-Aldrich, Bellifonte, PA, USA. HPLC grade methanol, acetonitrile and n-hexane and other chemicals of analytical grade were procured from Fisher Chemicals, USA. Standard stock solution of AFM₁ was prepared by diluting appropriate amount of reference standard in acetonitrile to achieve a final concentration of 10 mg/mL. Working standard solution of AFM₁ (0.01, 0.05, 0.1, 0.5, 1.0 mg/mL) were prepared fresh using mobile phase on the day of analysis. Water used was purified by ultra-pureMilli Q water purification system (Millipore Synergy UC France).

Instrumentation and chromatography

AflaM₁™, immunoaffinity columns (VICAM, Watertown, MA, USA) and 12 port vacuum manifold with disposable liners (Visiprep™, Sigma Aldrich, USA) were used for solid phase extraction of the samples. All the samples were analyzed for AFM₁ by Waters® high performance liquid chromatography system (Waters Alliance ® HPLC – e2695 Separation Module, USA) equipped with fluorescence detector (FLD) at excitation and emission wavelength of 355 and 435 nm, respectively. Waters® XBridge® BEH C-18 (250 × 4.6 mm, 5 μm) reverse phase analytical column maintained at 40 °C and isocratic mobile phase consisting of water: acetonitrile (67:33, v/v) with a flow rate of 1 mL/min were used for chromatographic separation of analyte. Instrument control and data analysis of samples was carried out by using Empower® 3 software (Waters Corporation, USA).

Sample preparation

The extraction of AFM₁ from milk (raw, pasteurized, UHT), powdered milk and butter milk samples was carried out according to the protocol recommended by immune-affinity column (IAC) manufacturer. Briefly, temperature of milk and butter milk samples was maintained at 37 °C by placing them in a water bath and then samples were centrifuged at 1000 × g for 5 min. After discarding the top fat layer, samples were filtered through fluted Whatman filter paper No. 1. Skimmed and filtered portion of the sample (50 mL) was then passed through IAC at a rate of about 0.5 mL/s. After washing the IAC with 20 mL of Milli-Q water, AFM₁ was eluted with 5 mL of acetonitrile. Final elute was evaporated to dryness using vacuum concentrator (Concentrator plus, Eppendorf™ AG, Germany). After complete evaporation of the organic solvent, elute were reconstituted in 1 mL of the mobile phase for further HPLC-FLD analysis.

For the extraction of AFM₁ from yogurt samples, the method described by Iqbal and Asi (2013) was followed with some modifications. Briefly, 10 g of yogurt and 10 g of Celite were blended with 80 mL of dichloromethane for 3 min. After centrifugation at 25,000 × g for 5 min, slurry was filtered through Whatman filter paper No. 1 and filtrate was collected and evaporated to dryness using vacuum concentrator. Thereafter, elute were reconstituted in 10 mL of methanol, water and n-hexane mixture (3:5:2, v/v/v) and aqueous phase was separated by using separator funnel. Finally, 5 mL of the aqueous phase of each sample was passed completely through IAC. After washing the IAC with 20 mL of Milli-Q water, AFM₁ was eluted with 5 mL of acetonitrile. Elute was evaporated to dryness in vacuum concentrator and re-dissolved in 1 mL of mobile phase for further HPLC-FLD analysis.

Method validation

The methods validation and quality control study was carried out and all performance parameters viz. sensitivity, trueness, precision, ruggedness, selectivity and specificity etc. were evaluated by taking into consideration the performance criteria and other requirements for analytical methods recommended by the European Commission decision 2002/657/EC (EC 2002). Linearity was evaluated by calculation of five-point linear plots with three replicates based on linear regression and coefficient of determination (R²). LOD and LOQ were determined on the basis of standard deviation of the blank by using following equations:

$$LOD=\frac{3.3X\sigma }{m}LOQ=3\times LOD$$

where, σ = residual standard deviation, and m = slope of calibration curve (Kumar et al. 2018).

Human health risk assessment

To evaluate the health risks from consumption of milk and milk products containing AFM₁ among consumer, health risk assessment was done. The risk assessment was performed by calculating estimated daily intakes (EDIs) based on daily consumption/per-capita availability of milk and milk products Chhattisgarh state i.e. 157 g/person/day (NDDB 2020), body weight of adults (60 kg) and child (15 kg) and the concentrations of AFM₁ detected in the samples using the following equation: $EDI=\frac{C\times R}{W}$

Wherein; C is the mean AFM₁ concentration in milk (µg/L), R is the milk consumption rate or per capita availability of milk, and W is mean human body weight.

The EDI so calculated was then compared with established TDI (Tolerable daily intakes). There is always a risk for liver cancer following the intake of AFM_1. Therefore, by considering cancer potency factors (CPF) of 3 × 10^–7 and 1 × 10^–8 per year per ng of AFM₁ per kg body weight per day in HBsAG⁻ (Hepatitis B surface antigen negative) and HBsAg⁺ (Hepatitis B surface antigen positive) adults and children, respectively, the additional hepatic cancer risk was assessed using the equation: $PopulationRisk=EDI\times Cancerpotencies$.

Statistical analysis

Statistical Package for the Social Sciences (SPSS, version 20.0) analytical software for windows was used for all statistical analysis. Concentrations of AFM₁ in milk and milk products samples were summarized using means, standard error together with minimum and maximum values. One-way ANOVA with Post-hoc Tukey’s test was applied to check the significant difference between levels of AFM₁ in various milk and milk products under study. A p-value of < 0.05 was considered as statistically significant.

Results and discussion

Method quality control

The five point AFM₁ standard solutions calibration curve for the HPLC-FLD method exhibited good linearity (R² = 0.999) over the concentration range of 0.01–1.0 µg/L. The limit of detection (LOD) and limit of quantification (LOQ) for the method were fund to be 0.009 µg/L and 0.027 µg/L, respectively. Both LOD and LOQ were found to be well below the MRLs established by FSSAI and EC for targeted AFM₁. Recovery experiments were carried out by conducting tests on each of known blank samples of milk, milk powder, yogurt and buttermilk (three replicates each) fortified with AFM₁ standard at 0.05, 0.1, 1.00 µg/L. The mean recovery percentages of AFM₁ in fortified milk, milk powder, yogurt and buttermilk samples were found to be 80–86%, 76–84%, 79–83%, and 80–85% with precision (%RSD) of 8–13%, 10–16%, 9–17%, and 6–14%, respectively, which are well within the EC criteria of 80–120% (recoveries) and < 20% (% RSD), respectively, (Table 1). Further, analyses of blank samples showed that there were no interference peaks around the retention time window of AFM₁ (Fig. 1, 2 and Online Resource 1).

Table 1.

Recovery percentage of AFM₁ in milk and milk products

Fortification levela (µg/L)	Milk		Milk powder		Yogurt		Buttermilk
	Recovery (%)	RSD (%)	Recovery (%)	RSD (%)	Recovery (%)	RSD (%)	Recovery (%)	RSD (%)
0.05	80	11	76	13	83	17	80	11
0.1	82	8	84	10	79	13	85	6
1.0	86	13	81	16	82	9	81	14

^aThree replicates at each level

Fig. 1.

Representative HPLC-FLD chromatograms of blank milk sample

Fig. 2.

Representative HPLC-FLD chromatograms of milk sample spiked with AFM₁

Since, the overall validation data exhibited acceptable results for the evaluated performance parameters, therefore, optimized method was applied for analyzing collected milk and milk product samples.

Detection of AFM₁ in real samples

The occurrence of mycotoxins in milk has become an emerging public health issue. In the current investigations, out of a total of 146 milk and milk product samples tested, 52 samples (35.6%) were found to be contaminated with AFM₁ with mean concentration of 60.9 ± 42.8 µg/L in positive samples. Out of 52 positive samples, 49 (94.2%) were found to contain AFM₁ above maximum permissible limits established by the European Commission. It was observed that all positive samples of pasteurized milk, UHT milk, milk powder, and fermented milk products exceeded the MPLs legislated by EC. However, as per FSSAI, only 21 (40.4%) positive samples exceeded the established limits. This is due to non-homogeneity on established limits among these food safety regulations. Since, European commission enforces more stringent food safety regulations, therefore, most of the positive sample exceeded the limits established by EC. However, even though the FSSAI have established relatively higher limits for AFM₁ occurrence in milk and milk products, the presence of AFM₁ above MPLs in approximately 40% of the samples is alarming and require interventions by the stakeholders.

In raw milk samples (n = 46) analyzed, AFM₁ were found in 41.3% samples (n = 19) with the mean concentration of 0.273 ± 0.092 µg/L with 84.2% and 31.6% samples exceeding the permissible limits recommended by EC and FSSAI, respectively (Table 2). Our findings are in agreement with the reports of Nile et al. (2016) from Maharashtra, India, where 45.3% of the raw cow milk samples were found to be contaminated with AFM₁. However, the detection frequency of AFM₁ in raw milk observed in the present study is relatively less in comparison to some of the previous studies conducted from India. Siddappa et al. (2012) reported that 100% of raw milk samples from Karnataka and Tamil Nadu contained AFM₁. From Punjab, AFM₁ was detected in 60% of the raw milk samples (Patyal et al. 2020). The higher frequency of detection in raw milk may be associated with poor storage of feed and fodder and their subsequent contamination with AFB₁. As, we know a good quality finished product can never be made from poor quality raw product. Therefore, the high contamination frequency of raw milk require immediate attention of veterinarians, public health professionals and food safety regulators to protect consumers.

Table 2.

Occurrence and levels of AFM₁ in milk and milk products

Sample type (n)	Positive samples (%)	Mean concentrations of AFM1 ± SE (µg/L)	Range (µg/L)	Samples exceeding EC limit (%)	Samples exceeding FSSAI limit (%)
Raw milk (n = 46)	19/46 (41.3)	0.273 ± 0.092a	ND-2.913	16/19 (84.2)	6/19 (31.6)
Pasteurized milk (n = 15)	6/15 (40)	0.278 ± 0.104a	ND-1.212	6/6 (100)	4/6 (66.7)
Plain UHT milk (n = 12)	5/12 (41.7)	0.416 ± 0.169a	ND-1.523	5/5 (100)	4/5 (80)
Flavored UHT milk (n = 40)	13/40 (32.5)	0.135 ± 0.037a	ND-0.730	13/13 (100)	6/13 (46.2)
Milk powder (n = 10)	2/10 (20)	0.486 ± 0.426a	ND-2.608	2/2 (100)	1/2 (50)
Yogurt (n = 10)	3/10 (30)	0.073 ± 0.048a	ND-0.303	3/3 (100)	0/3 (0)
Buttermilk (n = 13)	4/13 (30.8)	0.061 ± 0.503a	ND-0.308	4/4 (100)	0/4 (0)
Total (n = 146)	52/146 (35.6)	-	-	49/52 (94.2)	21/52 (40.38)

Mean values in the same column with same superscript letters denote non-significant differences (Tuckey post hoc test, p > 0.05)

n = number; SE: Standard Error; ND: Not detected

Among heat treated milk samples, 40% of pasteurized milk samples (with mean concentration of 0.278 ± 0.104 µg/L), 41.7% of plain UHT milk (0.416 ± 0.169 µg/L) and 32.5% of flavored UHT milk (0.135 ± 0.037 µg/L) samples were found positive for AFM₁ (Table 2). Indescribably, all the heat treated milk samples (100%) exceeded the maximum permissible limits established by EC. The results of present study are comparable with the findings of Siddappa et al. (2012), who reported AFM₁ contamination of 42.9% of pasteurized milk samples, 100% of plain UHT milk and 37.5% flavored UHT milk samples. In a similar study, Kanungo and Bhand (2014) analyzed 54 pasteurized milk samples from Goa, India and found 100% positive for AFM₁. Likewise, 41% of pasteurized milk samples and 47% plain UHT milk samples collected from Punjab, India were also found to be contaminated with AFM₁ (Patyal et al. 2020). The occurrence of AFM₁ in heat treated milk has also been reported from other countries such as Lebanon (El-Khoury et al. 2011), Egypt (Tahoun et al. 2017), Portugal (Duarte et al. 2013), Pakistan (Iqbal et al. 2017), Turkey (Kocak et al. 2015) and Iran (Mashak et al. 2016).

The presence of AFM₁ in heat-treated milk with concentration above maximum permissible limits may be attributed to heat stability of AFM₁. AFM₁ has been considered as one of the major important xenobiotic compounds detected in heat treated milk and milk products because of its high heat stability during different processing such as pasteurization and UHT treatment etc. (Prandini et al. 2009).

In milk powders, the AFM₁ resides have been detected earlier by Kanungo and Bhand (2014) from Maharashtra and Rastogi et al. (2004) from Lucknow, India. In concordance with the previous findings, our results also demonstrated that 20% of milk powder samples contained AFM₁ with mean concentration of 0.486 ± 0.426 µg/kg (Table 2). The present detection frequency is less in comparison to previous reports. However, the mean concentration in the range of nd-2.608 µg/kg was observed for milk powder samples which is highest among all the samples analyzed. This might be attributed to the concentrated form of milk powder.

Among the fermented milk products analyzed, 30% of yogurt samples and 30.8% of the buttermilk samples were found to be positive for AFM₁ with the mean concentration of 0.073 ± 0.048 µg/L and 0.061 ± 0.503 µg/L, respectively (Table 2). All the positive yogurt samples exceeded the EC MPL of 0.05 µg/L. Butter milk is popularly known as ‘Lassi’ in India, which is a traditional fermented dairy product prepared in Indian households and sold in markets of India. However, despite its huge sale and likeability by the Indian people, the maximum permissible limits have not yet been established for it. However, critical perusal of the results revealed that the mean concentration level are quite high and therefore not acceptable from human health perspective.

For comparisons of the results obtained for AFM₁ occurrence in fermented milk products, only a couple of international reports are available in published literature. Regarding the presence of AFM1 in yogurt, the results of current investigation corroborate with the previous studies by El-Khoury et al. (2011) who reported that 33% of yogurt samples in Lebanon contained AFM₁. In contrary, higher contamination frequency (75–95%) have been observed in earlier studies conducted from Brazil (Iha et al. 2011), Turkey (Sarica et al. 2015) and Qatar (Hassan et al. 2018).

Since. buttermilk is obtained after churning yogurt and no other procedure is involved in its preparation, therefore, statistically no significant difference was observed in their positive percentage. Results of current study also revealed that fermented milk products have comparatively lower AFM₁ level as compared to liquid milk and milk powder samples (Table 2). This may be attributed to the action of fermenting lactic acid bacteria present in it (Iqbal and Asi 2013). During fermentation process pH of fermented milk products decreases up to 4—4.5, thus that may lead to denaturation and coagulation of casein protein which can further affect the AFM₁ adsorption in these milk products (Fallah et al. 2011).

Human health risk assessments

The health risks associated with consumption of milk and milk products containing AFM₁ were evaluated by considering the residue analysis results obtained in the present study and daily consumption of milk and milk products in the study area. Hazard Index (HI) model was used to assess toxicological significance of human exposure to AFM₁ detected in samples, wherein, the calculated EDIs for both adults (body weight 60 kg) and children (body weight 15 kg) were compared with the established tolerable daily intake (TDI) value established by WHO/FAO organizations, as shown in Table 3.

Table 3.

Human health risk assessment

Sample type	Age Groupa	Risk Assessment
		EDI	Hazard Indexb	Carcinogenic riskc
				HBsAg +	HBsAg-
Raw milk	Adult	0.714	3.572	2.14E-07	7.14E-09
	Children	2.857	14.287	8.57E-07	2.86E-08
Pasteurized milk	Adult	0.727	3.637	2.18E-07	7.27E-09
	Children	2.910	14.549	8.73E-07	2.91E-08
Plain UHT milk	Adult	1.089	5.443	3.27E-07	1.09E-08
	Children	4.354	21.771	1.31E-06	4.35E-08
Flavored UHT milk	Adult	0.353	1.766	1.06E-07	3.53E-09
	Children	1.413	7.065	4.24E-07	1.41E-08
Milk powder	Adult	1.271	6.359	3.82E-07	1.27E-08
	Children	5.087	25.434	1.53E-06	5.09E-08
Yogurt	Adult	0.191	0.955	5.73E-08	1.91E-09
	Children	0.764	3.820	2.29E-07	7.64E-09
Butter Milk	Adult	0.160	0.798	4.79E-08	1.60E-09
	Children	0.638	3.192	1.92E-07	6.38E-09

^a Adult = 60 kg body weight, and children = 15 kg body weight

^b Hazard Index (HI) = EDI/TDI, TDI = 0.2 ng/kg b.wt/day

^c Carcinogenic risk = EDI × Cancer potency factor (1 × 10^–8 for HBsAg⁻and 3 × 10^–7 for HBsAg⁺consumers)

The EDIs of the AFM₁ for both adults and children were found to be higher than the ADIs i.e. hazard index more than unity, except for yogurt consumption in adults. This indicates that consumers especially the children are significantly exposed to health risk associated with AFM₁ through consumption of milk and milk products in study area. Onyemelukwe et al. (2012) in their study from Nigeria made similar observations regarding higher vulnerability of children to protein energy malnutrition after exposure to AFM1 through consumption of contaminated milk.

Furthermore, it is emphasized that there is always an additional risk for hepatic cancers associated with exposure to AFM₁. Therefore, carcinogenic risk among HBsAg⁻ (Hepatitis B surface antigen negative) and HBsAg⁺ (Hepatitis B surface antigen positive) was also assessed by considering cancer potency factor of 3 × 10^–7 and 1 × 10^–8 per year per ng of AFM₁ per kg body weight per day in adults and children, respectively.

The risk of hepatic cancer in HBsAg⁺ and HBsAg⁻ adults ranged from 4.79 × 10^–8 to 3.82 × 10^–7 and from 1.60 × 10^–9 to 1.27 × 10^–8, respectively. The liver cancer risk among children with HBsAg⁺ and HBsAg⁻ ranged from 1.92 × 10^–7 to 1.53 × 10^–6 and from 6.38 × 10^–9 to 45.09 × 10^–8, respectively (Table 3). Although, there is negligible risk of liver cancer in consumers of study area, but the presence of AFM₁ in milk and milk products may definitely acts as one of the contributory factor in predisposing people to future health risks. However, the current findings does not discourage people from consumption of milk and milk products. But, there is an urgent need for the consumers to be aware on consumption of only certified food products available in the market.

Conclusions

Present study was conducted to assess the contamination level of AFM₁ in milk and milk products and the potential risk posed to consumers. The investigation provides useful data on contamination status of milk and milk products in Chhattisgarh, India. The findings indicated that dairy product processing has non-significant effect on AFM₁ concentrations and therefore, higher levels of AFM₁ in raw milk may result in rejection of various value added milk products. This not only affects the dairy economy but also poses a significant human health risk. Therefore, there is a need for constant monitoring throughout the milk production chain in order to minimize health risk associated with AFM₁ present in milk and milk products.

Authors' contributions

SS and AP conceived the study design; DH and CC carried out the laboratory work; AK analyzed the data and performed health risk assessments; DH and CC drafted the manuscript; AK and AP revised and edited the manuscript. All authors read and approved the final manuscript.

Funding

Not applicable.

Declarations

Conflicts of interest

The authors declare that they have no competing interests.

Data availability

All data generated or analysed during this study are included in this published article [and its supplementary information files].