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Nutrition & Food

Sharaf, J Nutr Food Sci 2013, 3:2 http://dx.doi.org/10.4172/2155-9600.1000195

Sciences Research Article

Open Access

Aflatoxin M1 in Raw, Pasteurized and Baby Infant Formula Milk Available in Jordanian Market Omar S Sharaf* Department of Nutrition and Food Processing, Faculty of Agricultural Technology, Al-Balqa Applied University, Salt, Jordan

Abstract The incidence of contamination of aflatoxin M1 (AFM1) in milk samples collected from the Jordanian market was investigated by using the competitive Enzyme Linked Immunosorbent Assay (ELISA) technique. A total of 175 samples composed of raw cow milk (50), raw sheep milk (20), raw goat milk (20), raw camel milk (10), pasteurized cow milk (30), evaporated milk (10), full cream powdered milk (15), and baby infant formula milk (20), were collected during the 2011-2012 year. All tested samples were contaminated with various levels of AFM1, ranging from 9.71288.68 mg/kg. The concentration of AFM1 in 66% of fresh milk samples was higher than the maximum tolerance limit accepted by the European Union (50 mg/kg), and 23% higher than the maximum tolerance limit accepted by USA (500 mg/ kg). Percentages of contaminated raw cow, sheep, goat and camel milk exceeding the European tolerance limit were 60%, 85%, 75% and 0%, respectively. 12% of AFM1 contaminated pasteurized cow milk samples exceeded the European tolerance limit, with a range of contamination between 14.60 and 216.78 ng/kg. For baby infant formula samples, the average concentration of AFM 1 was 120.26 ng/kg.

Keywords: Aflatoxin M1; Raw animal milk; Pasteurized milk; Baby infant formula; ELISA; Jordan Introduction Aflatoxins are a group of naturally occurring toxins produced mainly by molds, such as Aspergillus flavus and Aspergillus parasiticus [1-3]. Aflatoxin M1 and M2 are the hydroxylated metabolites of aflatoxin B1 and B2 [4,5], and may be found in milk products obtained from livestock that have ingested contaminated feed [6-9]. This metabolite has been found to be a major excretion product in the milk of lactating animals exposed to dietary aflatoxin B1 [10-12]. The toxicity of aflatoxin M1 is about one order of magnitude less than that of aflatoxin B1 [2]. Aflatoxin M1 could be detected in milk, 12–24 h after the first aflatoxin B1 ingestion, reaching a higher level after a few days. As milk is the main source of nutrient for infants and children who are considered to be more susceptible to adverse effects of mycotoxins, the presence of aflatoxin M1 in milk is carcinogenic. On the other hand, milk is not only consumed as liquid milk, but also utilized for the preparation of infant formulas, yogurt, cheese, and milk-based confectioneries, including chocolate, and pastry. Therefore, it is important to determine aflatoxin M1 levels in milk and dairy products, in order to protect consumers in various age groups from its potential hazards [13-17].

Materials and Methods Sampling A total of 175 samples composed of raw cow milk (50), raw sheep milk (20), raw goat milk (20), raw camel milk (10), pasteurized cow milk (30), evaporated milk (10), full cream powdered milk (15), and baby infant formula milk (20), were collected during 2011 year. All milk samples were thawed gradually at 4°C, and then vigorously mixed.

Sample preparation For raw milk samples and evaporated milk, 5 ml was incubated for 30 min at 4°C, and centrifuged at 3000 g for 10 min. The milk serum, below the fat layer was sampled and direct assay for AFM 1, using a specific ELISA KIT (Romer Labs, 2011), was done. For full-cream milk powder and baby infant formula, 9.1 g of the powder was dissolved in 100 ml double-distilled water, the solution was warmed up to about 50°C, and homogenized using a magnetic stirrer. Then, the sample was prepared, as described above for raw milk sample.

Analysis of AFM1 in samples by the competitive ELISA Two types of ELISA kits were used; AgraQuant Aflatoxin M1fast (100/2000 mg/kg) (Romer Labs, Singapore) and AgraQuant Aflatoxin M1sensitive (25/500 mg/kg) (Romer Labs, Singapore). These kits were

*Corresponding author: Omar S Sharaf, Department of Nutrition and Food Processing, Faculty of Agricultural Technology, Al-Balqa Applied University, Salt, PO Box 36197, Jordan, E-mail: sharaf@bau.edu.jo

Milk and dairy products are considered as a part of the main nutrient in Jordan. However, the percentage of daily consumption may reach 100%, and sometimes changed, depending on the economic status of people. Therefore, it is important to determine not only aflatoxin M1 levels in certain milk samples, but also routine-monitoring surveys should be considered in this regard [18-20].

Received January 23, 2013; Accepted February 27, 2013; Published March 01, 2013

The aim of this study is to investigate the presence of Aflatoxin M1 in various types of milk samples consumed in Jordan, by Enzyme Linked Immunosorbant Assay (ELISA).

Copyright: © 2013 Sharaf OS. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

J Nutr Food Sci ISSN: 2155-9600 JNFS, an open access journal

Citation: Sharaf OS (2013) Aflatoxin M1 in Raw, Pasteurized and Baby Infant Formula Milk Available in Jordanian Market. J Nutr Food Sci 3: 195. doi:10.4172/2155-9600.1000195

Volume 3 • Issue 2 • 1000195


Citation: Sharaf OS (2013) Aflatoxin M1 in Raw, Pasteurized and Baby Infant Formula Milk Available in Jordanian Market. J Nutr Food Sci 3: 195. doi:10.4172/2155-9600.1000195

Page 2 of 3 stored at 2-8°C. Before their use, the kits were left for 1 h at room temperature to bring them to room temperature. The kits were used according to the manufacturer’s instruction (Romer Labs, 2005) as follows: Into an AFM 1-antibody-coated Microtiter plate, an AFM 1 standard (supplied with the KIT), were pippeted into each well (100 µl/wall/standard). Test samples were also pippeted in duplicate (100 µl/well/sample). The plate containing the samples was incubated at room temperature for 60 min, using a titer plate shaker, at a speed of approximately 100 RPM. Following a washing step with washing solution (supplied with the KIT), AFM1 conjugate was added to the wells, and the plate was incubated again at room temperature for 30 min on a Microtiter plate shaker, at a speed of approximately 100 RPM. The plate was washed with the washing solution, in order to remove the unbound conjugate. In 100 µl, all of substrate solution was added into the wells and the reaction was allowed to proceed in the dark for 40 min at room temperature, at the end of which a blue color is developed. The reaction was stopped by adding 100 µl all of stop solution to the wells, and the color changed from blue to yellow. The absorbance was measured at 450 nm in Multiskan Ascent ELISA Plate Reader (Thermo Labsystems, USA), and the absorption intensity was found to be inversely proportional to AFM 1 concentration in the samples. The log-logit AFM 1 sheet supplied with the Kit was used to generate a standard curve, and to calculate the concentration of AFM 1 in the samples (Tables 1 and 2).

Calculation of extrapolating values of AFB1 concentration in feeds The values of aflatoxin B1 (AFB1) in animal feeds were extrapolated from a back calculation of the values of AFM 1 obtained from analysis of milk samples. The calculation was based on the assumption that only 1.6% of ingested AFB1 is converted to AFM 1 by dairy animals [16,17]. The formula used for calculation was AFM1(mg / kg) ×100 AFB1µg / Kg = 1.6 ×1000

Results and Discussion Occurrence of AFM 1 in baby infant formula and other milk products All samples from baby infant formula, full cream powdered milk Sample

N Positive samples

Baby infant formula

20

100%

Full cream powdered milk 15

100%

Evaporated milk

100%

10

AFM 1 contamination (ng\kg) Range

Mean ± SD

16.55-154.14 120.26 ± 33.54 18.0-288.68

103.95 ± 76.56

149.39-264.82 195.91 ± 34.72

Table 1: Occurrence of AFM 1 in baby infant formula and other milk products. Samples

N

Mean ± SD ( mg\Kg)

Range (ng\kg)

Extrapolated AFB1 (µg\Kg)

Fresh cow milk

50

Fresh sheep milk

20

68.91 ± 23.15

9.71-129.79

0.6-8.1

70.25 ± 14.85

23.56-137.18

Fresh goat milk

1.5-9

20

60.25 ± 33.41

20.25-125.89

1.3-8.0

Fresh camel milk

10

37.15 ± 12.10

23.57-96.52

1.5-6

Pasteurized cow milk 30

59.45 ± 42.12

14.60-216.78

0.9-13.5

Table 2: Concentration of AFM 1 in different milk samples, and extrapolated AFB1 concentration in animal feeds.

J Nutr Food Sci ISSN: 2155-9600 JNFS, an open access journal

Sample category

Positive samples

Exceeding EC Regulation

Exceeding US Regulation 23%

Fresh animal milk

100%

66%

Pasteurized animal milk

100%

12%

0%

Baby infant formula

100%

85%

85%

Full cream powdered

100%

33.3%

0%

Evaporated milk

100%

100%

0%

Table 3: AFM 1 contamination in different kinds of milk samples, exceeding limits established by the EC/Codex and US regulations.

and evaporated milk were contaminated with AFM 1. The average of AFM 1 in each group was 120.26, 103.95 and 195.91 ng\kg, ranging from 16.55-154.14, 18.0-288.68 and 149.39-264.8 ng\kg, respectively. European Communities and Codex Alimentarius prescribe a limit of 50 mg/kg AFM1 in milk and 25 mg/kg for infant milk products. However, US regulation fixed the limit to a maximum of 500 mg/kg for milk and 25 mg/kg for infant milk products. In Austria and Switzerland, the maximum level is further reduced to 10 mg/kg for infant food commodities. (Codex Alimentarius Commission 2001; European Communities 1992; European Commission Regulation 2001). There are thus differences in maximum permissible limit of AFM 1 in various countries and many countries, including Jordan, have no legal limit for AFM 1 in milk and dairy products. In general, the amount of AFM 1 in 85% of baby infant formula samples, 33.3% of full cream powdered milk, and all evaporated milk was higher than the maximum tolerance limit accepted by EC/Codex limits. All tested samples were accepted according to US regulation recommended limit. Due to high toxicity and carcinogenic properties of AFM 1, its presence in milk is a concern. AFM 1 is resistant to thermal inactivation, pasteurization, autoclaving and other varieties of food processing procedures [1]. So, to produce high quality milk, it is essential to keep feeding free from contamination by AFB1 (Table 3) [17].

Concentration of AFM 1 in different milk samples All samples from cows, sheep, goats, camels and pasteurized cow milk were found to be contaminated with AFM 1. The range of contamination levels varied among different categories of milk samples. In total, 66% of fresh animal milk samples exceed EC regulation and 23%, unacceptable by U.S regulation. Pasteurized cow milk showed the highest range of contamination of 14.60-216.78 mg/kg, with a mean value 59.45 mg/kg. Pasteurization plants in Jordan usually collect milk from different farmers at different locations in the country, and collected milk is pooled and pasteurized. No testing of milk for contamination with AFM 1 is done, prior to pasteurization. AFM 1 contamination in camel milk was relatively lower than that in other animal milk samples. Camels in Jordan freely graze wild plants for the major part of the year; they are fed on stored grains for two to three months only. Other animals are fed on manufactured feedstuffs made of various stored grain products, and by products of agricultural industry. The latter type of feedstuffs is prone to fungal infection, and to subsequent contamination with aflatoxins during storage [4,7,20]. Results of extrapolation based on AFM 1 concentrations found in milk samples tested in the present study indicates the likelihood that feeds provided to dairy animals in Jordan contains higher concentrations of AFB1, than those prescribed by the European Communities, 5 mg/

Volume 3 • Issue 2 • 1000195


Citation: Sharaf OS (2013) Aflatoxin M1 in Raw, Pasteurized and Baby Infant Formula Milk Available in Jordanian Market. J Nutr Food Sci 3: 195. doi:10.4172/2155-9600.1000195

Page 3 of 3 kg. It is therefore, important to monitor the levels of AFB1 in feedstuffs of dairy animals in Jordan, and to devise mechanisms to improve their quality, in such a way that reduces AFM 1 contamination of milk and milk products [6]. The findings of this study are in close agreement with the results reported by previous investigators, confirming the presence of AFM 1 in milk and other dairy products [1,8,15,20-22]. Accordingly, to produce high quality milk, it is essential to keep feeds free from contamination by aflatoxin B1. In Jordan, the pasture is not widely available; feeding dairy animals with feedstuffs is more common, especially at milk industry. Therefore, it is important to inform producers and consumers about the toxicity potential of aflatoxins, in order to reduce their potential health risk and economic loss.

Conclusions It is important to minimize exposure of our infant to AFM 1, so protective diet oriented governmental strategies should be taken into consideration. AFM 1 incidence in milk appears to be a serious public health problem in Jordan. However, it is important to minimize exposure of milking animals to moldy feed contaminated with AFB1, and take necessary precautions to prevent fungal contaminations and growth, particularly during the storage of feed. Acknowledgments This research was supported by a Grant from the Deanship of scientific research at Al-Balqa Applied University. Salt, Jordan.

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Citation: Sharaf OS (2013) Aflatoxin M1 in Raw, Pasteurized and Baby Infant Formula Milk Available in Jordanian Market. J Nutr Food Sci 3: 195. doi:10.4172/2155-9600.1000195

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