ODREĐIVANJE Afm! U JOGURTU Toxicol Ind Health-2013-Tabari-72-6

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DOI: 10.1177/07482337124467292013 29: 72 originally published online 25 May 2012Toxicol Ind Health

Mahsa Tabari, Khashayar Tabari and Orod Tabariand high-performance liquid chromatography

atoxin M1 determination in yoghurt produced in Guilan province of Iran using immunoaffinity colu

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Article

Aflatoxin M1 determination inyoghurt produced in Guilan provinceof Iran using immunoaffinity columnand high-performance liquidchromatography

Mahsa Tabari1, Khashayar Tabari2 and Orod Tabari2

Abstract

The present study was aimed to determine the presence and levels of aflatoxin M1(AFM1) in 120 natural yoghurtsamples consisting of 80 samples of commercial and 40 samples of traditional yoghurt in Guilan province in thenorth of Iran. The occurrence and concentration range of AFM1in samples were determined by immunoaffinity

column extraction and high-performance liquid chromatography. Analysis of yoghurts showed that all the sampleswere contaminated with AFM1 in concentration levels ranging from 4.2 to 78.9 ng/kg. In general, 16 samples(13.33%) had higher AFM1 level than the maximum tolerance limit (50 ng/kg) accepted by European Union, but thecontamination level was lower than 500 ng/kg in all the samples, which is accepted by Codex Alimentarius andNational Standard. The concentration of AFM1in 26 samples (21.66) was lower than 10 ng/kg. The range of con-tamination of AFM1 was comparatively higher in traditional yoghurt (average concentration of 32.9 ng/kg) than thatin commercial yoghurt (average concentration of 21.6 ng/kg;p< 0.01). Because yoghurt is the most popular dairyproduct consumed in Iran, the AFM1contamination is a serious problem for public health. This study reports thedata of a first survey on the presence of AFM1 in yoghurt in Guilan, Iran.

Keywords

Yoghurt, aflatoxin M1, high-performance liquid chromatography, Guilan, Iran

Introduction

More than 300 mycotoxins had been identified but

only a limited number have been found to be present

in food and feedstuff at levels that could cause

problems. Aflatoxins are mycotoxins produced as

carcinogenic, teratogenic and mutagenic secondarymetabolites byAspergillus flavus,Aspergillus bomby-

cus, Aspergillus parasiticus, Aspergillus nomius,

Aspergillus ochraceoroseus, Aspergillus tamarrii,Aspergillus pseudotamarii,Aspergillus parviscleroti-

genus, Aspergillus rambellii and certain members

from Aspergillus subgenus nidulantes (Emericella)

(Frisvad et al., 2005). However, not all strains are able

to produce aflatoxins, and hence all strains are recom-

mended for screening of their aflatoxin production

abilities (Fente et al., 2001; Moss, 2002). Aflatoxin

B1 (AFB1) represents the highest degree of toxicity

for animals, followed by aflatoxin M1 (AFM1), G1

(AFG1), B2 (AFB2) and G2 (AFG2) (Gourama and

Bullerman, 1995). AFM1 may be found in the milk

of animals that have been fed with feed containing

AFB1.

The International Agency for Research on Cancer

(IARC) classifies aflatoxin B1 in group 1, as agents that

are certainly carcinogenic to human: evaluation of the

risk is well known because it is considered the most pow-

erful hepatocarcinogenic agent in mammals. AFM1, on

the other hand, is included in group 2, as an agent with

1Department of Food Science and Technology, Faculty of

Agriculture, Lahijan Branch, Islamic Azad University, Lahijan, Iran2Department of Mining Engineering, Lahijan Branch, Islamic Azad

University, Lahijan, Iran

Corresponding author:

Mahsa Tabari, Department of Food Science and Technology,

Faculty of Agriculture, Lahijan Branch, Islamic Azad University,

Lahijan, Iran.Email: [email protected]

Toxicology and Industrial Health

29(1) 7276

The Author(s) 2012

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DOI: 10.1177/0748233712446729

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possible carcinogenic effects to humans (IARC, 1993).

The formation of AFM1, a metabolite of AFB1, occurs

in the liverand it is excreted through the mammary gland

in the milk of dairy cows (Cathey et al., 1994). When

yoghurt was made from milk contaminated with AFM1,

AFM1 was carried over into this product.

Tabari (2010) also found that the AFM1 levelsdiffered with the milk products. Pasteurization pro-

cesses, even those using UHT techniques, do not affect

the AFM1 concentration drastically because of its heat

stability (Galvano et al., 1996; Tabari, 2010).

To protect consumers, particularly children, from con-

taminated milk and dairy products, several countries have

established legislation to regulate the levels of AFB1 in

feeds and AFM1 in milk. Thus, the European Union

(EU) has set a maximum admissible level of 0.05mg/kg

in raw milk, heat-treated milk and milk for the manufac-

ture of milk-basedproducts like yoghurt (European Com-mission, 2001), whereas the Food and Drug

Administration (FDA) has established an action level of

0.05mg/kg in whole, low-fat and skim milk (US FDA,1996). The importance of AFM1 as a food safety hazard

is reflected in the existence of a maximum permissible

level of 0.05mg/kg AFM1 in milk products like yoghurt

established by the European Commission (BS EN ISO

14501, 2007) and additional 17 national regulations

Union, but according to the Codex Alimentarius and

National Standard contamination level are lower than

500 ng/kg.

Guilan province in the northern part of Iran is moresubject to high temperature and high humidity. These

environmental conditions favor the contamination of

animal feedstuffs by Aspergillus fungi. Below-

normal soil moisture (drought stress) has also been

found to increase the number of Aspergillus spores

in the air. The increased load of spores in the airgreatly increases the chances of contamination. Tem-

peratures between 27C and 38C and relative humid-

ity of 85% (corresponding to 18% grain moisture) are

optimum for the growth ofAspergillus(Cassel et al.,

2001).The objective of this work was to evaluate the level

of AFM1in both industrial and traditional yoghurt in

the region. This study reports the data of a first survey

on the presence of AFM1 in Yoghurt in Guilan, Iran.

Materials and methods

Sampling

In this study in 2009, AFM1 was analyzed in a total of

120 yoghurt samples collected based on the Iranian

National Standard Sampling Method INS No.419. A

total of 120 samples (80 samples commercial and

40 samples traditional) of yoghurt were collected. The

industrial yoghurts were manufactured by dairy plants

and the traditional yoghurt samples were made by

local people, which offered their products to sell on

the markets of Gilan province.

Chemicals

All of the solvents like acetonitrile, methanol, dichloro-

methane and water were of high-performance liquid

chromatography grade and were purchased from Baker

(Deventer, Holland). Acetonitrile and methanol used in

ruggedness studies were purchased from Sigma Chemi-

cal Co. Ltd. (St. Louis, MO, USA). The reference stan-

dard of AFM1 (from A. flavus, 10mg) was purchased

from SigmaAldrich. We prepared an AFM1 stock solu-tion (5mg/ml) by dissolving 10mg of powder in 2 ml of

pure acetonitrile; this solution was stable for 9 months at

20C. The concentration was tested by a spectrophoto-metric method according to the European commission

decision (BS EN ISO 14501, 2007). Working standard

solutions (0.1, 0.5, 1 and 2.0 mg/l) were prepared by

appropriate dilution of pure acetonitrile and stored at

20C when not in use; these solutions were stable forat least 3 weeks.

InstrumentationAn Agilent 1100 Series (Waldbronn, Germany) consist-ing of an LC system equipped with a membrane degas-

ser, a quaternary pump, an autosampler, a 20 ml loop, a

thermostated column compartment and a fluorescence

detector set at 360 nm (emission) and 440 nm (excita-

tion) was used for the analyses. The LC column was a

ZORBAX Eclipse XDB-C18, 250 mm4.6 mm, par-ticle size 5 mm, purchased from Agilent. The mobile

phase consisted of water, acetonitrile and methanol

(55:15:30, v/v/v) flowed at 1.2 ml/min.

Chromatographic method for AFM1analysis inyoghurt

Chromatographic analysis of AFM1is preceded by a

sequence of complex general operations that include

sample preparation, extraction, purification and con-

centration of the extract obtained before the separa-

tion, quantitation and confirmation steps. Analysis

was carried out following the method described by

Tabari et al. (2011).

Tabari et al. 73

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Calculations

Calculations were made according to the following

equation

Wm Wa Vf=Vi 1=Vs

where Wm

amount of AFM1 in the test sample inmicrograms per liter; W

a amount of AFM1 corre-

sponding to area of AFM1 peak of the test extract(nanograms); Vf the final volume of redissolvedeluate (liters); Vi volume of injected eluate (liters)and Vs volume of test portion (yoghurt) passingthrough the column (milliliter).

Statistical analysis

All experiments were carried out in triplicate and data

in tables represent mean values + standard deviation

(n 3). Results were evaluated for statistical signifi-cance using one-way analysis of variance using SPSS

(Version 11.5). The confidence level for statisticalsignificance was set atp 0.01.

Results

A more recent advancement in quantitative extraction of

AFM1 and subsequent cleanup are the use of immunoaf-

finity columns. Modifications of the immunoaffinity-

based methods for AFM1 were subsequently published

and studied collaboratively, under the auspices of the

International Dairy Federation and AOAC International,

by a group mainly of European laboratories that coulddetermine AFM1 in yoghurt at concentrations of

0.05 mg/l. A collaborative study resulted in the approval

of AOAC method 2001 (Dragacci et al., 2001).

The standard solutions of concentration from 0.05

to 10mg/l AFM1 were used to find a calibration/stan-

dard curve as described by the following regression

equation:y 17.579x 1.520, where y area andx amount of AFM1. The results showed the linear-ity of the standard curve over the range studied. The

coefficient of determination (R2) was 0.9998.

AFM1 was detected in alltheexamined yoghurt sam-

ples by average concentration of 28.2 ng/kg. The results

on an amount of AFM1 are shown in Table 1. Sixteen

samples (13.33%) had higher AFM1 level than the

admissible level (50 ng/kg) for adult established by the

Commission of the European Communities (10% of

commercial yoghurts and 20% of traditional yoghurts

were contaminated higher than 50 ng/kg). In addition,33.33% of yoghurt sampleshad higher AFM1 level than

the acceptable level (25 ng/kg) for children (27.5% of

commercial and45% of traditional yoghurts).The range

of contamination level varied from 4.2 to 64.7 ng/kg and

from 7 to 78.9 ng/kg in commercial and traditional

yoghurt samples, respectively.

The results demonstrate that there were significant

differences (p < 0.01) in the levels of AFM1 in commer-

cialand traditional yoghurts. Contamination level in tra-

ditional yoghurts was higher than commercial ones.As it can be seen, 72.5% (58 samples) of commer-

cial yoghurts had lower AFM1 level than 25 ng/kg,and 25% (20 samples) had lower AFM1 level than

10 ng/kg (Table 2). In addition, 27.5% of these samples

had the higher contamination levels than 25 ng/kg, and

only 10% of samples had higher AFM1 level than

50 ng/kg. Average concentration of AFM1 was

21.6 ng/kg being lower than EU standard.

Twenty percent of AFM1-contaminated traditionalyoghurt samples exceeded the European tolerance

limit (50 ng/kg).The percentage of samples with the

lower level of AFM1 decreased in traditional yoghurts.

The concentration of AFM1 in six samples (15%) waslower than 10 ng/kg. In addition, 55% of yoghurt sam-

ples had lower AFM1 levels than 25 ng/kg. AFM1 was

found in 100% of examined traditional samples by a

mean concentration of 32.9 ng/kg.

Discussion

In order to address the problem of AFM1in milk and

dairy products, it is necessary to focus the attention on

the most sensitive steps of feedstuff production for

Table 1. Occurrence of AFM1 in yoghurt samples.

Type of yoghurt No. of samples

Positive samples (%) AFM1 concentration (ng/kg)

>25 >50 Mean + SD Minimum Maximum

Commercial yoghurt 80 27.5 10.0 21.6 + 2.18 4.2 64.7

Traditional yoghurt 40 45.0 20.0 32.9 + 5.08 7.0 78.9Total 120 33.33 13.33 28.2 + 4.01 4.2 78.9

AFM1: aflatoxin M1.

74 Toxicology and Industrial Health 29(1)



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lactating cows. In order to prevent losses of yield and

hazards for human and animal health, it is necessary

to take care of cultural phases that can represent a crit-

ical point for fungal growth and mycotoxin produc-

tion in an organic, low and a high-input farming

system (e.g. excessive nitrogen fertilization in a

high-input farming system; in organic and low-inputfarming system). To prevent future aflatoxins out-

breaks, it is necessary to communicate about the

potential risk deriving from unsuitable farming

managements that could lead to the development of

contaminated feeds and foods. However, only a few

surveys refer to the occurrence of AFM1 in yoghurt.

Some studies are shown in Table 3.

According to Galvano et al. (1996), of the 114 sam-

ples of yoghurt, 91 (80.0%) contained AFM1 with

levels ranging from 1 to 496.47 ng/kg. Later, Galvano

et al. (2001) reported that in 120 yoghurts analyzed,

73 (61.0%) samples were contaminated with AFM1at lower levels (132.1 ng/kg) when compared with

those of the previous survey of 1996. In Campinas,

Brazil, Sylos (1996) did not detect the presence of

AFM1 in 30 of the tested yoghurts. According to these

authors, the possible explanation for the absence of

AFM1 is that cows in the Campinas area graze all year

round. Another explanation could also be that the

methodology used for the detection of AFM1 was the

thin layer chromatography method (Sylos, 1996). In a

survey carried out by Kim et al. (2000) in the city of

Seoul, South Korea, the presence of AFM1 was

detected in 50% of the 60 yoghurt samples with levelsranging from 17 to 124 ng/kg. These results were in

agreement with the study by Martins and Martins

(2004) who observed that AFM1 ranging from 19 to

98 ng/kg was detected in 18 (18.8%) yoghurt samples,

and 78 samples (81.2%) did not reveal the presence of

the toxin. Furthermore, in Turkey, Gurbay et al.(2006) verified the presence of AFM1 ranging

between 62 and 366 ng/kg in 80% of 40 yoghurt

samples.

In other study, we determined AFM1 in Doogh

(Tabari et al., 2011). Doogh is a yogurt-based bever-

age and is a popular dairy product in Iran, owing to its

beneficial influence on human health and nutritional

value and also found in Afghanistan, Azerbaijan,

Armenia, Iraq, Syria, Turkey, as well as the Balkans.

We evaluate AFM1 contamination in 225 commer-

cial doogh samples composed of traditional (115 sam-ples) and industrial pasteurized doogh (110 samples)

obtained from popular markets of Tehran, Iran. AFM1

was found in 100% of the examined doogh samples by

average concentration of 49.7 ng/l. Aflatoxin M1 was

detected in 151 (67.1%) samples. In Tehran, 27 of the

doogh samples, 15 samples (12.8%) of traditional and

12 samples (10.8%) of industrial had higher AFM1

level than the maximum tolerance limit (50 ng/l)accepted by the EU, but the contamination level was

lower than 500 ng/l in all the samples, which is

accepted by Codex Alimentarius and National Stan-

dard. The results indicated that the contamination ofthe samples with AFM1 in such a level could not be

a serious public health problem at the moment. This

article represents the data of the first survey on the

occurrence of AFM1 in commercial doogh marketed

traditional and industrial in Tehran of Iran.

Funding

This research received no specific grant from any funding

agency in the public, commercial or not-for-profit sectors.

Table 3.Incidence and level of AFM1 in yoghurt samples.

Country No. of samples Incidence (%) Range (ng/kg)

Italy 114 80.0 1496.47South Korea 60 50 17124Italy 120 61.0 132.1Portugal 96 18.8 1998

Turkey 177 38.65 0150Turkey 40 80.0 62366

AFM1: aflatoxin M1.

Table 2.Level of AFM1 in yoghurt: comparison between commercial and traditional yoghurt.

Range of AFM1 concentration (ng/kg)Commercial yoghurt Traditional yoghurt TotalNo. of samples (%) No. of samples (%) No. of samples (%)

50 8 (10.0) 8 (20.0) 16 (13.33)

AFM1: aflatoxin M1.

Tabari et al. 75



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