Assessment of Milk Quality by Analyzing Aflatoxin M1 in Branded and Non Branded Milk Samples Collected from Different Shops/Areas in Lahore, Pakistan

Naseem Zahra1,2,*, Nadia Jamil1, Sajid Rashid Ahmad1, Imran Kalim2, Muhammad Khalid Saeed2, Ijaz Ahmed2, Qurat-ul-ain Syed2 and Soniya Munir1

1College of Earth and Environmental Sciences, University of the Punjab, Lahore

2Food and Biotechnology Research Centre, PCSIR Laboratories Complex, Ferozepur Road, Lahore-54600

ABSTRACT

In the present study a total of 80 samples were analyzed for Aflatoxin M1 using competitive ELISA kit method. Branded samples (n=40) were collected from different shops in Lahore city of Punjab Province while non-branded samples (n=40) collected from four selected towns of Lahore. Overall 58% and 100% of aflatoxin contamination in branded and non-branded milk samples were found, respectively. The quantities of AFM1 in 58% of branded milk samples and 95% of non-branded milk samples were found beyond permissible limits of 50 ppt as set by European Union. The most pathetic condition of non-branded milk samples was found in Shalimar Town of Lahore which showed highest levels of aflatoxin M1 i.e. 2735.08 ppt.

Article Information

Received 04 June 2017

Revised 12 May 2018

Accepted 04 February 2019

Available online 26 September 2019

Authors’ Contributions

NZ, NJ and SRA designed the study. NZ and IK conducted the experiments. MKS and IA collected the samples. QAS reviewed the article. SM analyzed the data. NZ wrote the article.

Key words

Aflatoxin B1, M1, Milk, Contamination, ELISA.

DOI: http://dx.doi.org/10.17582/journal.pjz/2020.52.1.sc2

* Corresponding author: [email protected]

0030-9923/2020/0001-0373 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

Aflatoxins are toxins produced by different species of Aspergillus especially flavus and parasticus. International Agency of Research on Cancer (IARC, 2002) has also placed aflatoxins among powerful carcinogens. Aflatoxin M1 is a hydroxylated metabolite of aflatoxin B1. Cytochrome oxidase system of P450 present in the microflora and animal cells is responsible for conversion of aflatoxin B1 to M1 (Fallah et al., 2009; Unusan, 2006).

In European Union countries, the allowable limits of AFM1 in milk are 0.05 ppb and 0.025 ppb consumed by adults and infants, respectively while in the United States, the permissible limit of aflatoxin M1 in milk is 0.5 ppb (Kyprianou, 2007; Battacone et al., 2009).

In present study the objective was to check the levels of aflatoxin M1 in different branded and non branded milk samples by using ELISA method.

Materials and methods

The non-branded samples were collected from retail milk shops of city Lahore, Pakistan. Forty samples of raw milk (500 ml each) were collected randomly from different markets of Samanabad Town, Iqbal Town, Shalimar Town and Gulberg Town during October, 2016 to March, 2017. 10 samples from each town were collected. Similarly, branded samples were also collected from different shops in Lahore. The milk samples were transported in sterilized polythene bags in ice-packed cooler to the aflatoxin laboratory at PCSIR Laboratories Complex, Lahore, where samples were kept at -20°C until analyzed for AFM1 detection (Muhammad et al., 2010). Samples were prepared by using Romer Labs Method #: PI-000047-1. Fresh milk sample (5 ml) was taken into test tubes and incubated at 4°C for 30 min. The samples were centrifuged at 3000 g for at least 10 min. Milk serum (0.4 ml) below fat layer was taken and mixed with 0.1mL of 100% methanol i.e. in the ratio is 4:1. ELISA kit (Romer Labs, Singapore), was used for the examination of M1 in milk samples (Kamkar et al., 2011).

Results and discussion

Aflatoxin M1 was determined by ELISA method in branded and non-branded milk samples supplied in Lahore city of Pakistan. The percent maximum absorbance at 450 nm was observed for aflatoxin M1.

In present study it was determined that 58% of branded samples collected from Lahore shops were infected with aflatoxin M1, respectively. In a similar studies the occurrence of AFM1 contamination in the raw milk (non branded) samples collected from Gulberg Town, Ravi Town, Gunjbaksh Town and Shalimar Town was 71%, 86%, 81% and 86%, respectively (Muhammad et al., 2010). The results of aflatoxin M1 detection in different 40 branded milk samples are given in Table I.

Table I.- Quantity of aflatoxin M1 in branded milk samples.

*ND, not detected; MQ (ppt) ± SD, mean quantity ± standard deviation.

The branded sample (B29) was found with maximum concentration (605.90 ppt) of aflatoxin M1 beyond EU permissible limits i.e. 50 ppt. It was observed that all 40 raw milk (non-branded) samples collected from different shops of selected Towns were contaminated with aflatoxin M1. 100% contamination of milk samples show miserable conditions of supplied milk at Lahore shops. The results of aflatoxin M1 analysis in non-branded milk samples are given in Table II.

The sample with maximum concentration 2735.08 ppt (exceeding permissible limits set by EU) of aflatoxin M1 was also collected from Shalimar Town. In comparison with present study of aflatoxin M1 in non-branded milk samples range between 17.34-2735.08 ppt, the analysis of randomly selected raw cow milk samples in North African countries (Elgerbi et al., 2004), were contaminated with AFM1 (range between 30 and 3130 ppt). These results are in accordance with Elgerbi et al. (2004) and El-Sayed et al. (2000) who reported high levels of AFM1 in bovine raw milk samples. The percentage analysis of non-branded milk samples regarding aflatoxin M1 contamination showed that all the samples were highly infected with aflatoxin M1 among which 95% of the samples had aflatoxin M1 levels beyond permissible limits i.e. 50 ppt.

Table II.- Quantity of aflatoxin M1 in non-branded milk samples.

MQ (ppt) ± SD, mean quantity ± standard deviation.

The percentage analysis of branded milk samples showed that 42% samples were not contaminated and found no aflatoxin M1 while 58% samples were aflatoxin M1 contaminated. Among these contaminated samples all the samples contained aflatoxin M1 levels beyond permissible limits.

In a study conducted on buffalo and cow milk samples collected from dairy farms of Faisalabad, Pakistan. 84% buffalo milk samples and 72% cow milk samples were exceeded the European Commission MRL of 50 ppt (Sajid et al., 2015). Fungal metabolite AFM1 is carcinogenic, hepatotoxic and immunosuppressive and is accountable for injurious effects on human as well as animal’s health (Williams et al., 2004). This adverse scenario has dragged the international trepidation over AFM1 contamination in milk supplied.

Conclusion

In current study the analysis depicts that 58% of branded samples while 100% of non-branded milk samples were found contaminated with Aflatoxin M1, respectively. HACCP system and analytical inspection are highly recommended to limit aflatoxin M1 contamination in order to fearless consumption of milk in Pakistan.

Statement of conflict of interest

The authors declare no conflict of interest.

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