An Assessment and Control of AFM₁ in Milk and Main Dairy Products in Lahore, Pakistan

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.

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 AFB_1, hydroxylation reaction is occurred on tertiary carbon of difuran ring system which yields AFM₁ [5, 6, 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 AFM_1. 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.

2. Material and methods

2.1 Sample collection

Sampling strategy is comprehensively discussed in Table 1. After collection, samples were transported in icebox to University of Veterinary and Animal Sciences, Lahore where these were stored at −4°C till further processing.

Sample	Sample category
Milk (n = 60)	10 ml unprocessed milk from local milk shop (n = 20)
	10 ml unprocessed milk local dairy farm (n = 20)
	10 ml processed milk sample from a commercial shop (n = 20)
Butter (n = 30)	50 g Processed butter samples (n = 15)
	50 g unprocessed butter samples (n = 15)
Cream (n = 30)	50 g processed cream samples (n = 15)
	50 g unprocessed cream samples (n = 15)
Cheese (n = 30)	50 g processed cheese samples (n = 15)
	50 g unprocessed cheese samples (n = 15)
Yogurt (n = 30)	50 g processed yogurt samples (n = 15)
	50 g unprocessed yogurt samples (n = 15)

Table 1.

Sampling strategy.

2.2 Sample processing

2.2.1 Processing of milk samples through rapid test kit

Rapid test kit detects AFM₁ to the limit of 0.5 ng/ml-ppb. The kit was used according to manufacturer instructions. Briefly, 200 μL milk samples were pipetted into reagent microwell and after mixing, these samples were incubated at room temperature for 2 minutes. Then dipstick was inserted in each sample and incubated for another 5 minutes at the same temperature. After that dipstick was taken out from each sample and samples were interpreted. There would be two lines i.e. test line (T) and control line (C) on dipstick. If T > = C then sample was considered negative while if T > C or there was no test line then it was considered as positive.

2.2.2 Processing of samples of dairy products through HPLC

1. Chemicals and Reagents:

   AFM₁ standards (10 μg/l in acetonitrile), Celite and HPLC grade acetonitrile of Sigma–Aldrich, Steinheim, Germany and Immunoaffinity column Afla^Tm of VICAM, USA were used.

   AFM₁ standard curve or linearity curve was prepared by diluting the standards with acetonitrile at 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 μg/ml concentrations and stored in caped vials in refrigerator at – 4 C.

2. Samples Extraction:

   5 g sample of each products and 5 g Celite mixed with 40 ml of dichloromethane in a 50 ml falcon tube. Then centrifuged at 21,000 rpm for 5 mins. After centrifugation the supernatant was separated and evaporated in water bath at 80 C. After evaporation the beaker was shifted in ultrasonic clean-up for 5 min. Then residues in beaker were dissolved in 10 ml mixture of methanol, water and n-hexane with the ratio of 3:5:2. Then 15 ml of this solution mixed by vortex mixture and again centrifuged at 21,000 rpm for 5 mins. After this, aqueous filtrate was passed through immunoaffinity column. The column was washed with 10 ml of water to remove toxins. After this column again washed with 2.5 ml of acetonitrile to get the final extract. This extract then dry under nitrogen steam at 40 C. After evaporation residues were dissolved with 2 ml mobile phase and mix by using vortex mixture. Finally, 20 μ/l sample was injected in HPLC for the analysis.

3. HPLC Conditions:

   The HPLC used for the analysis was a Shimadzu LC-10A series (Japan) with the fluorescence detector (HPLC-FLD) having excitation wavelength of 365 nm and emission wavelength of 435 nm.

2.3 Therapeutic trial

Three different toxin binders were used in respective groups A, B, and C each having 10 AFM₁ positive animals. The Table 2 showed types of toxin binder and their dose rates. Each toxin binder was used on the daily basis for 7 days.

Groups	Type of toxin binders	Dose rate
A (n = 10)	Clay based toxin binder	100 g/40 Kg feed
B (n = 10)	Whole yeast-based toxin binder	1 g/40 Kg feed
C (n = 10)	Yeast (75%) + Algae (25%)	10 g/40 Kg feed

Table 2.

Therapeutic trials in different groups.

2.4 Sample collection and processing after therapeutics

At days 2nd, 3rd, 4th, and 7th 10 ml milk samples from each animal were collected in plain vacutainers and serum was extracted through centrifugation. After that milk samples were checked by using AFM₁ rapid test kit.

2.5 Statistical analysis

Collected data will be statistically scrutinized by SPSS 20.0 software and t-test as well as Chi square test was used to analyze the results.

3. Results and discussion

The results of this study were comprehensively described in Tables 3 and 4. Figure 1 showed the linearity curve of AFM₁ standard concentrations of 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 μg/ml. The method showed linear response R² = 1.

Type of Sample	Group	Mean ± S.D	Range of Conc. of Aflatoxin M1 in ppb	Samples Exceeding EC limits (0.05 ppb)
Butter	Processed	.0900 ± .18000	.36	(5/30) 16.7%
	Local	.0000 ± .00000	.00
	Total	.0450 ± .12728	.36
Cheese	Processed	.3500 ± .60622	1.05	(10/30) 33.3%
	Local	.0000 ± .00000	.00
	Total	.1750 ± .42866	1.05
Cream	Processed	.0000 ± .00000	.00	(4/30) 13.3%
	Local	.5425 ± 1.08500	2.17
	Total	.2713 ± .76721	2.17
Yogurt	Processed	.5525 ± 1.00118	2.05	(8/30) 26.6%
	Local	.0000 ± .00000	.00
	Total	.2763 ± .71889	2.05

Table 3.

HPLC results of aflatoxin M₁ in dairy products.

Time of sampling	After 2 days
Groups	Positive	Negative	Percentage	p-value (p < 0.05)
A	10	0	100%	0.000
B	9	1	90%
C	3	7	30%
Time of sampling	After 3 days
Groups	Positive	Negative	Percentage	p-value (p < 0.05)
A	9	1	90%	0.000
B	7	3	70%
C	0	10	00%
Time of sampling	After 4 days
Groups	Positive	Negative	Percentage	p-value (p < 0.05)
A	9	1	90%	0.000
B	6	4	60%
C	0	10	00%
Time of sampling	After 7 days
Groups	Positive	Negative	Percentage	p-value (p < 0.05)
A	8	2	80%	0.000
B	6	40	60%
C	0	100	00%

Table 4.

Efficacy of different toxin binders in groups A, B, and C.

Figure 1.

Linearity curve of aflatoxin M₁ standard concentrations.

Table 3 shows the level of AFM₁ of all dairy products that exceeds the tolerable limits (0.050 μg/kg) established by European Commission Regulation.

Our results concluded that 33.3% of processed butter sample showed positive recovery of AFM₁ with range concentration above EU limits (0.050 μg/kg) while no local butter sample with AFM₁ toxicity was found (Table 3). These results are only in agreement with a study conducted by Fallah et al. [16] who analyzed 31 butter samples and got 25.8% AFM₁-positive ones with range above permissible limits established by EU.

The similar trend was found in case of processed cheese samples which were found contaminated (33.3%) with AFM₁ (Table 3) whereas in another study the AFM₁ concentration was found much higher i.e., 78% [15]. There are many other such studies having positive percentages of AFM₁ higher than our results [16, 17, 18].

Similarly, analysis of processed and unprocessed yogurt samples showed that 26.6% of former were contaminated with AFM₁ above EU permissible limit whereas no sample in latter was found affected with these aflatoxins. Our results showed low positive percentage than documented by Iqbal and Asi [15] who found 59 AFM₁-positive samples. Many other such studies also showed higher incidents of AFM₁ in yogurt samples than our study [19, 20, 21]. Lastly, results of unprocessed cream samples showed the highest concentration of AFM₁ whereas no aflatoxin was detected in processed cream samples as shown in Table 3. These results have not only local impacts but high impact at global level too. Because at global level, particularly in underdeveloped and developing countries, the topic of aflatoxins in dairy sector impacting humans as well as animals is a neglected topic.

During the second phase of the study, milk samples collected on 2nd, 3rd, 4th, and 7th day showed significantly different efficacies of three toxin binders in groups A, B, and C. It was found that toxin binder used in group C had significantly higher (<0.05) efficacy as compared to those used in groups A and B. This toxin binder C had yeast wall (75%) in combination with algae (25%) so it showed best results and eradicated AFM₁ from all the animals after 48 h. The reason behind is the main role of yeast wall in the whole yeast which binds with mycotoxin and its binding ability is catalyzed by algae so the product having this combination provided the best results. Whereas, the clay-based toxin binder used in group A showed comparatively worst results in controlling AFM₁ due to their lack of binding with these mycotoxins as described by Chestnut et al. [22]. Other disadvantages associated with clay-based toxin binders are their probable interaction with the essential nutrients [23] and high inclusion rates [24]. On the other hand, whole yeast-based toxin binder showed significantly lower efficacy as compared to group C and higher efficacy as compared to group A. The reason behind would be that whole yeast alone does not have good binding ability with mycotoxins so it also failed to control AFM₁, comparatively. The whole findings are summarized in Figure 2.

Figure 2.

Positive percentage in each group after offering toxin binders.

4. Conclusion and recommendations

The results of this study confirmed that processed and unprocessed milk and main dairy products i.e. butter, cheese, cream, and yogurt has significantly (p < 0.05) higher contamination of AFM₁ than EU permissible limit. Moreover, toxin binder having yeast wall (75%) and algae (25%) showed significantly (p < 0.05) higher efficacy to control AFM₁ in milk samples. The study depicts alarming situation of AFM₁ problem and stresses the need of establishing permissible limits of AFM₁ by the regulatory bodies to avoid their adverse effects on public health in Pakistan as well as in other developing countries. It is noted with grave concern that the issue of aflatoxins is one of the most discussed topic at global level but it neglected in underdeveloped countries. So, this study emphasizes the need of research on the impact and control of aflatoxins in milk, and other dairy products which are consumed by human beings.

Declaration

The authors declare no conflict of interest.