**Abstract**

The main objective of this study is to investigate the presence of Aflatoxin M1 (AFM1) in local and processed milk and main dairy products available in Lahore. Total 60 milk samples and 120 samples of dairy products including butter (n = 30), cheese (n = 30), cream (n = 30), and yogurt (n = 30) were collected. Milk samples were collected from 3 different sources i.e. unprocessed milk from local milk shop (n = 20) and a local dairy farm (n = 20), and processed milk sample from a commercial shop (n = 20) while samples of each dairy product were also different i.e. processed (n = 15) and unprocessed (n = 15). Milk samples were analyzed using kit method while dairy product samples were analyzed by high performance liquid chromatography (HPLC) technique equipped with fluorescence detector (HPLC-FLD) followed by immunoaffinity column clean up. In second phase of the study, efficacy of three different toxin binders was compared and analyzed. The results showed that AFM1 was detected in 16.7% of processed butter samples, 33.3% of processed cheese samples, 13.3% of local cream samples and 26.6% of processed yogurt samples and these samples exceeds European Union (EU) permissible limits of 0.05 ppb with mean concentration 0.090 ± 0.180 μg/kg and 0.000 ± 0.000 μg/kg for processed and local butter samples, 0.350 ± 0.606 μg/kg and 0.000 ± 0.000 μg/kg for processed and local cheese samples, 0.000 ± 0.000 μg/kg and 0.542 ± 1.085 μg/kg for processed and local cream samples and 0.552 ± 1.001 μg/kg and 0.000 ± 0.000 μg/kg for processed and local yogurt samples, respectively. Moreover, milk samples showed highest AFM1 (62%) in local unprocessed dairy farm followed by samples from local milk shop (51%) and commercial dairy farm (31%). In addition, therapeutic efficacy of three different types of toxin binders showed that the toxin binder which had yeast wall (75%) and algae (25%) is the best to control AFM1 under field conditions. Overall, results of this study are valuable for dairy farmers on one hand and law enforcement authorities on the other to comprehend and control AFM1 problem in milk and main dairy products.

**Keywords:** AFM1, Milk, Dairy products, HPLC, Toxin binders

### **1. Introduction**

Milk and dairy products both are vital part of human nutrition and ideal sources of nutritional components because of their biochemical complexity for supplying

essential mixture of proteins, vitamins, calcium, amino acids and antioxidants [1]. Pakistan is 4th largest milk producing country in the world and produces 45 billion liters per year [2]. The extensive and vast dairy industry of Pakistan faces a lot of problems including Aflatoxin M1 (AFM1). AFM1 are playing negative impacts on animal production as well as dangerous for human health [3, 4]*.*

AFM1 is a monohydoxylated product of Aflatoxin B1 (AFB1). When lactating mammals consume AFB1 contaminated feed then production of AFM1 becomes enhanced. After ingestion of AFB1, hydroxylation reaction is occurred on tertiary carbon of difuran ring system which yields AFM1 [5–7]. The biotransformation frequency of AFB1 to AFM1 in excreted milk is different in all lactating animals. But AFM1 start producing in milk within 12 – 24 hours after AFB1 ingestion from feed [8].

80% people especially children consume dairy products as an important part of their diet [9]. But it is a dejected reality that dairy products are compromised badly because of mycotoxins. These fungal toxins not only destroy these dairy products but also produce dreadful disease which causes chronic diseases in the consumer. The World Health Organization (WHO) has endorsed the depletion of Aflatoxins in food by establishing tolerable limits for Aflatoxins to fulfill Farm-to-Fork principle.

International Agency for Research on Cancer (IARC) which is specialized cancer agency of World Health Organization (WHO) has categorized aflatoxins B1 and M1 into group 1 carcinogens [10, 11]. Therefore, different international organizations and countries have established standards for AFM1. The European Commission Regulation 1881/2006 set permissible limits for AFM1 in milk and dairy products of 0.050 μg/kg [12, 13]. According to Codex Alimentarius Commission permissible level of AFM1 in butter is 50 μg/kg and in cheese is 250 μg/kg [14]. So, permissible limits of AFM1 vary in milk and dairy products in different countries. But many countries including Pakistan also have no proper safety and regulatory limits and levels for AFM1 in milk and dairy products [15] which may be due to oversight of policy makers with negligible research on aflatoxins.

Hence, this study was designed to gauge AFM1 problem in milk and dairy products in and around Lahore from different sources. In the second phase of the study, efficacy of three different toxin binders were compared in a local dairy farm. The outcomes of the study will help dairy farmers on one hand and law enforcement agencies on the other to understand the gravity of AFM1 problem in milk and main dairy products, and formulate strategies to control it.
