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A survey of aflatoxin M1 in somecommercial milk samples and infantLizy Kanungoa & Sunil Bhanda

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

To cite this article: Lizy Kanungo & Sunil Bhand , Food and Agricultural Immunology (2013):A survey of aflatoxin M1 in some commercial milk samples and infant, Food and AgriculturalImmunology, DOI: 10.1080/09540105.2013.837031

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A survey of aflatoxin M1 in some commercial milk samples and infant

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 formulamilk samples in the Goan market, India, was conducted using high performance liquidchromatography, association of analytical communities approved commercial kit and asensitive 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 theEuropean Communities recommended limits (50 ng/L) and 75% of the samplesexceeded Codex Alimentarius, Food Safety and Standards Authority of India (FSSAI)and US Food and Drug Administration recommended limits (500 ng/L). The range ofcontamination of AFM1 was found lower in infant milk formula (501–713 ng/L) thanliquid 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 AFM1contamination in milk.

Keywords: aflatoxin M1; milk; contamination; ultrasensitive; ELISA

Introduction

Milk and milk products in several countries have been widely surveyed for the naturaloccurrence of aflatoxin M1 (AFM1). AFM1 is the metabolite of aflatoxin B1 (AFB1) andis found in milk when lactating animals are fed with contaminated feedstuffs. Whenanimals consume AFB1-contaminated feedstuffs, the toxin is metabolised in the liver andexcreted as AFM1 via milk and urination (Henry et al., 2001). AFM1 is bound to milkproteins, 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 ofAFM1 in milk possess a major risk for humans especially infants as it can haveimmunosuppressive, mutagenic and teratogenic effects. Studies show that, AFM1 isrelatively stable during milk pasteurisation, storage as well as during the preparationof various dairy products (Badea et al., 2004; Codex Committee on Food Additives andContaminants, 2001). The toxic and carcinogenic effects of AFM1 led WHO-InternationalAgency 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 worldwideconcern 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: sunilbhand@goa.bits-pilani.ac.in

Food and Agricultural Immunology, 2013http://dx.doi.org/10.1080/09540105.2013.837031

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2005), several countries have set or proposed legal regulations for AFM1 levels in milkand dairy products. These regulations vary in different countries and are often based oneconomic considerations (Stoloff, Van Egmond, & Parks, 1991). The European Commun-ity legislation imposes maximum permissible limit for AFM1 concentration as 50 ng/L formilk and 25 ng/L for infant formulae (Henry et al., 2001). Recently the Food Safety andStandards Authority of India (FSSAI) have set the permissible limit for AFM1 concentrationin milk and milk products at 0.5 µg/kg (FSSAI, 2011).

The literature describes the various methods in which milk may be analysed directlyor 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 AFM1which, in addition, requires organic solvents. Among other established methods, immuno-chemical techniques are very popular for mycotoxins analysis with many literaturesreporting 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 suitabletool for quick and sensitive analysis with high sample throughput but also cost-effective andrequires only a little sample volume for analysis (Parker & Tothill, 2009; Pei, Zhang,Eremin, & Lee, 2009; Sawaf, Abdullah, & Sheet, 2012). Among the established ELISAtechniques, sandwich-type immunoassay is an effective bioassay due to the high specificityand sensitivity (Knopp, 2006). Recently, we have reported a novel approach where a highlysensitive microplate sandwich ELISA was developed and integrated with magnetic nanoparticles (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 milksamples (Bakırdere, Yaroğlu, Tırık, Demiröz, & Karaca, 2012; Iqbal, Asi, & Ariño, 2011;Kawamura et al., 1994; Rohani, Aminaee, & Kianfar, 2011). In India, a survey found that87.3% 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 majorconcern 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 ofAFM1 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 inthe Karnataka and Tamil Nadu region of India (Siddappa, Nanjegowda, & Viswanath,2012). It has been surveyed that varieties of packaged milk samples are available in theIndian market for consumption without any food safety certification. There is a need forroutine screening of such packaged samples which projects a vital need for simple,robust, low-cost bioanalytical methods for AFM1 detection in milk and milk productsthat can be used in the laboratories of developing countries.

In the present work, a survey was conducted to check the occurrence of AFM1 incommercial milk samples and infant formula milk samples of Goa, India. Herein, weanalysed 15 milk brands and 3 infant formula milk brands (total 72 samples) to quantifythe AFM1 level as recommended by the FSSAI, Codex, US Food and DrugAdministration (USFDA) and European Union (EU) guidelines. One of the milk sampleswas artificially contaminated with known concentrations of AFM1, and these weredetected and validated by high performance liquid chromatography (HPLC) forconfirmation of the presence of the toxin. This AFM1 analysis using HPLC was carriedout in SGS India Pvt. LTD., Chennai, India. The commercial milk and infant formula

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samples were tested by two commercial kits (Art. No.: R1121 & R5802) bought fromRidascreen® (approved by association of analytical communities [AOAC]) and asensitive 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, <0.02 µg/kg) were purchased fromSigma-Aldrich (USA). Hydrogen peroxide (H2O2) 30% (w/v), acetonitrile (ACN) HPLCgrade, sodium chloride (NaCl), methanol (99% pure) was purchased from Merck(Germany). Rat monoclonal [1C6] primary antibody (1°Ab) of AFM1 and HRPconjugated secondary antibody (2°Ab) raised from rabbit were purchased from Abcam(UK). Sodium hypochlorite (4%) solution was purchased from Fisher Scientific (India).Milk samples were centrifuged by minispin plus centrifuge purchased from Eppendorf(Germany) and shaking of the samples were done by Spinix shaker, purchased fromTarsons (India). White 384 well polystyrene microtiter plates were purchased from Nunc(Denmark). Multichannel automatic pipette from Eppendorf was used for multi plateassay for AFM1 analysis in real sample. For CL measurement, VictorX4 2030 optiplatereader from Perkin Elmer (USA) was used. Glove box, Cole Parmer (USA), was used forthe handling of AFM1 standard solution. Water produced in a Milli-Q system (Millipore,Beford, MA, USA) was used for preparing all the solutions. pH metre from Seven Multi

Sample collection Pre-treatment : Centrifugation & filtration

HPLC Approved commercial Kit CL Sandwich ELISA

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

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Mettler Toledo, 8603, Switzerland, was used to prepare buffers. The Ridascreen® AFM130/15 test kit with dynamic range 0–80 ng/L (Kit-1) and Ridascreen® Fast AFM1 test kitwith dynamic range 0–2000 ng/L (Kit-2) were purchased from R-Biopharm AG,Darmstadt, Germany. Milk samples and infant formula milk powders were purchasedfrom the local markets of Goa, India. For HPLC analysis, CRM milk sample was spikedwith AFM1, and the HPLC was carried out in an accredited Lab.

Preparation of buffers, AFM1 standard solutions and antibody solutions

Carbonate buffer (CB; pH 9.6), Phosphate buffered saline (PBS; pH 7.4), PBST bufferand blocking solution (BSA in PBS) were made by the same protocol as described in ourearlier paper (Kanungo et al., 2011). All buffer solutions were stored at 4°C when not inuse. All the AFM1 solutions were prepared in a Glove box under inert (N2) atmosphere.Working standard solutions in the range of 2.5–10,000 ng/L were prepared by dilutingthe stock with 5% ACN. [Safety Note: Aflatoxins are highly carcinogenic and should behandled with extreme care. Aflatoxin contaminated lab wares should be decontaminatedwith an aqueous solution of sodium hypochlorite (4%)]. From the stock solution of ratmonoclonal [1C6] 1°Ab, working 1°Ab solution was prepared prior to the experiment.The working Ab dilutions were prepared by serial dilution in double distilled water as1:1000, 1:2000, etc., and then added with equal volume of CB (1:1). Similarly, from thestock solution of 1 mg (2 mg/mL) rabbit polyclonal to rat IgG-H and L (HRP) 2°Ab,working 2°Ab solution was prepared prior to the experiment by serial dilution in PBS.

Milk sample collection and pretreatment

Both milk and infant formula milk powders were randomly collected from the markets ofGoa. Totally, 15 popular liquid milk and 3 infant formula milk brands were analysedcomprising 54 and 18 samples, respectively. Powder-based samples (formula milk food)were suspended in warm deionised water as per the instructions written on the packets.The packaged milk samples as well as the formula milk samples were centrifuged at 6000g for about 10 min. After centrifugation, the upper fat layer was completely removed. Thedecanted aqueous layer was filtered through a syringe filter using 0.22 µ filter paper andused for the analysis.

Immunoassay procedure

To investigate the presence of AFM1, the milk samples were analysed by ELISA. First,the samples were analysed by sandwich ELISA. Subsequently, they were also tested byAOAC-approved commercial kits from Ridascreen® where competitive ELISA wasperformed as per the protocol provided in the literature (R1121 and R5802).

CL sandwich ELISA

Sandwich ELISAwas performed in 384 microwell plate. We followed the same protocol asdescribed in our earlier paper (Kanungo et al., 2011) with reduced incubation time of 1 h.

Competitive ELISA (using commercial Kit-1, dynamic range 0–80 ng/L)

The quantitative analysis of AFM1 in the samples was first performed by competitiveELISA using Ridascreen AFM1 30/15 test kit (Kit-1) as per the instructions. Theabsorbance was measured after addition of stop solution at 450 nm by the plate reader.

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Competitive ELISA (using commercial Kit-2, dynamic range 0–2000 ng/L)

We have also analysed AFM1 in milk samples by Ridascreen® Fast AFM1 test, whichhas two types of antibodies and a wider dynamic range of antigens (0–2000 ng/L) as perthe instructions. The absorbance was measured by the plate reader at 450 nm. Theintensity of absorbance was inversely proportional to the concentration of AFM1 insamples. The AFM1 concentration results from the ELISA assay were then analysed.

Results and discussion

In HPLC analysis, the peaks were studied for the confirmatory test of AFM1 (Figure 2).In Figure 2, the peaks are shown for the concentrations such as 0.625, 2.5, 5 and 10 ng/mL, respectively, that were spiked in CRM milk samples and were analysed by anaccredited laboratory.

AFM1 standard calibration curve

The calculations for CL sandwich ELISA were made by simple observations of signalintensities or photon counts plotted against various AFM1 concentrations. The signalintensity or photon counts were generated from the luminol/peroxidase reaction. Thestandard curve for AFM1 detection by CL sandwich ELISA is shown in Figure 3a. Aconcentration-dependent decrease in signal intensity was observed for AFM1. CRM(ERM-BD282) was reconstituted to a liquid form, which was later spiked with knownamounts of AFM1 solutions in different concentrations. The assay was performed forthree times, and the error values were plotted as a standard calibration curve. The dose–response curve showed the photon count in terms of signal intensity. From the standard

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Figure 2. HPLC peaks confirming AFM1 presence in the analysed milk samples.

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calibration curve, we obtained the [AFM1] in tested infant formula milk powders as wellas milk samples. The error bar indicates the standard deviation (n = 3), where ‘n’ is anindependent assay by the proposed method. The SD and R2 were calculated to be 1.69and 0.89, respectively. The calculations used for quantitative analysis by Ridascreen testkits were followed from the kit protocol. For the same, the percentage absorbance wasplotted against the standard concentrations of AFM1 provided in the kit. The absorptionintensity was found to be inversely proportional to AFM1 concentration in the sample.The calibration curve was then used to analyse several milk samples to detect AFM1contamination levels.

The standard curve for competitive assay by commercial kit was obtained and isdepicted in Figure 3b. A concentration-dependent decrease in percentage absorbance wasobserved for AFM1. The standard curve was used for calibration of unknown milksamples. The error bar indicates the standard deviation (n = 6), where n is an independentassay by the method. The IC50 was 817.8 pg/mL. The SD and R2 were calculated to be2.24 and 0.91, respectively. From all the measurements, it was observed that all sampleswere contaminated with AFM1.

Recovery of AFM1 from spiked and CRM milk samples

The sandwich ELISAwas validated with CRM (ERM-BD 282, zero level of AFM1). Themilk powder was reconstituted as indicated in the certification report supplied by theIRMM, Belgium. To test the accuracy of the assay, AFM1 concentrations ranging from 2.5to 10,000 ng/L were added to the CRM milk sample and assayed by both CL ELISA andRidascreen Fast kit. CRM milk sample containing zero-level AFM1 was compared with asample deliberately contaminated with known amounts of AFM1. Recovery was assessedby spiking AFM1 with the BD282 reconstituted material and presented as in Table 1. Thefortified (2.5, 6.25, 12.5, 25, 50, 100, 200, 300, 600, 1250, 5000, 10,000 ng/L of AFM1)milk samples were interpolated from the calibration curve performed using reconstitutedCRM. The precision and reliability of the CL ELISA are notable from the data presented in

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Figure 3. Standard calibration curve of AFM1: (a) by CL Sandwich ELISA; (b) by commer-cial kit.

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Table 1. The resultant data showed an excellent percentage of recovery, close to 100% forCRM. The precision was determined by calculating the relative standard deviation (RSD%) for the replicate measurements and the accuracy (RE%) was calculated by assessing theagreement between measured and nominal concentration of the fortified samples.

Relative error ðREÞ % ¼ ðmeasured value - true valueÞ/true value� 100

RSD% ¼ standard deviation/mean� 100; n¼ 3

AFM1 contamination level in commercial milk samples

The comparison of AFM1 contamination levels in real samples with EU, Codex, FSSAI andUSFDA standard is summarised in Table 2. It was observed that 100% of all the samplesexceeded EU standard, and around 75% of the samples exceeded Codex, FSSAI and USFDAstandards. By CL sandwich ELISA method as well as by commercial kit, it was found that,out of 18 samples of infant formula milk analysed, 100% would not pass the EU regulations,while this number is reduced to 66.6% and 33.3%, respectively, if the USFDA, Codex or

Table 1. Recovery studies of AFM1 using CL sandwich method and commercial kit.

AFM1 found(ng/L) R.S.D % R.E. % Recovery %

MilkSamples

AFM1 added(ng/L)

CLELISA

Kitassay

CLELISA

Kitassay

CLELISA

Kitassay

CLELISA

Kitassay

IF*1 50.00 47 47.5 2.12 1.05 −6 −5 94 95500.00 486 480 1.02 0.41 −2.8 −4 97.2 96

M**1 50.00 57.5 47 4.31 2.1 15 −6 115 94500.00 508 534 1.18 0.56 1.6 6.8 101.1 106.8

Table 2. Comparison of AFM1 contamination levels in packaged milk samples with EU, Codex,FSSAI and USFDA standards.

Exceeding EUregulations (Infantfeed >25 ng/kg)(Liquid milk>50 ng/kg)

Exceeding Codex,FSSAI and USFDAregulations (Liquidmilk >500 ng/kg)

Assay typeSamplecategory

Samplesanalysed

Positivesamples Number*

Range(ng/kg) Number*

Range(ng/kg)

CL SandwichELISA

IF*1 18 18 18 (100) 160–713 12 (66.6) 501–713

M**1 54 54 54 (100) 172–809 42 (77.7) 511–809CompetitiveELISA (AOACapproved kit)

IF*1 18 18 18 (100) 150–500 6 (33.3) 500–730

M**1 54 54 54 (100) 178–820 42 (77.7) 160–820Total 72 72 72 150–820 48 (66.6) 160–820

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FSSAI regulations are applied. On the other hand, out of 54 liquid milk samples 100%exceeded EC regulations and 77.7% surpassed USFDA, Codex and FSSAI regulations.

The figures of merit of the CL sandwich ELISA were compared with Ridascreen kitand presented in Table 3. The parameters such as detection limit, analysis time,sensitivity, sample throughput and cost per sample were compared. The dynamic rangeand the upper limit of detection for the milk samples using the Ridascreen kit were foundinferior when compared against the sandwich assay. Although the analysis time of the kitwas less than our assay, the detection limit and sensitivity of the CL sandwich assay wasfound to be more promising than the kit. The higher analysis time of CL sandwich assaycan be compensated against the number of real samples per assay (n = 128) in triplicate asagainst the commercial kit wherein 48 samples (Kit-1) and 24 samples (Kit-2) per assaycan be analysed. Moreover, the use of 384 microwell plate facilitates reduction in reagentconsumption and volume of toxic waste. The comparison result suggests that thesandwich assay is better suited for ultra-sensitive analysis of AFM1 contamination inmilk samples and can be easily adapted for routine analysis.

Conclusions

Based on the random sampling and analysis of commercial milk samples and infantformula milk samples, it is evident that the all the analysed samples were found tocontain AFM1 concentrations exceeding permissible limits of EU standard. Theseobservations strongly suggest that it is necessary to pay attention to this subject. In ourreport, both CL sandwich and competitive ELISA have been shown to be simple anduseful analytical techniques that can be used to monitor low-level AFM1 contamina-tion in milk. Moreover the sandwich assay could detect AFM1 contaminaiton as lowas 60 pg/mL, whereas commercial ELISA could detect 110 pg/mL. From the survey, itwas found that all milk samples were contaminated with AFM1, but 75% of thesamples exceeded the Codex, USFDA and FSSAI regulation level. The detectedlevels of AFM1 in the conducted survey of samples show a serious health alarm inregards to the safety limits for AFM1 levels in infant formula and milk samples ofIndian market.

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 0–80 ng/L 0–2000 ng/L 0–10,000 ng/LLimit of detection 5 ng/L, 50 ng/L <367 ng/L 2.5 ng/LLimit of quantification 25 ng/L 500 ng/L 5 ng/LRecovery rate withcoefficient ofvariation(CV)

95% (CV = 14%) 78–115% (CV=20%) 94–115% (CV = 20%)

Time requirement 1.5 h/48 samples 0.5 h/24 samples 3h/128 samplesHigh throughput 96 48 384Cross reactivitywith AFM2

30% – 65%

Sample volume 100 µL 50 µL 40 µLCost per sampleanalysis

4.5 (EUR) 2.3 (EUR) 0.88 (EUR)

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AcknowledgementL.K. acknowledges NAIP for the award of Research Associate Fellowship.

FundingThis work is funded by National Agriculture Innovation Project (NAIP) No. C4/C10125, 2008–14,Indian Council of Agriculture and Research and the World Bank.

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