A survey of Aflatoxin M1 in some commercial milk ...

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A survey of Aflatoxin M1 in some commercial milk samples and infant formula milk samples in Goa, India a

a

Lizy Kanungo & Sunil Bhand a

Department of Chemistry, BITS, Pilani-K. K. Birla Goa Campus, Goa, India Published online: 19 Sep 2013.

To cite this article: Lizy Kanungo & Sunil Bhand (2014) A survey of Aflatoxin M1 in some commercial milk samples and infant formula milk samples in Goa, India, Food and Agricultural Immunology, 25:4, 467-476, DOI: 10.1080/09540105.2013.837031 To link to this article: http://dx.doi.org/10.1080/09540105.2013.837031

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Food and Agricultural Immunology, 2014 Vol. 25, No. 4, 467–476, http://dx.doi.org/10.1080/09540105.2013.837031

A survey of Aflatoxin M1 in some commercial milk samples and infant formula milk samples in Goa, India

Downloaded by [Birla Institute of Technology and Science] at 09:38 06 October 2014

Lizy Kanungo and Sunil Bhand* Department of Chemistry, BITS, Pilani-K. K. Birla Goa Campus, Goa, India (Received 23 November 2012; accepted 19 August 2013) A survey of aflatoxin M1 (AFM1) contamination in packaged milk and infant formula milk samples in the Goan market, India, was conducted using high performance liquid chromatography, association of analytical communities approved commercial kit and a sensitive chemiluminescent sandwich enzyme-linked immunosorbent assay (ELISA). A total of 72 samples of infant formula milk food (18) and packaged milk samples (54) was analysed. One hundred per cent of the analysed samples exceeded the European Communities recommended limits (50 ng/L) and 75% of the samples exceeded Codex Alimentarius, Food Safety and Standards Authority of India (FSSAI) and US Food and Drug Administration recommended limits (500 ng/L). The range of contamination of AFM1 was found lower in infant milk formula (501–713 ng/L) than liquid milk (511– 809 ng/L). The methods were also compared for their performance, and ELISA was found to be most suitable for analysis of low-level AFM1 contamination in milk. Keywords: aflatoxin M1; milk; contamination; ultrasensitive; ELISA

Introduction Milk and milk products in several countries have been widely surveyed for the natural occurrence of aflatoxin M1 (AFM1). AFM1 is the metabolite of aflatoxin B1 (AFB1) and is found in milk when lactating animals are fed with contaminated feedstuffs. When animals consume AFB1-contaminated feedstuffs, the toxin is metabolised in the liver and excreted as AFM1 via milk and urination (Henry et al., 2001). AFM1 is bound to milk proteins, especially casein, which leads to its presence in dairy products (Prandini et al., 2009). AFM1 is known for its hepatotoxic and carcinogenic effects. The presence of AFM1 in milk possess a major risk for humans especially infants as it can have immunosuppressive, mutagenic and teratogenic effects. Studies show that, AFM1 is relatively stable during milk pasteurisation, storage as well as during the preparation of various dairy products (Badea et al., 2004; Codex Committee on Food Additives and Contaminants, 2001). The toxic and carcinogenic effects of AFM1 led WHO-International Agency for Research on Cancer (IARC) to change its classification from group 2 to group 1 (IARC, vol. 82, 2002). Occurrence of AFM1 in milk and milk products is a worldwide concern since these products are consumed by all groups of the population (Fallah, 2010). Due to the carcinogenic and toxic nature of the AFM1 in milk and milk derivatives (Oveisi, Jannat, Sadeghi, Hajimahmoodi, & Nikzad, 2007; Sassahara, Netto, & Yanaka, *Corresponding author. Email: [email protected] This article was originally published with an error. This version has been corrected. Please see Erratum (http://dx.doi.org/10.1080/09540105.2013.849051). © 2013 Taylor & Francis


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2005), several countries have set or proposed legal regulations for AFM1 levels in milk and dairy products. These regulations vary in different countries and are often based on economic considerations (Stoloff, Van Egmond, & Parks, 1991). The European Community legislation imposes maximum permissible limit for AFM1 concentration as 50 ng/L for milk and 25 ng/L for infant formulae (Henry et al., 2001). Recently the Food Safety and Standards Authority of India (FSSAI) have set the permissible limit for AFM1 concentration in milk and milk products at 0.5 µg/kg (FSSAI, 2011). The literature describes the various methods in which milk may be analysed directly or after simple and limited pretreatment (Badea et al., 2004; Farjam, De Vries, Lingeman, & Brinkman, 1991; Micheli, Grecco, Badea, Moscone, & Palleschi, 2005; Parker & Tothill, 2009). The analysis of dairy products still involves time-consuming extraction of AFM1 which, in addition, requires organic solvents. Among other established methods, immunochemical techniques are very popular for mycotoxins analysis with many literatures reporting the use of a commercially developed enzyme-linked immunosorbent assay (ELISA) (Devi et al., 2002; Lopez, Ramos, Ramadan, & Bulacio, 2003; Rastogi, Dwivedi, Khanna, & Das, 2004; Rodriguez, Delso, & Escudero, 2003). ELISA is not only a suitable tool for quick and sensitive analysis with high sample throughput but also cost-effective and requires only a little sample volume for analysis (Parker & Tothill, 2009; Pei, Zhang, Eremin, & Lee, 2009; Sawaf, Abdullah, & Sheet, 2012). Among the established ELISA techniques, sandwich-type immunoassay is an effective bioassay due to the high specificity and sensitivity (Knopp, 2006). Recently, we have reported a novel approach where a highly sensitive microplate sandwich ELISA was developed and integrated with magnetic nano particles (MNPs) which could detect ultra trace amount of AFM1 in milk (Kanungo, Pal, & Bhand, 2011). There are many reports available in the view of detection of AFM1 residues in milk samples (Bakırdere, Yaroğlu, Tırık, Demiröz, & Karaca, 2012; Iqbal, Asi, & Ariño, 2011; Kawamura et al., 1994; Rohani, Aminaee, & Kianfar, 2011). In India, a survey found that 87.3% of the milk-based samples analysed were contaminated with AFM1; of these 99% were much above European permissible limits (Rastogi et al., 2004). This is a major concern considering that India is the largest producer of milk in the world (Devi et al., 2002; Parker & Tothill, 2009; Rastogi et al., 2004), and there are very scarce reports of AFM1 analysis. One recent report describes about the occurrence of AFM1 in raw, pasteurised and ultra-high temperature treatment of milk of the major brands prevalent in the Karnataka and Tamil Nadu region of India (Siddappa, Nanjegowda, & Viswanath, 2012). It has been surveyed that varieties of packaged milk samples are available in the Indian market for consumption without any food safety certification. There is a need for routine screening of such packaged samples which projects a vital need for simple, robust, low-cost bioanalytical methods for AFM1 detection in milk and milk products that can be used in the laboratories of developing countries. In the present work, a survey was conducted to check the occurrence of AFM1 in commercial milk samples and infant formula milk samples of Goa, India. Herein, we analysed 15 milk brands and 3 infant formula milk brands (total 72 samples) to quantify the AFM1 level as recommended by the FSSAI, Codex, US Food and Drug Administration (USFDA) and European Union (EU) guidelines. One of the milk samples was artificially contaminated with known concentrations of AFM1, and these were detected and validated by high performance liquid chromatography (HPLC) for confirmation of the presence of the toxin. This AFM1 analysis using HPLC was carried out in SGS India Pvt. LTD., Chennai, India. The commercial milk and infant formula

Food and Agricultural Immunology

Sample collection


HPLC

469

Pre-treatment : Centrifugation & filtration

Approved commercial Kit

CL Sandwich ELISA

Figure 1. Schematic diagram of analysis of AFM1 in milk by HPLC, AOAC-approved kits, CL ELISA method.

samples were tested by two commercial kits (Art. No.: R1121 & R5802) bought from Ridascreen® (approved by association of analytical communities [AOAC]) and a sensitive chemiluminescent (CL) technique based sandwich ELISA (Figure 1). Experimental procedure Materials and methods Chemicals and instrumentation AFM1, bovine serum albumin (BSA), tween 20, luminol, certified reference material (CRM) ERM-BD282 (AFM1 in whole milk powder, 25 ng/kg) (Liquid milk >50 ng/kg) Assay type CL Sandwich ELISA Competitive ELISA (AOAC approved kit)

Exceeding Codex, FSSAI and USFDA regulations (Liquid milk >500 ng/kg)

Sample category

Samples analysed

Positive samples

Number*

Range (ng/kg)

Number*

Range (ng/kg)

IF*1

18

18

18 (100)

160–713

12 (66.6)

501–713

M**1 IF*1

54 18

54 18

54 (100) 18 (100)

172–809 150–500

42 (77.7) 6 (33.3)

511–809 500–730

M**1 Total

54 72

54 72

54 (100) 72

178–820 150–820

42 (77.7) 48 (66.6)

160–820 160–820

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Table 3. Comparison of CL sandwich method vs Ridascreen Kits. Figures of merit

Ridascreen 30/15 kit

Ridascreen Fast kit

CL Sandwich Assay

Dynamic range Limit of detection Limit of quantification Recovery rate with coefficient of variation(CV) Time requirement High throughput Cross reactivity with AFM2 Sample volume Cost per sample analysis

0–80 ng/L 5 ng/L, 50 ng/L 25 ng/L 95% (CV = 14%)

0–2000 ng/L