IMTIAZ HUSSAIN INSTITUE OF CHEMISTRY UNIVERSITY OF...

Micro-Analysis of Aflatoxin M1 in Dairy Products at

Trace Levels and Its Elimination

A THESIS SUBMITTED TO

UNIVERSITY OF THE PUNJAB

IN FULFILMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

CHEMISTRY

BY

IMTIAZ HUSSAIN

INSTITUE OF CHEMISTRY

UNIVERSITY OF THE PUNJAB

LAHORE, PAKISTAN

July, 2009

1

CERTIFICATE

This is to certify that the research work described in this thesis entitled “Micro-

Analysis of Aflatoxin M1 in Dairy Products at Trace Levels and Its Elimination” by

Imtiaz Hussain is the original work and has been carried out under my supervision. I

have gone through all the data/ results/ materials reported in the manuscript and certify

their correctness/ authenticity. I further certify that the material included in this thesis has

not been used in part or full in a manuscript already submitted or in the process of

submission in partial/ complete fulfillment of the award of any other degree from this or

any other institution. I also certify that thesis has been prepared under my guidance

according to the prescribed format and I endorse its evaluation for the award of PhD

degree though the official procedures of the university.

Research Supervisor:

Professor Dr. Jamil Anwar . . . . . . . . . . . . . . . . . . . . . . . .

Institute of Chemistry

University of the Punjab

Lahore

Dated: ……………………

i

CERTIFICATE

I, as a Co-supervisor certify that the research work described in this thesis entitled

“Micro-Analysis of Aflatoxin M1 in Dairy Products at Trace Levels and Its

Elimination” by Imtiaz Hussain is the original work. I have gone through all the data/

results/ materials reported in the manuscript and certify their correctness/ authenticity. I

further certify that the material included in this thesis has not been used in part or full in a

manuscript already submitted or in the process of submission in partial/ complete

fulfillment of the award of any other degree from this or any other institution. I also

certify that thesis has been prepared under my guidance according to the prescribed

format and I endorse its evaluation for the award of PhD degree though the official

procedures of the university.

Research Co-supervisor:

Dr. Munwar Ali Munwar . . . . . . . . . . . . . . . . . . . . . . . .

Institute of Chemistry

University of the Punjab

Lahore

Dated: ……………………

ii

DEDICATED TO

MY SWEET MOTHER

& MY LOVING FATHER

iii

ACKNOWLEDGEMENTS

Allah Almighty, the most merciful and compassionate, the most gracious and

beneficent, is the Lord of lords and reserves all praises and I bow my head in His

gratitude for enabling me to achieve one of the goals of my life. Million and million

times peace (Drood-o-Salam) upon the Holy Prophet Hazrat Muhammad (Peace of

Allah be upon him), who is blessing for humanity as a whole and for whole of the

universe and whose life and teachings is a source of eternal success.

I deem it a great honour and privilege to record my profound gratitude and

indebtedness to my worthy supervisor, Prof. Dr. Ch. Jamil Anwar, Professor Institute of

Chemistry, University of the Punjab, Lahore for his sympathetic and inspiring attitude,

patronizing supervision, generous assistance, erudite suggestions, and unfailing patience

through out the course of study and research and encouragement during the write up of

the manuscript. I express my deep gratitude to my honourable Co- supervisor Dr.

Munawar ali Munawar, Associate Professor Institute of Chemistry, University of the

Punjab, Lahore for his beneficial suggestions and constructive criticism during the course

of this study. I am thankful to Prof. Dr. Saeed Ahmad Nagra, Director Institute of

Chemistry, for providing research facilities.

I wish to express my most sincere thanks to Dr. Muhammad Rafique Asi, Senior

scientist Nuclear Institute for agriculture and Biology (NIAB), Faisalabad for his

technical and skilful guidance and unlimited cooperation from the beginning to the end of

my research work. I extend my thanks to Mr. Muhammad Aslam, Accountant Institute

of Chemistry, University of the Punjab, Lahore and Mr. Muhammad Aslam, In-charge

store, Institute of Chemistry, University of the Punjab, Lahore, for their best cooperation

in the departmental work.

I am highly thankful to Higher Education Commission (HEC), Islamabed for

financial support for this project without which the present cherished goal would have

merely been a dream. I do not find appropriate words to express my thanks to Dr. Abu-

Saeed Hashmi, Eminent Scientist, UVAS, Lahore, for his sincere guidance. With sincere

gratitude, I offer my thanks to Dr. Khushi Muhammad, Chairman Microbiology

Department, University of Animal and Veterinary Science (UVAS), Lahore for his open

hearted cooperation in research work. I am cordially thankful to Dr. Muhammad

iv

Zargham, Professor Department of Veterinary Pathology, Faculty of Veterinary Science,

University of Agriculture, Faisalabad, for his sincere cooperation.

I feel pleasure to express my gratitude to the administration and members of

CENUM, Mayo Hospital, Lahore, especially Mrs. Affia Tabassam, Principal Scientist,

Dr. Shan Elahi, Senior Scientist, and Mr. Hamid Rashid, technologist for their

cooperation and providing facilities for ELISA analysis.

I extend my special thanks to Mr. Fayyaz Ahmad Shafique, Chief Executive

SNAM PHARMA, Lahore, for his valuable services in obtaining literature and chemicals

relating to aflatoxin analysis. I extend my thanks to Mr. Ibrar Minhas, Chief Executive

AM Traders for providing materials and chemicals required for research purposes.

Its pleasure for me in expressing gratitude for Dr. M. Ishaq Khalid Consultant

Psychiatrist (UK) and Dr. Tayyaba Khalid (UK) who always encouraged me for the

higher ideals of life. I am especially thankful to my college colleagues Prof. Dr. Khalid

Zafar-Ullah, Prof. Bashir Ahmad, Prof. Sajid Asdullah, Prof. Tabassam Rasool,

Prof. Imran Zaidi, and Prof. Iftikhar Haider Malik for their timely help. I appreciate

my genius students Kashif, Qamar, Bilal, and Osman for their cooperation in research

activities and proof reading of the manuscript. My special thanks are for my friends Dr.

Akhlaq Ahmad, Prof. Ahmad Sher Awan, Ch. Ashiq Ali, Mian Ghulam Mustafa,

Mr. Hafeez-Ullah, Kari Abdullah, Mr. Khalid Anjum, Mr. Sajjad Baig and Mr.

Shahbaz Nazir for their moral support.

I am also indebted to my loving wife Tahira Yasmin and my lovely sons

Muhammad Najm-os-Saqib and Muhammad Aqib, who always prayed for me and

whose love remained all the time with me in this whole run.

I finally express my great gratitude and my deepest affection for my parents, my

cousin Javed Yaqoob, elder brother Ijaz Hussain, sweet nephews Rizwan and Abdur

Rehman, for their love, good wishes, inspiration and whose hands always rose in prayer

for me

IMTIAZ HUSSAIN

v

Table of Contents

1. INTRODUCTION ......................................................................................................... 1

1.1. Mycotoxins and Aflatoxins ....................................................................................... 1

1.2. Aflatoxin Biosynthesis .............................................................................................. 6

1.3. Toxicity of Aflatoxins ............................................................................................... 7

1.4. Aflatoxin M1 and Dairy Products ............................................................................. 8

1.5. Aflatoxin Determination Techniques ...................................................................... 10

1.5.1. (1) Sample Preparation Techniques ................................................................. 10

1.5.1.1. Liquid-Liquid Separation .......................................................................... 11

1.5.1.2. Solid Phase Extraction (SPE) .................................................................... 11

1.5.1.3. Immunoaffinity Columns (IACs) .............................................................. 11

1.5.1.4 MycosepTM Columns ................................................................................ 12

1.5.2. (2) Detection Techniques ................................................................................. 12

1.5.2.1. Analytical Methods ................................................................................... 13

1.5.2.1.1. Thin-Layer Chromatography .............................................................. 13

1.5.2.1.2. High Performance Thin-Layer Chromatography ............................... 14

1.5.2.1.3. High Performance Liquid Chromatography ....................................... 14

1.5.2.1.4. Liquid Chromatography with Mass Spectrometric Detection ............ 15

1.5.2.2. Immunological Methods ........................................................................... 16

1.5.2.2.1. Enzyme-Linked Immunosorbent Asssay (ELISA) ............................ 16

1.6. Management and Control of Aflatoxin Hazards ..................................................... 17

1.6.1. Pre-Harvest Management ................................................................................. 17

1.6.1.1. Cultural Control ......................................................................................... 17

1.6.1.2. Genetic Resistance of Cultivars ................................................................ 18

1.6.1.3. Use of Plant Breeding for Development of Host Resistance .................... 18

1.6.1.4. Genetic Engineering .................................................................................. 18

1.6.1.5. Bio-Competitive Agents ............................................................................ 19

1.6.2. Post-Harvest Management ............................................................................... 19

1.6.3. Detoxification ................................................................................................... 19

1.6.3.1. Chemical Detoxification ........................................................................... 20

1.6.3.2. Physical Detoxification ............................................................................. 20

1.6.3.2.1. Thermal Inactivation .......................................................................... 20

1.6.3.2.2. Irradiation ........................................................................................... 21

1.6.3.2.3. Use of Aflatoxin Binders .................................................................... 21

1.7. Legislation ............................................................................................................... 22

vi

2. REVIEW OF LITERATURE ..................................................................................... 23

2.1. Toxicological Studies .............................................................................................. 23

2.2. Methodology Studies ............................................................................................... 25

2.3. Carry-Over of Aflatoxin in Milk ............................................................................. 30

2.4. Survey of AFM1 in Milk and Milk Products .......................................................... 31

2.4.1. Survey of AFM1 in Milk .................................................................................. 31

2.4.2. Survey of AFM1 in Cheese .............................................................................. 51

2.4.3. Survey of AFM1 in Yoghurt ............................................................................ 63

3. MATERIALS AND METHODS ................................................................................. 68

3.1. Materials and Instruments ....................................................................................... 68

3.1.1. Milk Samples ................................................................................................... 68

3.1.2. Samples of Cheese and Yoghurt ...................................................................... 68

3.1.3. Feed Samples ................................................................................................. 68

3.1.4. Chemicals and Standards ................................................................................. 69

3.1.5. Instruments ....................................................................................................... 69

3.2. Methods ................................................................................................................... 70

3.2.1. Determination of Aflatoxin M1 with Fluorometer ........................................... 70

3.2.2. Determination of Aflatoxin M1 by HPLC ....................................................... 70

3.2.2.1. Extraction Procedure ................................................................................. 70

3.2.2.2. LC Determination with Fluorescence Detection ....................................... 71

3.2.2.3. Calculations ............................................................................................... 71

3.2.3. Determination of Aflatoxin B1 by HPLC ........................................................ 71

3.2.3.1 Extraction and Clean-up Procedure ............................................................ 71

3.2.3.2. Aflatoxin Derivatization ............................................................................ 72

3.2.3.3. LC Determination with Fluorescence Detection ....................................... 72

3.2.3.4. Calculations ............................................................................................... 72

3.2.4. Determination of Aflatoxin M1 in Cheese and Yoghurt by ELISA ................ 73

3.2.4.1. Sample Preparation ................................................................................... 73

3.2.4.2. Test Procedure ........................................................................................... 73

3.2.4.3. Calculations ............................................................................................... 74

3.2.5. Determination of Milk Fat Percentage ............................................................ 74

3.2.6. Determination of Milk Protein ....................................................................... 74

3.2.6.1. Test Portion Preparation ............................................................................ 74

3.2.6.2 Digestion .................................................................................................... 75

3.2.6.3. Distillation ................................................................................................. 75

3.2.6.4. Calculations .............................................................................................. 75

3.2.7. Statistical Analysis ........................................................................................... 76

vii

4. RESULTS AND DISCUSSIONS ................................................................................ 79

4.1. Aflatoxin M1 Contamination in Raw Milk- A General Survey .............................. 79

4.2. Aflatoxin M1 Contamination Variation with Respect to Localities and with Respect to Herd-Size of Cattle ....................................................................................... 90

4.3. Aflatoxin M1 Contamination in Milk of Different Species .................................. 100

4.4. Aflatoxin M1 Contamination in Milk Products .................................................... 115

4.5. Aflatoxin B1 Contamination in Dairy Feed .......................................................... 120

4.6. CONCLUSIONS ................................................................................................... 126

4.6.1. Implications of Study Results and Elimination of AFM1 Contamination in Milk and Milk Products ........................................................................................... 127

REFERENCES ............................................................................................................... 131

APPENDIX .................................................................................................................... 156

LIST OF PUBLICATIONS ......................................................................................... 156

viii

LIST OF TABLES

Table No. Title Page No.

1

Target organs of some mycotoxins along with their primary

health effects, feed suitable for growth, and fungi producing

them

3

2Distribution of aflatoxin M1 (µg/ L) in raw milk samples by

month-wise and district-wise in the Punjab82

3Distribution of aflatoxin M1 (µg/ L) in raw milk samples by

month-wise83

4Distribution of aflatoxin M1 (µg/ L) in raw milk by district-wise

and season-wise85

5Concentration of aflatoxin M1 (µg/ L) in the contaminated raw

milk samples of urban buffaloes93


milk samples of semi-urban buffaloes94


milk samples of rural buffaloes94


milk samples of urban cows94


milk samples of semi-urban cows95


milk samples of rural Cows95

11

Concentration of aflatoxin M1 (µg/ L) in the contaminated raw

milk samples of urban buffaloes belonging to the small herd-size

category

96

12


milk samples of urban buffaloes belonging to the medium herd-

size category

96

13


milk samples of urban buffaloes belonging to the large herd-size

category

97

14


milk samples of semi-urban buffaloes belonging to the small

herd-size category

97

15 Concentration of aflatoxin M1 (µg/ L) in the contaminated raw 97

ix

milk samples of semi-urban buffaloes belonging to the medium

herd-size category

16


milk samples of semi-urban buffaloes belonging to the large

herd-size category

98

17


milk samples of rural buffaloes belonging to the small herd-size

category

98

18


milk samples of rural buffaloes belonging to the medium herd-

size category

98

19


milk samples of rural buffaloes belonging to the large herd-size

category

98

20

Aflatoxin M1 (AFM1) level (µg L-1) in buffalo milk and cow

milk in urban, semi-urban and rural areas along with variation of

aflatoxin M1 concentration in buffalo milk with respect to herd-

size.

99

21Concentration of aflatoxin M1 (µg/ L) in the contaminated milk

samples of five species103

22 Fat% in buffalo milk samples 10423 Fat% in cow milk samples 10424 Fat% in goat milk samples 10525 Fat% in sheep milk samples 10526 Fat% in camel milk samples 10527 Protein% in buffalo milk samples 10528 Protein% in cow milk samples 10629 Protein% in goat milk samples 10630 Protein% in sheep milk samples 10631 Protein% in camel milk samples 106

32Composite result of AFM1 (µg/ L) contamination, fat%, and

protein% in milk of the five species107

33Aflatoxin M1 (µg/ L) contamination, fat%, and protein% in

buffalo milk109

34Aflatoxin M1 (µg/ L) contamination, fat%, and protein% in cow

milk110

35Aflatoxin M1 (µg/ L) contamination, fat%, and protein% in goat

milk111


sheep milk112

x


camel milk113

38Pearson correlation analysis of aflatoxin M1 (µg/ L)

concentration, fat%, and protein% in the milk of five species114

39Concentration of aflatoxin M1 (µg/ kg) in the contaminated

cheese samples117

40Concentration of aflatoxin M1 (µg/ kg) in the contaminated

yoghurt samples118

41Aflatoxin M1 (AFM1) contamination (µg/ kg) in cheese and

yoghurt118

42Concentration of aflatoxin B1 (µg/ kg) in the contaminated

cottonseed cake samples123


concentrate samples124

44Concentration of aflatoxin B1 (µg/ kg) in the contaminated wheat

bran samples124

45 Concentration of aflatoxin B1 (µg/ kg) in bread samples 124


paddy straw samples124

47 Aflatoxin B1 (AFB1) level (µg/ kg) in different feedstuffs 125

LIST OF FIGURES

Fig. No. Title Page No.

1 Structures of aflatoxins 52 The regression line 773 The linear equation 784 District-wise distribution of AFM1 in raw milk samples 84

5Distribution of AFM1 season-wise and district-wise in raw milk

samples86

6 Season-wise distribution of AFM1 in raw milk samples 877 Month-wise distribution of AFM1 in raw milk samples 88

8Comparison of HPLC chromatograms: (A) standard (B) sample,

for AFM1 in milk92

9Calibration curve of standard solutions of AFM1 by HPLC

analysis93

10Area-wise comparison of aflatoxin M1 in milk of buffaloes and

cows95

11 Calibration curve of standard solutions of AFM1 with 101

xi

concentrations of 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 µg/ L by

HPLC analysis

12Comparison of HPLC chromatograms: (A) standard (B) sample,

for AFM1 in milk of five species 102

13Comparison of AFM1 contamination%, fat%, and protein% in

milk of the five species108

14 Standard curve from ELISA analysis for cheese 11615 Standard curve from ELISA analysis for yoghurt 117

16HPLC chromatograms: (A) Standard (B) Sample, for aflatoxin

B1

122

17The linear standard curve of AFB1 standards with concentrations

from 0.5µg/ L to 15µg/ L123

xii

LIST OF PICTURES

Picture No. Title Page No.

1Buffaloes grazing at island in the Ravi river near Lahore, Pakistan

89

2 The growth of moulds on bread 125

xiii

LIST OF ABBREVIATIONS

Abbreviation WordA AspergillusAFB1 Aflatoxin B1

AFB2 Aflatoxin B2

AFM1 Aflatoxin M1

AFM2 Aflatoxin M2

ANNEX AnnextureANOVA Analysis of VarianceAOAC Association of Official analytical ChemistsAOCS American Oil and Chemical Society AVN Averantinbw Body weight°C Degree Celsius (centigrade)CEC Commission of European Communitiescm Centimeter(s)Co A Co-enzyme ACV Coefficient of varianceC8 Carbon 8C18 Carbon 18Da DaltonDAD Diode array detectorDHOMST Dihydroxy O-methylsterigmatocystinEC European commission/ communitiese.g. For exampleELISA Enzyme linked immunosorbent assayet al. And others (co-workers)etc. (ETCETERA) Other (things)EU European UnionF F ratio (statistics)F FusariumFAO Food and Agriculture OrganizationFDA Food and Drug AdministrationFig. FigureFLD Fluorescence Detectorg Gram(s)g Gravity(in centrifuging)GIT Gastrointestinal tractHAVN Hydroxy averantinHPLC High-performance liquid chromatographyHSCAS Hydrated sodium calcium aluminosilicateIAC Immunoaffinity columnsIARC International agency for Research on Cancerid inner diameteri.e. That isIUPAC International Union of Pure and Applied ChemistryJECFA The Joint Expert Committee on Food Additiveskg Kilogram(s)kGy Kilogray

xiv

L Litre(s)LC Liquid chromatography; Liquid chromatographLtd. LimitedM Molar (applied to concentration of solution)min Minuteml Millilitremm MillimeterMS Mass spectrometry; Mass spectrometerN Normal (applied to concentration of solution)N2 NitrogenND Not detectedng Nanogram(10-9 g)NGO Non government organization(s)nm Nanometer(10-9 m)No. NumberNOR NorsoloricOMST O-methylsterigmatocystinOTA Ochratoxin Ap p value (statistics)PA Penitrem APBS Phosphate buffer salinePCR Polymerase chain reactionppb Parts per billionppt Parts per trillionPSI Per square inchPSQCA Pakistan Standards and Quality Control Authority® Trademark name (registered)Rf Retardation factor (Distance spot moved/distance solvent moved, TLC) rpm revolutions per minuteRP-HPLC Reversed phase high performance liquid chromatographySD Standard deviationSE Standard errorSIM Selection-ion-moniteringSPE Solid phase extractionST SterigmatocystinStd. Standardtech. TechnologyTFA Tri-fluoro acetic acidTLC Thin-layer chromatographyTM TrademarkUAE United Arab EmiratesUHT Ultra high-temperature treatedUSA United States of America USDA United States Department of AgricultureUV UltravioletVAL VersiconolVERA Versicolorin AVRB Versicolorin Bvs VersusWHO World Health Organization

xv

WTO World Trade Organizationλ Lambda(for wavelength)µg Microgram(s) (10-6 g)µL Microlitre(s) (10-6 L)µm Micrometer(s) (10-6 m)% Percent (parts per hundred); percentage< Less than; under; below (used with numbers only)> More than; greater; above; exceeds (used with numbers only)

xvi

ABSTRACT

Milk is a complex mixture of fat, protein, carbohydrate, and mineral components

and it has been a source of human food since the recorded history. Aflatoxin M1 is

excreted in milk of those lactating animals which have ingested aflatoxin B1 contaminated

feed. Aflatoxin B1 (AFB1) is metabolized to aflatoxin M1 in liver and then excreted in

milk and urine. Aflatoxin B 1 is a potent carcinogen and aflatoxin M1 (AFM1), being the

metabolite of AFB1, has toxic properties similar to AFB1. Several researches have

demonstrated the potential toxicity of exposure to AFM1. Aflatoxin M1 is present in milk

and milk products.

This study includes the determination of contamination of aflatoxin M1 in milk

and milk products and contamination of aflatoxin B1 in dairy feed in the Punjab province

of Pakistan. The analytical techniques used in the determination of AFM1 were high

performance liquid chromatography (HPLC), fluorometry (using Fluorometer), and

enzyme linked immunosorbent assay (ELISA). For the determination of AFB1, HPLC

was used. Immunoaffinity columns were used to accomplish cleanup step during HPLC

and fluorometric determination. A total of 977 samples of milk, cheese, and yoghurt

were analyzed for AFM1 contamination. Whereas a total of 260 samples of feed

commodities (concentrate feed, cottonseed cake, wheat bran, bread, paddy straw, and

wheat straw) were analyzed for AFB1 contamination.

In the first phase of study 168 sample of raw milk from fourteen districts, were

analyzed by using immunoaffinity columns and Fluorometer. All the samples were found

contaminated with AFM1, however in 96.4% samples the level of contamination was

below the US tolerance limit of 0.5 µg/ L. Only 3% samples showed AFM1

contamination higher than the US tolerance limit. While considering EU maximum

permissible limit (0.05 µg/ L), 99.4% samples exceeded this limit. Seasonal effect was

also studied on the presence of AFM1 contamination in milk. ANOVA analysis indicated

significant difference (p < 0.01) in AFM1 concentration in milk in different seasons. The

AFM1 contamination was higher in winter as compared to summer and this was supported

by previous studies.

During the study of AFM1 contamination in raw milk taken from different

localities, variation in levels of AFM1 was found in raw milk from different localities in

the central areas of the Punjab, Pakistan. Total 480 milk samples of buffaloes and cows

xvii

from different localities (urban, semi-urban, and rural) were analyzed by using HPLC

with prior clean-up step applying immunoaffinity columns. The percentage of AFM1

contamination in buffalo and cow milk was 42.5% and 52.5% respectively. In both types

of milk, level of AFM1 contamination was higher in milk samples obtained from urban

and semi-urban areas and it was minimal in milk samples taken from rural areas. The

AFM1 contamination in buffalo milk was studied statistically with respect to herd-size

variation also. The results showed significant variations with respect to herd-size (F=

6.631, p= 0.001). Milk samples in case of small herd-size (1-5 cattle) and medium herd-

size (6-10 cattle) showed higher AFM1 concentration as compared to large herd-size

(more than 10 cattle).

Another study was conducted to investigate the AFM1 contamination in the milk

of five mammalian species namely buffalo, cow, goat, sheep, and camel from the area of

Faisalabad district of the Punjab province, Pakistan. Analysis was made by using HPLC

with fluorescence detection. Immunoaffinity columns, which are based on the principle

of affinity chromatography, were used for clean-up purposes. Total 169 milk samples

were analyzed. The percentage of AFM1 contamination in buffalo milk, cow milk, goat

milk, and sheep milk was found to be 34.5%, 37.5%, 20%, and 16.7% respectively.

AFM1 contamination was not detected in camel milk in this area.

Although there is massive use of fresh milk in Pakistan, but still significant

consumption occurs after milk has been processed. As AFM1 concentration is not

affected by normal milk processes, AFM1 is also present in milk products like cheese and

yoghurt. The milk product samples including 80 cheese samples and 80 yoghurt samples

were analyzed by using ELISA technique. The percentage of AFM1 contamination was

found to be 87.5% and 70% in cheese and yoghurt samples respectively.

Because of the possibility of presence of aflatoxin B1, feed plays a major role in

the occurrence of aflatoxin M1 in milk. The monitoring of AFB1 contamination in dairy

feed is compulsory to ensure safety of milk consumers. The study on the contamination

of AFB1 in the dairy feed samples showed high contamination of AFB1 in cotton-seed

cake samples and concentrate feed samples. Total 260 samples of different commodities,

used as dairy feed, were analyzed for AFB1 contamination by HPLC. The average AFB1

contamination levels in cottonseed cake, concentrate feed, wheat bran, bread pieces, and

paddy straw were found to be 242, 176, 98, 23, and 37 µg/ Kg respectively. The

contamination level was high as compared to US tolerance i.e., 20 µg/ Kg.

xviii

The study revealed that the menace of AFM1 concentration in milk and milk

products is present in the area. It is imperative to take measures to control and reduce the

AFM1 contamination in milk and milk products in the area. Contamination of AFM1 was

found in milk and milk products, but only a small percentage of contaminated samples

exceeded the US tolerance limit. This can be controlled by taking precautionary

measures. The study conducted for AFB1 contamination determination showed the high

concentration of AFB1 in cottonseed cake and concentrate feed. The use of these two

commodities must be controlled in the feed regimen of milking animals. Moreover, the

proper use of toxin binders can reduce the menace of AFM1 contamination in milk and

milk products in the area. The feed, straw, bread and other commodities contaminated

with moulds must not be used as a feed for milking animals. Availability of sufficient

moisture is the most critical factor in mould growth. A relative humidity of 70% to 90%

is suitable for growth of moulds and production of mycotoxins. Long storage of dairy

feed and feedstuffs should not be practiced or these should be stored in a proper way,

especially at low moisture content, to avoid the growth of moulds and to eliminate the

contamination of aflatoxins. Low moisture content and low humidity percentage can be

achieved by aeration. The concerned authorities should set a legal limit for AFM1 in

dairy products and AFB1 in dairy feed. Furthermore, establishment of sophisticated

laboratories for aflatoxins’ analysis and arrangement of surveillance programs for

aflatoxin contamination will prove to be of a great help in controlling aflatoxin

contamination.

xix

1. INTRODUCTIONContamination of aflatoxin M1 in milk is a matter of serious concern, because

AFM1 is a carcinogen. Milk is a part of common diet for adults as well as children which

are more susceptible to the hazardous effects of toxins. As AFM1 is heat stable and no

reduction has been observed in toxin level during pasteurization process, AFM1 has been

found in UHT milk (Diaz et al., 1995). It passes from raw milk to milk products. In

some milk derivatives like cheese and yoghurt, it is associated with the protein fraction

and hence there is 3-5 fold enrichment over that in milk (Bracket and Marth 1982; Pietri

et al., 1997). Aflatoxin M1 is an important member of aflatoxins, which are a group of

mycotoxins.

1.1. Mycotoxins and Aflatoxins

Mycotoxins are secondary metabolites, produced by filamentous fungi that can

pose a serious threat for human and animal consumers. The word mycotoxin literally

means “poison from fungi” (myco = fungus, toxin = poison), but all toxic compounds

produced by fungi are not considered to be mycotoxins. Mycotoxins are toxic to

vertebrates and other animal groups even in low concentrations. On this basis, other low-

molecular-weight fungal products like ethanol, toxic only in high concentrations, are not

termed as mycotoxins (Bennett, 1987). Mycotoxicology, the study of mycotoxins,

however arbitrarily excludes mushroom poisons, definitely deadly fungal metabolites,

from its area of study. The distinction between a mycotoxin and a mushroom poison is

made on two different levels; one is the size of the fungus producing them and other is the

human intention of exposure to these. The former is a production of micro-fungi

(moulds) and its exposure is almost accidental, whereas the latter is a macro-fungi

production and humans can be exposed to it because they may wrongly take these to be

delectable species (Moss, 1996). Fungal productions that are toxic to bacteria are known

as antibiotics and others which are fatal for plants are known as phytotoxins. Although

over 300 different types of mycotoxins have been identified but only a few are of

importance as far as human beings are concerned. Five genera of fungi, namely

Aspergillus, Fusarium, Penicillium, Alternaria, and Claviceps are responsible for the

production of a majority of mycotoxins (Geisen, 1998; D’Mello et al., 1998). The

mycotoxins produced by Aspergillus fungi are aflatoxin B, G, M, ochratoxin A (OTA),

1

sterigmatocystine and cyclopizonic acid. Penicillium toxins are patulin, OTA, citrinin,

penitrem A (PA) and cyclopiazonic acid. Fusarium toxins are deoxynivalenol (DON),

nivalenol, zearalenone, T-2 toxin, diacetoxyscipenol, fumonisins and moniliformin.

Alternaria toxins are tenuazonic acid, alternariol and alternariol methyl ether. Ergot

alkaloids are produced by claviceps fungi (Steyn, 1995).

The classification of mycotoxins has always been a troublesome question.

Different modes of classification have been proposed. The experts and researches of

different areas of study classify mycotoxins according to the mode and mood of their

subject. Organic chemists classify them according to their chemical structure (e.g.,

lactones, coumarins) and biochemists arrange them according to their biosynthetic origin

(e.g., polyketides, amino acid-derived). On the other hand, clinicians arrange them

according to the organ they affect (e.g., hepatotoxins, nephrotoxins, neurotoxins, and

immunitoxins), whereas the physicians use the illness associated with them to name them

(e. g., St. Anthony’s fire, stachybotryotoxicosis) and cell biologists put them into generic

groups (teratogenes, carcenogenes, mutagens, and allergens). Mycologists propose yet

another way to classify mycotoxins by classifying them according to the fungi producing

them (e.g., Aspergillus toxins, Penicillium toxins). All the above mentioned

classifications have certain shortcomings and none of these is comprehensive and entirely

satisfactory.

Mycotoxins can be hazardous for human and animal health because they are

capable of producing illness and death in humans and animals. Humans can be exposed to

mycotoxins by ingestion, inhalation, and dermal exposure (Pitt, 2000; Hendry and Cole,

1993). The diseases caused by mycotoxins in humans and animals are termed as

mycotoxicoses- characteristic symptoms of toxic effects of mycotoxins. The intensity of

these diseases ranges from acutely toxic to immunosuppressive or carcinogenic. All this

does not conclude that mycotoxins are only dangerous for humans. They have been

proven to be beneficial by the formation of antibiotics (penicillins), immunosuppressant

(cyclosporine), and in control of postpartum hemorrhage and migraine headaches (ergot

alkaloids) (Etzel, 2002).

Target organs of some mycotoxins along with their primary health effects and

fungi producing them are given in table 1 (D’Mello and MacDonald, 1997; Peraica et al.,

1999).

2

Table 1: Target organs of some mycotoxins along with their primary health effects, feed

suitable for growth, and fungi producing them.

Mycotoxin Food/Feed Symptoms/Primary

health effects

Target

organ

Main fungus

producingAflatoxins Cearls,

maize,

cottonseed,

ground nut

Acute hepatits,

haemorrhagic

desease, death

Liver Aspergillus

flavus, A.

paraciticus

Ergot alkaloids Cereals,

grasses

Gangrenous

necrosis, nervous

seizures,

reproductive failure

Peripheral

vascular

system

Claviceps

purpurea, C.

paspali

Fumonisins Maize Apathy, depression,

confusion, dullness,

hyper excitation

Esophagus Fusarium

proliferatum, F.

moniliforme, A.

ochraceusOchratoxin Cereals Nephritis Kidney A.ocharaceus,

Penicillium

viridicatum, P.

cyclopium, P.

verrucosum Trichothecenes Cereals Haemorrhage,

gastroenteritits,

dermal-mucosal

necroses

Mucoses F. culmorum,

F.

graminearum,

F. cerealis, F.

sporotrichioide

sZearalenone Cearls,

maize, corn

comb, maize

silage,

sorghum

Fertility disorder,

enlargement of

mammary glands

Urogenital

tract

F. culmorum,

F.

graminearum,

F.

sporotrichioide

s

3

Aflatoxins belong to the group of mycotoxins and are toxic metabolites,

produced by certain fungi. Their first appearance on the stage was quite mysterious and

fatal. In 1960, more than 100,000 turkeys died in England from an apparently new

disease that was termed as “Turkey X disease” (Blowt, 1961). Later on the same sort of

problem appeared in ducklings and young pheasants. Because of the importance of the

matter an extensive research was launched to probe into the matter. The early

investigations showed that the disease was associated with feed, namely Brazilian peanut

meal. It was established by the researchers that this peanut meal was highly toxic to

poultry and ducklings. It was speculated that the toxin might be of fungal origin. Finally,

in 1961, the toxin-producing fungus was identified to be Aspergillus flavus and was given

the name Aflatoxin. The word ‘aflatoxin’ is an acronym and in this word (A-fla-toxin),

the “A’ is for Aspergillus and “fla” for the species flavus along with word toxin. Now it

is known that aflatoxins are produced not only by Aspergillus flavus but a number of

other species like Aspegillus parasiticus, Aspegillus niger and Aspergillus nomius also

produce aflatoxins. Fungi, behaving in an opportunistic manner, get a chance to produce

aflatoxins by growing on poorly managed crops, appearing during crop drying and

processing, and while the commodity is either in storage or in transit.

More than a dozen different types of aflatoxins have been identified and the four

prominent members are B1, B2, G1, and G2. Others like M1 and M2, P1 and P2, and

aflatoxicol are produced as a result of animal and microbial metabolism. The structures

of important aflatoxins are shown in the Fig. 1. A. flavus fungus produces only B

aflatoxins, while the other species produce both B and G aflatoxins (D’Mello and

MacDonald, 1997). Aflatoxins M1 and M2 are the hydroxylated metabolites of aflatoxins

B1 and B2 respectively and are found in milk and milk products obtained from livestock

which have ingested contaminated feed. The aflatoxin M1 and M2 were first isolated from

milk of lactating animals that were fed aflatoxin preparations; therefore the M designation

is used for the aflatoxins which are present in milk. The B designation of aflatoxin B1

and B2 denotes the capacity to exhibit blue fluorescence under UV-light (365) and the G

designation refers to the yellow-green fluorescence of the relevant aflatoxins under UV-

light. The aflatoxin B1, after being ingested by taking contaminated commodities, is

converted into aflatoxin M1 by the action of certain enzymes found in the liver of animals

and humans. After its formation, AFM1 is excreted in the urine and milk of dairy cattle

and other lactating mammals.

4

Fig. 1: Structures of aflatoxins.

The commodities which are susceptible to the attack of fungi and which are

being contaminated with aflatoxins are peanuts and other nuts, cereals, spices, and

cottonseed. Usually in areas with higher temperature and humidity, food and feed are

contaminated with aflatoxins. The ideal conditions for the growth of Aspergillus and for

production of toxin are an equilibrium relative humidity of 80-85 per cent, equilibrium

moisture content of 17 per cent and temperature within the 24-35°C. The optimum

temperature for A. flavus is 28-30 °C and the minimum moisture content is 8-10 % in

peanuts (Hohler, 2000). Attack of fungus and thus in turn aflatoxin contamination

O O

O

O O

OCH3

Aflatoxin B1

O O

O

O O

OCH3

Aflatoxin B2

O O

O

O O

OCH3

Aflatoxin M2

OH

O O

O

O O

OCH3

Aflatoxin M1

OH

O O

O

O

OCH3

Aflatoxin G1

O

O

O O

O

O

OCH3

Aflatoxin G2

O

O

5

becomes more feasible when there is some sort of stress in plants. The stress may be in

the form of drought (below-normal soil moisture) that weakens the plant system,

extended periods of high temperature, damage from insects or birds, high crop density or

competition from weeds. These conditions weaken the host and provide a means of entry

to the fungus spores to establish a foothold in or on the host.

1.2. Aflatoxin Biosynthesis

Aflatoxins are a group of polyketide-derived furanocoumarins. The

biosynthesis of aflatoxins is a complex process and multi-enzymatic reactions are

involved in it and it is governed by genes maintained in a cluster. With the discovery of

the structures of aflatoxins, many attempts started to decipher the aflatoxin biosynthetic

pathway. The studies have determined that aflatoxins are synthesized in two stages from

malonyl CoA, first with the formation of hexanoyl CoA, followed by formation of

decaketide anthraquinone. A series of highly organized oxidation-reduction reactions

then allows formation of aflatoxins (Bhatnagar et al., 1992; Dutton, 1988; Townsend,

1997). The currently accepted scheme (Yu et al., 2002) for aflatoxin biosynthesis is:

hexanoyl Coa precursor → norsolorinic acid, NOR → averantin, AVN →

hydroxyaverantin, HAVN → versiconal hemiacetal acetate, VHA → versiconal, VAL →

versicolorin B, VERB → versicolorin A, VERA → demethyl-sterigmatocystin, DMST →

sterigmatocystin, ST → O-methylsterigmatocystin, OMST → aflatoxin B1 and aflatoxins

G1. Aflatoxin B2 and aflatoxin G2 are formed from dihydro O-methylsterigmatocystin,

DHOMST. Several specific enzymes with conversions in the aflatoxin pathway have been

partially purified (Bhatnagar et al., 1992; Dutton, 1988; Yabe et al., 1991a; Yabe et al.,

1991b; Bhatnagar et al., 1989; Bhatnagar et al., 1991), whereas others such as

methyltransferases (Bhatnagar et al., 1988) have been purified to homogeneity. Many

other enzymes, which are involved in aflatoxin biosynthesis such as a reductase and a

cyclase (Lin and Andeson, 1992) have also been purified from A. parasiticus. A

desaturase has been found in cell-free fungal extracts (Yabe et al., 1991a). Two

versiconal hemiacetal acetate reductases involved in toxin synthesis have been purified

and characterized by Matsushima et al. (1994). Kusumoto and Hsieh (1996) purified to

homogeneity an esterase that converts VHA to versiconal. Bhatnagar et al. (1991) and

Chatterjee and Townsend (1996) stated that in the later stages of AFB1 and AFB2

6

synthesis, independent reactions and formation of different chemical precursors are

catalyzed by common enzyme system (Bhatnagar and Cleveland, 1991).

There are a number of nutritional and environmental factors, such as

temperature, pH, carbon and nitrogen source, stress factors, lipids and trace metal salts,

which affect the production of aflatoxins by toxigenic Aspergilli. The molecular

mechanisms for these effects are still not clear, although numerous studies exist (Payne

and Brown, 1998; Bennett et al., 1979). As many nutritional and environmental factors

affect aflatoxins formation, it is likely that one or more signal transduction pathways

affect aflatoxins formation. Genetic connection between fungal development and toxin

formation also appears (Bennett and Papa, 1988).

1.3. Toxicity of Aflatoxins

Aflatoxins are the most toxic and carcinogenic compounds among the known

mycotoxins. Aflatoxins have been shown to be immunosuppressant, mutagenic,

teratogenic and hepatocarcinogenic in experimental animals. Aflatoxins are acutely and

chronically acting poisons. Main target is liver cells and these are able to cause cancers

in this vital organ even at very low concentrations. Livestock ingesting even minute

amounts of aflatoxins in contaminated feed suffer sickness, disease and mortality. The

contamination and toxicity may be transferred down the food chain to consumers in meat,

eggs and dairy products. The disease condition caused by the action of aflatoxins is

known as aflatoxicosis. It is primarily a hepatic disease. The effect of aflatoxins on

animals varies depending on species, age, sex and nutritional status. The young of a

species are more prone to aflatoxicosis. Gastrointestinal dysfunction, reduced

reproductivity, reduced feed-utilization, reduced efficiency, anemia, and jaundice are the

clinical signs of aflatoxicosis.

Acute toxicity of aflatoxins is less likely than chronic toxicity. Ducklings and

trout are more susceptible to acute poisoning by aflatoxins. When aflatoxins invade liver,

the principal target organ, lipids infiltrate hepatocytes and leads to necrosis or liver cell

death. This is the result of negative reaction of aflatoxin metabolites with different cell

proteins, which leads to inhibition of carbohydrate and lipid metabolism and protein

synthesis. The decrease in liver function results in derangement of the blood clotting

mechanism, jaundice, and decrease in essential serum proteins synthesized by liver.

Other signs of aflatoxicosis are edema of the lower extremites, abdominal pain and

7

vomiting. Chronic toxicity, with sub-lethal quantities of aflatoxin for several days or

weeks, includes moderate to severe liver damage. There will be a decrease in growth

rate, lowered milk or egg production and immunosuppression. Immunosuppression is

due to the reaction of aflatoxins with T-cells, decrease in vitamin K activities, and a

decrease in phagocytic activity in macrophages.

Aflatoxins cause carcinogenicity and AFB1 is the most potent carcinogen.

Carcinogenesis has been mainly observed in ducks, trout, rats and mice. Trots are the

most susceptible and 1ppb AFB1 will cause liver cancer in trout. The occurrence of

carcinogenesis results due to the formation of 8,9-epoxide, which binds to DNA and

alters gene expression. Presence of aflatoxins enhances the chances of liver cancer in

individuals that are hepatitis B carrier.

Ruminants and non-ruminants have similar effects of aflatoxicosis. With the

continuous ingestion of toxic meal in calves, there is reduction in growth rate followed by

un-thriftiness and loss of appetite. An aflatoxin dose of 0.2 mg/ Kg body weight can

cause a decrease in weight gains. This can be due to poor feed utilization and a dramatic

increase in alkaline phosphate in the rumen. In adult ruminants, chronic aflatoxicosis can

cause anorexia, drying and peeling of the skin on the muzzle, rectal prolapse, and

abdominal edema. Aflatoxicosis also causes decreased fertility, abortion and lowered

birth weights in sheep.

1.4. Aflatoxin M1 and Dairy Products

Milk cheese and yoghurt are prominent dairy products. Milk is a lacteal

secretion of healthy dairy animals. In the United States the term “milk” refers to the milk

of cows. For the milk of other species, the name of that species precedes “milk”, for

example goat milk, sheep milk, camel milk, and buffalo milk etc. The Food and Drug

Administration of the US department of Agriculture defined milk in August, 1926 that

milk is the whole fresh, clean, lacteal secretion obtained by the complete milking of one

or more healthy cows, properly fed and kept, excluding that obtained 15 days before and

5 days after calving, or such longer period as may be necessary to render the milk

practically colostrums free (Meyer, 1987). Now, according to the Pasteurized Milk

Ordinance and Code recommended by the United States Public Health Service, 1985,

milk is defined as the lacteal secretion, practically free from colostrums, obtained by the

8

complete milking of one or more healthy cows, containing not less than 8.25% milk

solids-not-fat, and not less than 3.25% of milk fat (Eskin, 1990).

The aflatoxin B1 (AFB1) is the most commonly occurring and the most acutely

toxic among the aflatoxins (Etzel, 2002; Carnaghan et al., 1963) and is

hepatocarcinogenic (Creppy, 2002; Wogen & Newberne, 1967). After the AFB1 has been

ingested, it is bio-activated by cytochrome P-450 to a genotoxic epoxide, a DNA reactive

metabolite that forms N7- guanine adducts and it is also able to bind with proteins (Cupid

et al., 2004) that ultimately can cause cancer. AFB1 is degraded in the rumen of dairy

cows and minor but important part is re-sorbed and metabolized into AFM1 (oxygenated

derivative of AFB1) in liver. AFM1 is relatively stable and after circulation in blood it is

excreted in milk, urine or bile. The aflatoxins were declared as human carcinogens in

1987 by the International Agency for Research on Cancer (IARC) and the classification

was confirmed by re-evaluation in 1992. Later, on the demonstrated toxic and

carcinogenic effects of AFM1, the toxin initially classified by IARC as a Group 2B human

carcinogen (IARC, 1993), has now moved to Group 1 (IARC, 2002). Aflatoxin M1 has

the molecular formula C17H12O7 with relative molecular mass of 328 Da and its structure

is shown in Fig. 1.

AFM1 in milk subsequently contaminate milk products like cheese and yoghurt.

Cheese is actually coagulated casein, where as yogurt is a fermented product. Generally,

concentration of AFM1 is higher in cheese and lower in yoghurt than that of in milk from

these are prepared. When milk was artificially spiked with AFM1 in small scale

manufacture of cheese, 60% was detected in whey while 40% remained in the cheese

(Lopez et al., 2001). Prasongsidh et al. (1999) found that when Cheddar cheese was

prepared from AFM1 spiked milk, there was average three-fold increase in the curd and

the enrichment factor in cheese was about 2.3-3.4 times. Govaris et al. (2001) spiked

milk with AFM1 to produce Telemes cheeses and studied and studied distribution and

stability of AFM1 during processing, ripening and storage. Telemes cheeses were

allowed to ripen for two months and stored for an additional four months. Concentrations

of AFM1 in the curds produced were 3.9-4.4 times higher than in the milk, while

concentrations were lower in whey than in milk and curds. Aflatoxins M1 concentrations

fell in the cheese during the ripening process. It was also shown by Bakirci (2001) that

levels of AFM1 in certain cheese in Turkey were higher than in bulk milk, while AFM1

levels in cream and butter were reduced. Govaris et al. (2002) showed that when cows’

9

milk was fermented to produce yogurt, concentration of AFM1 fell between 13 and 22%

and by 16 and 34% after storage of yoghurts of pH 4.6 and 4.0 respectively.

1.5. Aflatoxin Determination Techniques

Different highly efficient and sophisticated techniques have been developed in

the recent years for the determination of mycotoxins in different commodities. The

factors which cause the selection of a specific technique include type of mycotoxin,

available time and equipment, specificity, and sensitivity. The process of analysis

involves the steps; namely sampling and sample preparation, extraction, clean-up

(purification), and detection (measurement). The analytical details may be discussed in

two sub-groups: (1) Sample preparation techniques and (2) Detection techniques.

1.5.1. (1) Sample Preparation Techniques

Sampling and sample preparation is of utmost importance in the analytical

identification of aflatoxins. It certainly affects the final conclusion. For the

determination of aflatoxins at the parts-per-billion level, the systematic approaches to

sampling, sample preparation and analysis are absolutely necessary. European Union has

formed specific plans for certain commodities e.g. corn and peanuts. Due to

homogeneous distribution of AFM1 in liquid milk, there is less uncertainty in AFM1

measurement in milk. In case of sampling the entire primary sample must be ground and

mixed so that the analytical test portion has the same concentration of toxin as the

original sample. After proper sampling, there are the steps of extraction and clean-up.

Sometimes extraction and clean-up is the same step and sometimes extraction is different

step and clean-up is different step. Extraction of samples, together with effective clean-

up step, is an essential step in the analysis of aflatoxins. The analyte migrates into the

extraction solvent. The interfering compounds are removed by clean-up step. Common

extraction solvents for aflatoxins are acetonitrile/water and methanol/water.

In addition to conventional technique of liquid-liquid extraction, there was need

to develop new techniques due to its time consuming and tedious to apply nature. The

new approaches have been developed to lessen the problems. A number of clean-up

columns, using different principles such as solid phase extraction and immunoaffinity

techniques, have been developed. The new techniques are easy to use and easily

available. The immunoaffinity columns enhance selectivity, as only the analyte is

10

retained in the column which can be eluted easily. On the other hand, in Mycosep

columns the analyte is passed and all the other interfering contaminants are retained.

1.5.1.1. Liquid-Liquid Separation

The liquid-liquid separation is a conventional process and it is based on the

partition of organic compounds between aqueous phase and immiscible organic solvent

which may be non-polar or slightly polar. Hexane and cyclohexane are frequently used

for compounds with aliphatic properties, whereas dichloromethane and chloroform are

used for medium polar contaminants. This is simple procedure and involves inexpensive

equipment. Its disadvantages include contamination and loss of sample by adsorption to

the glassware, as there are several steps. Large volumes of solvents are used and have to

be disposed and these create pollution problems. In trace analysis, solvents with high

purity have to be used which are highly costly.

1.5.1.2. Solid Phase Extraction (SPE)

Solid phase extraction is suitable for the analysis of aqueous samples. It can be

performed on-line as well as off-line. Solid phase extraction process starts with

conditioning of the column by activating it with the solvent. The sample is then applied

and the analyte is trapped in the column. The interferences are removed by rinsing step.

Finally, the analyte is eluted and then pre-concentration step is employed by evaporating

excess solvent with nitrogen. A number of samples can be prepared simultaneously with

the use of vacuum manifold. Most frequently C8 and C18 bonded silica columns are used

and these are very pressure resistant and give reproducible results. There is no significant

drawback in case of SPE as compared to liquid-liquid separation. Its advantages include

the consumption of less solvent, less time, and the possibility of automation.

1.5.1.3. Immunoaffinity Columns (IACs)

Immunoaffinity columns have become increasingly popular in recent years for

clean-up purposes, because these offer high selectivity and are easy to use. These can be

applied for purification of samples that are contaminated with different mycotoxins.

Mycotoxins are low weight molecules and they are only immunogenic if they are bound

to a protein carrier. Antibodies are produced for mycotoxins. These antibodies are bound

to an agarose, sepharose, or dextran carrier and packed in a column. The analyte

11

molecules (aflatoxins) are bound selectively to the antibodies in the column. The matrix

components do not interact with the antibodies and a rinsing (washing) step removes most

of the possible interferences. The toxin can be eluted with a solvent causing antibody

denaturation. Immunoaffinity columns have higher recovery than liquid-liquid

partitioning. Single analyte columns are available and multifunctional columns for

simultaneous determination of a number of mycotoxins are also available. Major

disadvantages include the high costs and the fact that a column can be used once due to

the denaturation of antibodies during elution step. Columns are available commercially.

1.5.1.4 MycosepTM Columns

The MycosepTM multifunctional clean-up columns (Romer Labs Inc., Union,

MT, USA) consist of a number of adsorbents (charcoal, celite, ion exchange resins and

others) which are packed in a plastic tube. On the lower end of the MycosepTM column,

there is a rubber flange, a porous frit and one-way valve which allow the extract to force

through the packing material, when the column is inserted into the culture tube (glass

tube). The purified extract appears on the top of the plastic tube with in seconds. Almost

all interfering substances are retained on the column, whereas the analyte does not show

significant affinity to the packing material. No additional washing steps are required as in

solid phase extraction. Columns are available for a range of mycotoxins and are usually

suitable for one analyte.

1.5.2. (2) Detection Techniques

After the extraction of the analyte (aflatoxin) from the sample and applying a

clean-up procedure to remove interferences, then comes identification and quantification

in the last in the analytical methodology. For the detection of aflatoxins, three main types

of assays have been developed. These include biological, analytical and immunological

methods. The biological methods were used when analytical and immunological methods

were not available for routine analysis. Biological assays are non-specific and time

consuming and are qualitative in nature.

12

1.5.2.1. Analytical Methods

Many analytical methods have been developed and are available for estimation

of aflatoxins in agricultural commodities. These include thin layer chromatography, high

performance thin layer chromatography, and high-performance liquid chromatography.

1.5.2.1.1. Thin-Layer Chromatography

Thin layer chromatography is also known as flat bed chromatography or planar

chromatography and is one of the most widely used techniques in aflatoxin analysis.

TLC is a chromatographic technique which is used for the separation, purity assessment

and identification of aflatoxins. TLC can identify and quantify aflatoxins at levels as low

as 1ng/ g. Thin-layer chromatography consists of a stationary phase immobilized on a

glass or plastic plate and a solvent acting as a mobile phase. The sample, either liquid or

dissolved in a volatile solvent, is applied in the form of a spot on the stationary phase.

Then the chromatographic plate is placed vertically in a solvent reservoir and the solvent

moves up the plate by capillary action. When the solvent front reaches a certain limit of

the stationary phase, the plate is removed from the solvent reservoir. The separated spots

are then visualized with ultraviolet light or by spraying with a suitable reagent. The

contents of a sample can be identified by running standards simultaneously with the

unknown spots. The different components in a mixture move up the plate at different

rates due to differences in their partitioning behavior between the mobile liquid phase and

the stationary phase. The Rf value for each spot is calculated. It is the ratio of the distance

(cm) from start to centre of sample spot and distance (cm) from start to solvent front. Rf

stands for “ratio of fronts” or “retardation factor”. It is characteristic for a given

compound on the same stationary phase using the same mobile phase under same

conditions of development of the plate. For identification purposes, Rf values of

standards are compared to those of unknown samples. A number of methods have been

developed for the determination of aflatoxins by TLC. Silica plates are mostly used with

a number of solvent mixtures. Mostly the solvent systems are based on chloroform and

small amounts of methanol or acetone. Now-a-days, less toxic and environmental

friendly solvent mixtures (e.g. toluene/ethyl-acetate or acetone/ iso-propanol) are also

employed. Aflatoxins are strongly fluorescent (exitation λ= 365 nm, detection or

emission λ= 430 nm) themselves and can easily be detected by fluorodensitometry.

13

Thin layer chromatography is the standard AOAC method for aflatoxin analysis

since 1971, AOAC Official Method 971.24, First Action 1971 and Final Action 1988

(AOAC Official Method 971.24, 2000). TLC separation of aflatoxins provided basis for

sensitive analytical techniques. TLC quantification method gives a reasonable level of

selectivity and sensitivity to separate aflatoxins from other interfering compounds. TLC

is the method of choice for rapid screening of aflatoxins and for situations where

advanced techniques equipments are not available.

1.5.2.1.2. High Performance Thin-Layer Chromatography

There is lack of precision associated with TLC procedures due to the

introduction of possible errors during the sample application, plate development, and

plate interpretation steps. High performance thin-layer chromatography methods improve

the precision by automating the sample application and plate interpretation steps.

1.5.2.1.3. High Performance Liquid Chromatography

High performance liquid chromatography is a very precise and highly automated

quantification technique for aflatoxins analysis with high selectivity and sensitivity.

Now-a-days, HPLC methods are widely used because of their superior performance and

reliability as compared with TLC. HPLC methods have been developed for all major

mycotoxins in cereals and other agricultural commodities. In the field of analysis of

aflatoxins, HPLC is mainly used for final separation and detection of the analyte of the

interest and extraction and clean-up techniques have to be applied prior to detection with

HPLC.

In HPLC a liquid mobile phase or solvent is used to move the sample through the

column. An immobilized liquid stationary phase is packed in the column. The analyte is

then partitioned between the two phases as it passes through the column and thus leading

to the separation of compounds due to different partitioning coefficients. Two types of

HPLC methods are commonly used i.e., normal phase chromatography and reversed

phase chromatography. In normal phase chromatography, a polar stationary phase e.g.

silica gel and a non-polar solvent e.g. hexane are used. Whereas reversed phase

chromatography (RP-HPLC) employs non-polar stationary phase e.g. C8 or C18

hydrocarbons and polar mobile phase e.g. water, methanol or acetonitrile. In HPLC,

detection is mainly accomplished by using a UV detector, diode array detector (DAD) or

14

a fluorescence detector (FLD). Fluorescence detection utilizes the emission of light (435

nm) from molecules that have been excited to higher energy levels by absorption of

electromagnetic radiation (365 nm) for aflatoxins. Fluorescence detection has superior

sensitivity than other detection systems and sometimes derivatization of the analyte has to

be performed which enhances the sensitivity. Fluorescence detection is possible in the

range of microgram/Kg. Choice of detector usually depends on the nature of the sample.

RP-HPLC is commonly performed for determination of aflatoxins in foods.

Stationary phase for aflatoxins include C18 material. Pre- or post-column derivatization

is necessary for low-level detection. For aflatoxins derivatization is performed with

strong acids or oxidants e.g. Br2, I2 or trifluoro-acetic acid. This results in increase of

fluorescence by a factor 20. Sometimes, a pre-column is employed to avoid heavy

contamination or subsequent blocking of main separation column.

1.5.2.1.4. Liquid Chromatography with Mass Spectrometric Detection

Liquid chromatography with mass spectrometric detection (LC-MS) is fairly a

recent development in aflatoxins detection and it is one of the most advanced techniques.

It is time-consuming and requires expert knowledge. In mass spectrometric detection,

extraction and clean-up techniques have to be applied before detection. In LC-MS, the

HPLC effluent enters an ionization chamber via a nebuliser. There are several techniques

for ionization, namely electrospray, thermospray, chemical and fast atom bombardment.

Fragmentation takes place in a collision chamber. The fragments then enter the high

vacuum region of the MS where detection takes place. Several set-ups are available for

optimal identification and quantification. Ion trap instruments are more suitable for

identification than triple quadruple instruments (higher MSn power), whereas triple

quadruple instruments provide better information for quantification with faster scanning

and higher sensitivity. There are also available hybrid instruments that provide a linear

ion trap in a triple quad instrument to get the best results out of both set-ups. LC-MS

methods have their applications in determination of aflatoxins in corn, milk and samples

of other commodities. In Selection-Ion-Monitoring (SIM) mode, detection can be made

at levels as low as pico-grams.

15

1.5.2.2. Immunological Methods

Immunological methods are based on the affinities of the monoclonal or

polyclonal antibodies for aflatoxins. Due to the advancement in biotechnology, highly

specific antibody-based tests are now commercially available for measuring aflatoxins in

foods in less than ten minutes. There are two major requirements for immunological

methods. First requirement is high quality antibodies and second is methodology to use

the antibodies for the estimation of aflatoxins. Being low molecular weight molecules,

aflatoxins cannot stimulate the immune system for the production of antibodies. Such

molecules of low molecular weight, which cannot evoke the immune system, are called

haptens. Therefore, before immunization, aflatoxins must be conjugated to a carrier

molecule which is a larger molecule like proteins. Bovine serum albumin (BSA) is most

commonly used as a carrier protein and hapten is conjugated with it. The three types of

immunochemical methods are: immunuaffinity column assay (ICA), radioimmunoassay

(RIA), and enzyme-linked immunosorbent assay (ELISA). Immunoaffinity columns are

mainly used for clean-up purposes and RIA has limited use in aflatoxins analysis. ELISA

is most commonly used for the estimation of aflatoxins.

1.5.2.2.1. Enzyme-Linked Immunosorbent Asssay (ELISA)

ELSA is most widely used test to detect aflatoxins, due to its simplicity,

sensitivity and adaptability. There are two types of ELISA, which are direct competitive

ELISA and indirect competitive ELISA. In direct competitive ELISA method, specific

antibody is coated to a solid phase such as a microtiter plate, whereas in indirect

competitive ELISA method, toxin-protein conjugate is coated onto the microtiter plate.

In aflatoxin analysis, direct competitive ELISA is used. ELISA is detection and

quantification of an antigen (aflatoxin) in a sample by using an enzyme labeled toxin and

antibodies specific to aflatoxin. ELISA is based on antigen-antibody reaction (Aycicek et

al., 2005). Antigen is that substance which can elicit production of antibodies when

introduced into warm blooded animals. Whereas antibodies are glycoproteins which are

produced as a result of an immune response, after introduction of antigens, leading to the

production of a specific antigen-antibody complex. In the direct competitive ELISA,

specific antibodies for aflatoxin are coated on to the wells in the microtiter strip. The

milk test samples or AFM1 standards are added to the wells. After incubation and

washing, enzyme conjugate (a conjugate of aflatoxin and bovine serum albumin is

16

attached with an enzyme molecule, such as, horseradish peroxidase or penicillinase or

alkaline phosphatase) is added to the wells. Free AFM1 and AFM1 enzyme conjugate

compete for the AFM1 antibody sites in the wells. Washing step removes any unbound

enzyme conjugate. Then substrate/chromogen is added to the wells and incubated. The

bound enzyme conjugate converts the colorless chromogen into a blue product. The stop

solution is added which leads to color change from blue to yellow. Then measurement is

made photometrically at 450 nm. The absorbance is inversely proportional to the AFM1

concentration in the sample i.e., the lower the absorbance, the higher the AFM1

concentration.

1.6. Management and Control of Aflatoxin Hazards

Aflatoxins affect adversely the economy of developing countries and public health

and hence there is strict need to control contamination of food and feed grains with

aflatoxins. Mainly, there are three methods for the management of aflatoxins hazard

namely pre-harvest management, post-harvest management, and detoxification.

1.6.1. Pre-Harvest Management

Preventing fungal growth is obviously the best method for prevention of toxin

production. Pre-harvest prevention of aflatoxins contamination is probably the best

strategy and has been most widely explored. Several recent advances in aflatoxins

elimination research, such as identification of resistant genotypes in corn and peanut

through plant breeding, result in the preeminence of this strategy. Advances in pre-

harvest control are occurring rapidly in aflatoxins as compared to other mycotoxins.

1.6.1.1. Cultural Control

Cultural control includes all the environmental and agronomic factors that

influence pod and seed infection by A.flavis and aflatoxins production. These factors may

vary considerably from one location to another location and between seasons in the same

location. Growing of same crop continuously on the same land should be avoided

because it may lead to build up of high populations of A. flavis. To avoid the

contamination of the crop, it is very important to select the cultivar which should fit a

particular growing season and mature at the end of the rainy season so that post harvest

field drying can be done under suitable conditions. It is necessary to establish optimum

plant populations, since too high population may lead to severe drought stress where rain

17

fall is sub optimal in a growing season. Hot and arid environments favour the growth of

A. flavus in the soil. In order to ensure adequate soil moisture, irrigation should be done

during the last 4-6 weeks of crop growth to prevent pre harvest aflatoxins contamination.

The individual plants which die due to the attack of pests and diseases should be lifted

separately as these are likely to be contaminated with aflatoxins. To harvest the crop at

optimum maturity is very important to control contamination. These are some main

cultural control practices which are used to prevent aflatoxin contamination.

1.6.1.2. Genetic Resistance of Cultivars

The aflatoxins problem was initially linked more to the post harvest period than to

the period of pod development in the soil. The aflatoxins contamination problem could

be solved if cultivars of different varieties of plants could be identified or bred which are

immune to seed infection by aflatoxin producing fungi or once infected do not support

aflatoxins production.

1.6.1.3. Use of Plant Breeding for Development of Host Resistance

The use of resistant varieties should be considered as a part of an integrated

aflatoxins management program incorporating cultural and crop handling procedures

which suitable to different agro ecological situations. Many breeding lines with

resistance to Aspergillus flavus colonization of seeds comparable to that of resistance

sources and with greater yield potential have been bred. However, when we are dealing

especially with resistance to natural seed infection in the field, resistance mechanisms

may operate at the pod surface, with in the testa / cotyledons. There are different genes in

conferring resistance to seed colonization, pre-harvest seed infection and aflatoxins

production by toxigenic fungi.

1.6.1.4. Genetic Engineering

Genetic engineering may be utilized to develop host resistance through addition or

enhancement of antifungal genes. Gene, native or foreign, provoking resistance must be

identified that expresses inhibitory activity against Aspergillus flavus. The selection must

be done for gene promoters which will regulate the desired type of expression of anti

fungal genes at a desired time.

18

1.6.1.5. Bio-Competitive Agents

One option of pre-harvest management is the utilization of micro-organisms as

agents of control for aflatoxins contamination. The atoxigenic strains of Aspergillus

flavus would be the best bio-competitive agent to control Aspergillus flavus in the field,

because these strains as compared to other potential microbial bio-competitive agents

would be adaptable to environmental conditions identical to the toxigenic strains and

would be biological active at same time as well. However, aflatoxin contamination is a

complex process and in most cases a combination of approaches will be required to

control this problem.

1.6.2. Post-Harvest Management

Aflatoxins are naturally occurring toxicants and are unavoidable and pose serious

challenge to food safety. Mycotoxins contaminate nearly 25% of the world’s food crops

every year. Pre-harvest management is the best strategy for controlling aflatoxin

contamination, however even then contamination occurs, post-management strategy must

be applied to manage the hazards associated with aflatoxins in foods or feedstuffs.

Aflatoxin contamination can be successfully prevented by good practices of harvesting,

drying and storage. Aspergillus flavus needs moisture content of 18-19.5% in cereal

grains for growth and toxin production. If commodities are dried below this range of

moisture content, these would be resistant to invasion by Aspergillus flavus. Storage is

the most important procedure in post-harvest management of aflatoxins. Storage of food

grains under clean, dry conditions with low kernel moisture content (about 8-10%) and at

low temperature and with protection from insect infestation is extremely important.

1.6.3. Detoxification

Foodstuffs and feedstuffs may be contaminated with levels above the acceptable

limit. In samples contaminated with toxins, all seeds are not contaminated and in many

cases toxicity limits to very small number of seeds. These contaminated seeds can be

separated from the normal healthy looking seeds depending on kernel size, color etc. The

sorting of contaminated kernels by visual examination may not be affected when healthy

appearing kernels have concealed damage through mold growth between the cotyledons.

The main objective of removal of toxic kernels is to reduce the aflatoxin levels and if the

segregation of toxic kernels cannot be effectively carried out or is partially successful,

19

there still remains the possibility of destroying the aflatoxins in foods and feedstuffs by

chemical or physical treatments.

1.6.3.1. Chemical Detoxification

One of the methods to destroy mycotoxins is chemical detoxification. It is

important that chemical detoxification or chemical treatment should be economically and

technically viable. It should meat the criteria of FAO/ WHO. These criteria include that

the process of detoxification (a) should destroy or inactivate the toxin, (b) should not

produce toxic or carcinogenic products in the finished product, (c) should destroy fungal

spores and mycelia that could proliferate and produce the toxin, (d) should preserve the

nutritive value and acceptability of the product, and (e) should not significantly alter

important technological properties of the product. Ammoniation is the method which is

most commonly used for the detoxification of the contaminated animal feed. This

procedure is used with agricultural commodities in many countries. Nixtamalization,

which is the traditional alkaline heat treatment of corn used in the manufacture of

tortillas, reduces significantly the levels of aflatoxins. It has been shown that sodium

bisulfite reacts with aflatoxins under various conditions of temperature, concentration,

and time to form water soluble products.

1.6.3.2. Physical Detoxification

Physical treatments include application of heat, irradiation with micro-waves,

gamma-rays, X-rays, UV light, and adsorption on toxin binders/ inorganic clays, to

manage the hazards of aflatoxins.

1.6.3.2.1. Thermal Inactivation

This method is good for products that are usually heat processed. However many

mycotoxins are chemically stable at processing temperatures. Aflatoxins are stable up to

their melting point around 250 °C. Aflatoxins are not completely destroyed by boiling

water, autoclaving, or a variety of food and feed processing procedures. There may be

partial destruction of aflatoxins by oil and dry roasting of peanuts.

20

1.6.3.2.2. Irradiation

Ultra violet (UV) radiation and gamma (γ) radiation would be effective in

reducing aflatoxin levels in foods. A study made by Ferreira-Castro et al. (2007) showed

the efficacy of γ–irradiation as a method of decontamination of maize containing

Fusarium verticillioides under controlled conditions. Maize grains inoculated with a

spore suspension of Fusarium verticillioides were irradiated to 2, 5, and 10 kGy. The

irradiated and control samples were analyzed for the presence of fumonisins. Their viable

cells were counted and their morphology was investigated by electronic microscopy. It

was found possible to decrease the risk of exposure to fumonisins by irradiating maize to

5 or 10 kGy, while at the dose of 2 kGy, the survived fungi (36%) can produce more

fumonisins than the fungi in the control un-irradiated samples under the same conditions.

In the same way, gamma radiations could be studied to reduce aflatoxin levels in foods

and feeds.

1.6.3.2.3. Use of Aflatoxin Binders

Adsorption and binding of aflatoxins to non-biological/ mineral materials is a very

good method and applicable method for aflatoxins decontamination. The adsorbent

materials are readily available and are a part of normal processing operation in oil

refineries. Recent researches have shown that the addition of certain adsorbents to toxin

contaminated diets can greatly reduce the bioavailability of toxins in the gastrointestinal

tract. Mineral binders have been shown to be effective in vito and in vivo. Some toxin

adsorbents are: silicate products (montmorillonite, bentonite and hydrated sodium

calcium aluminosilicate, zeolites and clinoptilolite), carbon products (activated or

supervactivated charcoal), inorganic polymers (cholestyramine, polyvinylpyrrolidone).

Among all these adsorbents, hydrated sodium calcium aluminosilicate (HSCAS) has been

the most extensively studied in vitro and was selected for extensive in vivo application in

a varied number of farm animals. HSCAS adsorb and retain 95% of aflatoxins. HSCAS

are activated by heat drying process. These inorganic clays are thought to act by ion

exchange interactions between free radicals on the clays and potentially charged groups

on the toxins. That is why the clay binders are most effective against the polar toxins

such as aflatoxins.

21

1.7. Legislation

Mycotoxins have severe impacts on health and economy, especially, in developing

countries. The Joint Expert Committee on Food Additives (JECFA), a scientific advisory

body, of the World Health Organization and the Food and Agriculture Organization has

partly evaluated the hazard for some mycotoxins (ochratoxin A, patulin, aflatoxins).

JECFA provides a mechanism for assessing the toxicity of additives, veterinary drugs

residues and contaminants (Van Egmond, 2002). Food legislation act as a safeguard to

the health of consumers and to economic interests of food producers and traders. Since

the emergence of aflatoxins in the 1960s, regulations have been established in many

countries to protect the consumers from the harmful effects of mycotoxins in foods and

feedstuffs. There are many factors that play role in the process of setting limits for

mycotoxins. These include the availability of toxicological data, survey data, availability

of analytical methods, legislation in other countries and need for sufficient food supply.

International enquires on existing mycotoxin legislation for foods and feedstuffs have

been carried out several times and details about tolerance limits, legal basis, responsible

authorities, official protocols of analysis and sampling have been published (Schuller et

al., 1983; Van Egmond, 1989; Stoloff et al., 1991; Van Egmond and Dekker, 1995).

FDA’s action levels for a flatoxins (FDA, 1994) in human food, milk, beef cattle’ feed,

feed of swine over 100 lbs, feed of breeding beef cattle or swine or mature poultry, feed

of immature animals and feed of dairy animals are 20, 0.5, 300, 200, 100, 20, and 20 µg/

kg respectively. Mycotoxin regulations have been established in about 100 countries

(Wagacha and Muthomi, 2008). National maximum tolerance level for aflatoxins in

human food in Australia, China, European Union (total aflatoxins), European Union (only

AFB1), Germany, Kenya, Guatemala, Ireland, India, and Taiwan is 5, 20, 4, 2, 4, 20, 20,

30, 30, and 50 µg/ kg (Felicia, 2004). The EU tolerance level is the strictest in standard

worldwide and considerably more precautionary than any national or international

standard.

22

2. REVIEW OF LITERATURE

Food products which could be contaminated with aflatoxins include cereal such as

maize, sorghum, pearl millet, rice, wheat (Zheng et al., 2005; Attala et al., 2003; Abbas

et al., 2002; Aly, 2002; Vergas et al., 2001; Rojas et al., 2000; Kpodo et al., 2000),

oilseeds like, groundnut, soybean, sunflower, cottonseed (Thomas et al., 2003;), spices

like chilies, black pepper, coriander, turmeric, ginger (Taguchi et al., 2002; Reddy et al.,

2001; Martins et al., 2001) , nuts like, almond, pistachio, coconut, peanut (Yu et al.,

2004; Zhang et al., 2000; Diop et al., 2000) and milk (Calaresu et al., 2006). Growing

evidence indicates the presence of aflatoxin M1 in dairy products. Recently there is

increasing need for analytical methods for the trace level identification and quantification

of aflatoxin M1. It is a potent hepato-carcinogen and there is also need to develop

strategies for its reduction and elimination in dairy products.

2.1. Toxicological Studies

Aflatoxin M1 is a cytotoxic, as demonstrated in human hepatocytes and its acute

toxicity is similar to AFB1. It was reported by Allcroft and Carnaghan (1963) that milk

from aflatoxin-fed cows contained a toxic substance which caused lesions typical of

aflatoxins poisoning in one-day-old ducklings. De Iongh et al. (1964) stated that an

extract of the toxic milk, obtained from mammals fed on aflatoxins containing diet,

contained a blue-violet fluorescent substance (later called aflatoxin M) which was

responsible for the toxicity of the milk. A substance with similar chromatographic

characteristics was recovered from the urine of sheep which were fed aflatoxin (Allcroft,

et al., 1966) and from the liver of rats dosed with aflatoxins (Butler and Clifford, 1965).

The presence of a substance in mouldy peanuts with the same characteristics as aflatoxin

M was described by De Iongh et al. (1965). Aflatoxin M was isolated from the urine of

sheep and from mouldy peanuts and was found to be consisted of two components,

named as aflatoxin M1 and aflatoxin M2 (Holzapfel, et al., 1966). These two components

are hydroxy derivatives of aflatoxin B1 and aflatoxin B2 respectively. Holzapfel et al.,

reported the acute oral LD50s of aflatoxin M1 and aflatoxin M2 and described the

histopathology of the lesions caused by AFM1 and AFM2. Newly hatched ducklings were

shown to be extremely sensitive to both AFB1 and AFM1 with LD50 values of 12µg/ bird

23

and 16µg/ bird respectively (Purchase, 1967). In case of AFM1, histopathological

examination showed liver lesions similar to those caused by AFB1 and necrosis of the

renal tubules. AFM1 and AFB1 act by a similar mechanism in causing acute toxicity and

sub-cellular changes, such as changes in liver parenchymal cells, dissociation of ribosome

from the rough endoplasmic reticulum, and proliferation of the smooth endoplasmic

reticulum. Moreover, only the naturally occurring isomer of each aflatoxin is biological

active.

Van Egmond (1994) described the results of long-term studies of toxicity of

aflatoxins. In one study by Sinnhuber et al. (1974), rainbow trout were given diets

containing aflatoxin B1 at 4 µg/ kg or AFM1 at 0, 4, 16, 32, and 64 µg/ kg for 12 months

and then were given control diet. To determine the effect of maturation on tumour

development, selected groups were held for 20 months. Some trout were fed AFM1 at 20

µg/ kg of diet for 5-30 days to determine the effect of limited oral take of the toxin.

There was significantly higher mortality rate at maturation (16-20) months with AFM1-

induced hepatomas in female trout than in male trout. The trout, which were given AFM1

at 20 µg/ kg of diet, had a 3-12% incidence of hepatoma with in 12 months. It was

concluded in the study that AFM1 is a potent liver carcinogen but less potent than AFB1.

In another study by Canton et al. (1975), rainbow trout were given diets containing AFM1

at 0, 5.9, or 27 µg/ kg and AFB1 at 5.8 µg/ kg for 16 months. The study was made by

killing fish after 5, 9, and 12 months. Degeneration of the liver was seen in all the three

groups, but no timours or preneoplastic changes were found. However, at 15 months, the

fish fed with diet containing AFB1 5.8 µg/ kg had a 13% incidence of hepatocellular

carcinoma and a 23% incidence of hyperplastic nodules, and those fed the diet with AFM1

27 µg/ kg had a 2% incidence of hepatocellular carcinoma and a 6% incidence of

hyperplastic nodules. It was concluded that AFM1 is less carcinogenic in trout than

AFB1.

Van Egmond in 1994 described two further studies in rats. In the first study,

made in 1974, weanling Fischer rats were fed 25µg/ day of synthetic AFM1 by

intubations on 5 days/week for eight consecutive weeks. Under similar conditions, a

second group of rats was given natural AFB1 at the same concentration and a control

group was also included. Among the AFM1 fed rats, only one rat (3%) developed a

hepatocellular carcinoma and 28% rats had liver lesions (preneoplastic lesions). All the

rats, receiving AFB1, developed tumors. The controls showed no significant liver lesions.

It was concluded that the carcinogenic potency of AFM1 is much lower than that of AFB1.

24

In the second study, made by Cullen et al. (1987), Fischer rats were fed on diets

containing AFM1 at 0, 0.5, 5, or 50 µg/ kg and were killed between 18 and 22 months.

Hepatocellular carcinoma was detected in 5% rats and neoplastic nodules in 15% rats fed

diets with AFM1 50 µg/ kg between 19 and 20 months. No nodules or carcinomas were

observed at the lower dose of AFM1. Among the rats fed the diet containing AFB1 50 µg/

kg, 95% rats developed hepatocellular carcinomas. Only few rats fed the diet containing

AFM1 at 50 µg/ kg developed intestinal carcinomas. The greater polarity of AFM1 than

AFB1 might be associated with the higher incidence of intestinal tumours. It was

concluded that AFM1 was a hepatic carcinogen, but with potency 2-10% that of AFB1.

Thus AFM1 is toxic in a similar manner as AFB1, but lower in magnitude.

The ability of AFB1 and AFM1 in inducing genotoxicity and DNA damage was

tested in Drosophila melanogaster in vivo in the mei-9a mei-41D5 DNA repair test and

the mwh/flr3 wing spot test. Larval stock consisting of meiotic recombination-deficient

double-mutant mei-9a mei-41D5 males and repair-proficient females was exposed to the

test agent in the repair test. Evidence of DNA damage was taken by preferential killing

of the mutant larvae. Aflatoxin M1 was found to be a DNA-damaging agent, but its

activity was about one-third of AFB1. In the wing spot test larval flies, trans-heterzygous

for the somatic cell markers mwh and flr3, were treated. The wings were inspected at

adulthood for spots manifesting the phenotype of the marker. The geotoxicity of AFM1

and AFB1 was found similar. It was concluded that AFM1 is genotoxic in vivo

(Shibahara, et al., 1995).

2.2. Methodology Studies

Much research work has been devoted over the last 40 years for developing

methods for detection and determination of mycotoxins in foods and agriculture

commodities (Chu, 1991; Holcomb, et al., 1992). This effort is continuing and keeping

pace with the progress in analytical chemistry. Methods for mycotoxins are required to

meet the legislation, monitoring and survey work, and for research. Presently, the most

commonly used methods for detection of aflatoxin M1 are HPLC-, TLC-, ELISA-methods

(Lee et al., 2009; Stubblefield and Shannon, 1974a) and FLUOROMETERIC method

(Hansen, 1990). All analytical procedures include the steps: sampling, extraction, clean-

up (purification) and determination (separation, identification and quantification). As the

distribution of aflatoxin M1 in liquid milk is reasonably homogeneous, therefore, there is

25

less uncertainty and sampling of liquid milk for aflatoxin M1 is more accurate than

sampling of granular feed products. The performance of sampling plans for aflatoxin in

granular feed products, such as shelled maize (Park, et al., 2000; Johansson, et al., 2000a;

Johansson, et al., 2000b; Johansson, et al., 2000c) and cotton seed (Whitaker et al.,

1976) has been evaluated, while there has been little evaluation of sampling plans to

detect aflatoxin M1 in milk.

Extraction, in most cases, involves conventional procedures using acetone,

chloroform and methanol etc. Small amounts of water give better extraction efficiencies.

The most significant recent improvement in the purification step is the use of solid-phase

extraction (SPE). The use of solid-phase extraction (SPE) with C18 cartridges, or

immunoaffinity columns (IAC) is now well established in aflatoxin determination.

MycocepTM columns, which remove matrix components with efficiency and can produce

a purified extract within a short time, are also available. Conventional clean-up with silica

columns is also in use. Test extracts are cleaned up before instrumental analysis (thin

layer or liquid chromatography) to remove co-extracted materials that often interfere with

the determination of target substance. The immunoaffinity clean-up procedure was

expanded in order to encompass successfully the determination of aflatoxin M1 in milk by

thin layer chromatography. Thin layer chromatography (TLC), can be used easily to

identify and determine aflatoxins as low as 1 ng/ g. Among the chromatographic

techniques applied to mycotoxins, TLC is by far the most widely used in the detection,

analysis and characterization of fungal toxins. Reviews and book chapters on

chromatography of mycotoxins in general and on TLC in particular have been published

since late 1970s (Heathcote and Hibbert, 1978; Betina, 1984). In case of detection of

AFM1, once purified extracts are obtained, the concentration of aflatoxin M1 may be

determined in several ways. Mostly the quantitative methods involve TLC or HPLC.

Aflatoxin M1 is a weakly polar component and can be extracted with solvents such as

methanol, acetone, chloroform or combination of these solvents with water. Practically,

the choice of solvent depends on the clean-up and the separation procedure. The

quantitative methods that have been developed and validated for aflatoxin M1 in milk

products were originally designed for the analysis of milk powder. Milk powder is

prepared to increase shelf life and to reduce sample bulk. The various mixtures of

methanol – water (Masri, et al., 1968; Masri, et al., 1969; Fehr, et al., 1971), acetone -

water and acetone - chloroform – water (Purchase and Steyn, 1967) were used to extract

aflatoxin M1 from milk powder.

26

Methanol and water were used as the extraction solvents in the first effective

method for the determination of aflatoxin M1 in fluid milk (Jacobson, et al., 1971). This

method was modified by McKinney (1972) and others. Stubblefield and Shannon

(1974a) accomplished extraction with acetone and water, precipitation with lead acetate

solution to de-proteinize the milk, and a de-fating step with hexane. TLC with

fluorescence detection was applied for ultimate separation, detection, and quantification.

The collaborative study proved the method to be successful (Stubblefield and Shannon,

1974b) and the method became an official AOAC method for aflatoxin M1 (AOAC

Official Method 974.17, 1990).

In another method, extraction of aflatoxin M1 from liquid milk was made with

chloroform in a separating funnel and then extract was cleaned-up over a small silica gel

column. Finally the separation was made by TLC and detection was made with

fluorescence. Aflatoxin M1 spots were quantified by visual or densitometric estimation

(Stubblefield, 1979). After modifications, this method was applied for determination of

aflatoxin M1 in cheese, in which two-dimensional TLC was applied to improve separation

of the aflatoxin M1 spots from the background. An AOAC/ IUPAC collaborative study

evaluated the method (Stubblefield, et al., 1980) and it became an official AOAC method

for aflatoxin M1 in milk and cheese (AOAC Official Method 980.21, 2000).

Van Egmond et al. (1978) confirmed the identification of aflatoxin M1 on thin

layer plate by reacting aflatoxin M1 with trifluoroacetic acid (TFA). In the method the

plate was developed with chloroform-methanol-acetic acid-water (92+8+2+0.8) mixture.

The Rf value of the blue fluorescent derivative was compared with of the AFM1 standard.

Cohen et al. (1984) proposed a liquid chromatographic (LC) method for the

determination of aflatoxin M1 in milk. The samples were initially extracted with

acetonitrile-water mixture followed by purification using a silica gel cartridge and a C18

cartridge. Final analysis by LC was achieved using a radial compression module equipped

with 5 micron C18 column and a fluorescence detector. The method was successfully

applied to samples at levels of 10 to 0.08 ng/ g added aflatoxin M1 with recoveries in the

range of 70-98%.

Tyczkowska et al. (1984) modified the official AOAC method for aflatoxin M1 by

replacing cellulose column chromatography with cartridge chromatographic clean-up and

replacing thin layer chromatographic (TLC) determination with liquid chromatographic

(LC) quantification to yield a new method for bovine and porcine milk.

27

Bijl et al. (1987) proposed a simple and sensitive method for the determination of

aflatoxin M1 in cheese. The ground cheese sample is extracted with acetone-water

mixture (3+1). Acetone is evaporated under vacuum, and the aqueous phase is passed

through a C18 disposable cartridge. After cartridge is washed with acetonitrile-water

mixture (1+9), the toxin is eluted with acetonitrile. The extract is then cleaned up on a

silica cartridge. Final analysis is performed by two dimentional thin layer

chromatography (TLC) combined with fluorodensitometry or by liquid chromatography

on a reverse phase C18 column with fluorescence detection. Recovery is greater than

90%, the coefficient of variation is 6% or less. The detection limit is in the range 10

ng/kg. The identity of aflatoxin M1 is confirmed by formation of the M2a or acetyl-M1

derivative and re-chromatography.

Analytical laboratories moved away from TLC to HPLC determination with

advances in HPLC methods in 1980s. Moreover, factory-prepared solid-phase extraction

columns became available for the purification of milk extracts. The method of Ferguson-

Foos and Warren (1984) combined these two developments successfully and originally it

was developed for normal-phase HPLC. This method was modified by using reversed-

phase HPLC, with the preparation of trifluoroacetic acid (TFA) derivatives of aflatoxins

M1 and M2, and evaluated in an AOAC collaborative study (Stubblefield and Kwolek,

1986). The method was declared as AOAC official method (AOAC Official Method

986.16, 2000)

Many rapid tests have been introduced which use specific antibodies for isolation

and detection of mycotoxins in food (Newsome, 1987; Groopman and Donahue, 1988;

Pestka, 1988). ELISA systems, using enzyme-linked antibodies for detection, are very

simple to use, but there are interferences from sample components and variability due to

test conditions (Scott, 1988). Use of immunoaffinity cartridges is a more recent advance

in quantitative extraction of aflatoxin M1. Monoclonal antibodies specific for aflatoxin

M1 are immobilized on Sepharose® and packed into small cartridges. The method of

Mortimer et al. (1987) was the first published method for aflatoxin M1 with

immunoaffinity columns. For AFM1 determination, a milk sample is loaded onto the

affinity column. The antigen i.e. AFM1 is selectively complexed by the specific

antibodies on the solid support to form antigen-antibody complex. Then, the column is

washed with water to remove all other matrix components of the sample. A small volume

of pure acetonitrile is used to elute AFM1 and the eluate is concentrated and analyzed by

HPLC coupled with fluorescence detection.

28

Immunoaffinity-based methods for aflatoxin M1 were modified and subsequently

published and studied collaboratively under the auspices of the International Dairy

Federation and AOAC international by groups of mainly European laboratories that could

determine aflatoxin M1 in milk at concentrations equal to 0.05 µg/ L. The collaborative

study of Tuinstra et al. (1993) led to International Dairy Federation Standard 171.

Another collaborative study (Dragacci et al. 2001) was conducted to evaluate the

effectiveness of an immunoaffinity column clean-up liquid chromatographic for

determination of AFM1 in milk at proposed European regulatory limits. The procedure

included centrifugation, filtration, and application of the test portion to an immunoaffinity

column. Then the column was washed with water and aflatoxin was eluted with pure

acetonitrile. Aflatoxin M1 was separated by reversed-phase liquid chromatography and

detection was made with fluorescence detector. Liquid milk samples (frozen), both

naturally contaminated with AFM1 and blank samples for spiking, were sent to 12

collaborators in 12 different European countries. Test portions of milk samples were

spiked at 0.05ng AFM1 per ml. After the removal of two non-compliant sets of results, the

mean recovery of AFM1 was 74%. The relative standard deviation for repeatability

(RSDr) ranged from 8 to 18%, based on results of spiked samples (blind pairs at 1 level)

and naturally contaminated samples (blind pairs at 3 levels). The relative standard

deviation for reproducibility (RSDR) ranged from 21 to 31%. As evidenced by

HORRAT values at the low level of AFM1 contamination, the method showed acceptable

within and between laboratory precision data for liquid milk. The collaborative study

resulted in approval of AOAC Official Method 2000.08 (AOAC Official Method

2000.08, 2005).

Manetta et al. (2005) developed a new HPLC method with fluorescence detection

using pyridinium hydrobromide perbromide as a post-column derivatizing agent to

determine aflatoxin M1 in milk and cheese. The detection limits for milk and cheese were

1 ng/ kg and 5ng/ kg respectively. The calibration curve was linear from 0.001 to 0.1 ng

injected toxin. The method includes a preliminary C-8 (SPE) clean-up. The average

recoveries of aflatoxin M1 from milk and cheese, spiked at levels of 25-75 ng/ kg and

100-300 ng/ kg, respectively, were 90 and 76%. The precision (RSDr) ranged from 1.7 to

2.6% for milk and from 3.5 to 6.5% for cheese. The method is rapid and easily

automatable and therefore is useful for accurate and precise screening of aflatoxin M1 in

milk and cheese.

29

2.3. Carry-Over of Aflatoxin in Milk

The importance of carry-over of aflatoxin in milk is above board. When AFB1 is

consumed through feedstuffs, a part of it is degraded in the rumen of dairy animals. The

remaining part of AFB1 is metabolized into AFM1 in liver. Aflatoxin M1 is a stable

compound and it circulates in the blood until it is secreted in milk and urine. Several

studies have been performed to investigate the carry-over of aflatoxin from feedstuffs to

milk. Among the early studies are those of Sieber and Blanc (1978), Applebaum et al.

(1982), and Van Egmond (1989). In these studies the results are quite variable. The

carry-over of the consumed AFB1 to AFM1 in milk varied between 0.18 and 3.94% of the

consumed quantity. The variation in results may be attributed to the low quality of earlier

analytical methods for aflatoxins.

A number of reports on carry-over of aflatoxins have been published after 1985.

Among these the prominent are those of Price et al. (1985); Frobish et al. (1986); Fremy

et al. (1987); Munksgaard et al. (1987); Pettersson et al. (1989); Harvey et al. (1991);

Veldman et al. (1992); Veldman (1992); Galvano et al. (1996). Some of these are very

accurate and extensive investigations. The carry-over % values in the studies of Price et

al., Frobish et al., Fremy et al., Munksgaard et al., Pettersson et al., Harvey et al.,

Veldman et al., Veldman and Galvano et al., were 4.07, 2.33, 0.32, 1.54, 2.60, 0.63, 6.20,

2.7, and 0.53 respectively. These studies show variations of carry-over of aflatoxin

between 0.32 and 6.2 %. The mean value of these reports is 1.81 %. Considering these

studies, a carry-over of 1.63 % is obtained when cows are fed a maximum of 500 µg of

AFB1 per day. A number of reasons are responsible for variations. These may include

experimental techniques, species, production level of milking animals, and feeding and

milking routines. The production level of milking animals is important. The study of

Pettersson et al., 1989 in Sweden and that of Veldman et al., 1992 in Netherlands, with

high milk producing dairy cows, showed the highest carry-over of 2.6% and 6.2 %

respectively.

30

2.4. Survey of AFM1 in Milk and Milk Products

In the present era large number of surveys in various countries are undertaken to

find out the incidence of AFM1 contamination in milk and milk products due to its

detrimental effects on human health.

2.4.1. Survey of AFM1 in Milk

A limited survey of was carried out by Nuryono et al., (2009) in Indonesia in

2006. They analyzed 113 fresh milk samples by ELISA for the contamination of AFM1.

Concentration of AFM1 in 48 samples (42.5%) was less than 0.005µg/ L and in 31

samples (27.4%) AFM1 concentration was found between 5 and 10 µg/L. The

concentration of AFM1 in 34 samples (30.1%) was above 10 µg/ L and none of the

contaminated samples exceeded the EU regulation limits of 0.025 and 0.05 µg AFM1/ L

for adult and infant consumption.

Shundo et al., (2009) investigated, from September to November 2006, the

presence of AFM1 in 125 samples of powdered milk, pasteurized milk and UHT milk in

the city of Sao Paulo in Brazil and estimated the average daily intake of AFM1 among the

children enrolled in day-care centres and elementary schools.. Analysis for determination

of AFM1 was done with HPLC-RP along with immunoaffinity columns for cleanup. The

quantification limit was 0.01 µg/ kg and AFM1 was found in 119 (95.2%) samples at the

levels of ranging from 0.01 to 0.2 µg/ kg with mean concentration of 0.031 µg/ kg. It

was estimated that the average daily intake of AFM1 was 0.001 µg/ kg bw per day for

children and 0.000188 µg/ kg bw per day for adults.

Herzallah (2009) examined raw and pasteurized milk of sheep, cow, and goat and

also examined eggs and beef samples for the presence of AFB1, AFB2, AFG1, AFG2,

AFM1, and AFM2. The samples were collected from different local markets in Jordan

during a period of 5 months (January-May 2007) and analyzed with HPLC using UV and

fluorescence detectors. The milk samples collected in January were found to contain 0.56

µg/ L AFM1 and 0.1 µg/ L AFM2 whilst the concentration of AFM1 and AFM2 were less

than 0.05 µg/ L for milk samples collected from March to May.

The incidence of contamination of AFM1 in milk samples collected from the

Syrian market was investigated by Ghanem and Orfi (2009) with ELISA technique. The

31

analysis of a total of 126 samples of raw milk, pasteurized milk, and powdered milk

showed that 80% samples were contaminated with various levels of AFM1 ranging

from0.020 to 0.765 µg/ L. The AFM1 contaminated samples of exceeding the American,

Syrian, and EU tolerance limits were 22%, 38%, and 52% respectively.

Lee et al. (2009) investigated the level of AFM1 in raw milk, produced in South

korea, using immunoaffinity column chromatography and HPLC with fluorescence

detector. Raw milk samples (100) were collected from 100 cattle ranches located in three

different provinces of South Korea. Out of 100, forty eight raw milk samples contained

AFM1 at low level (0.002-0.08 µg/ L) with mean value of 0.026 µg/ L. Out of the AFM1

contaminated raw milk samples, 29 samples contained only traceable amount of AFM1

below the LOQ 0.02 µg/ L. None of the samples exceeded the maximum level (0.5 µg/

L) of Korean regulation for AFM1 in milk. The limit of detection was 0.002 µg/ L and

the result of recovery test with 0.5 µg/ L AFM1 in raw milk sample was 96.3% (SD 3.6, n

= 5).

Sadeghi et al. (2009) studied the exposure of infants to and of lactating mothers

AFB1 to, using AFM1 in breast milk as a biomarker for exposure to AFB1. An enzyme-

linked immunosorbent assay was modified the analysis of AFM1 in breast milk samples

from women in Tehran, Iran. Out of 160 samples, AFM1 was detected in 157 samples by

average concentration of 8.2 ± 5.1 ng/ kg (range 0.3-26.7 ng/ kg). The concentration of

AFM1 in one sample was higher than the maximum tolerance limit accepted by EU and

USA (25 ng/ kg) and in 55 samples was higher than the maximum concentration

recommended by Australia and Switzerland (10 ng/ kg). Logistic regression did not show

significant correlation between AFM1 and gestational age, education, postnatal age,

gender, nationality, clinical condition, the number of family members, the number of

children, type and amount of dairy consumption, vegetable, fruit, oil, and meat. But there

was significant relation to the cereal consumption, also to the height at birth.

Tajkarimi et al. (2008) analyzed 319 raw milk samples collected from dairy forms

and milk collecting centers of 15 dairy plants in 14 Iranian states in winter and summer

during February and August 2004. The samples were analyzed for AFM1 with a validated

HPLC method and the recovery at 0.05 µg/ kg was 68% and coefficient of variation (CV)

was 15%. The recovery at 0.5 µg/ kg was 81% and CV was 25%. The 54% of the field

samples were found contaminated with AFM1. The mean concentration of AFM1 was

0.057 µg/ kg with 0.014 SD and sample median 0.039. The 44% of the samples had

levels <0.01 µg/ kg and 77% had levels <0.05 µg/ kg. In industrial and traditional dairy

32

forms the levels of contamination were equal, but the season had an indirect effect (α ≤

0.05) on the levels of contamination on the farms. The level of contamination in winter

was significantly (α ≤ 0.1) higher than in summer.

A survey on the presence of AFM1 and OTA in leading brands of infant formulas

marketed in Italy was conducted by Meucci et al. (2008). Infant formulas constitute an

important or often sole source of food for newborns and infants during the first months of

life. These formulas have a special role in the neonate diet because they should serve as

substitutes of human milk. The analysis of AFM1 and OTA was performed by

immunoaffinity column clean-up and HPLC with fluorescence detection. Aflatoxin M1

was found in 2 out of 185 samples but at levels that are below the European legislation

limit of 25 ng/ L.

Oveisi et al., (2007) determined natural occurrence and level of AFM1in

pasteurized liquid milk, infant formula, and milk-based cereal weaning food consumed in

Tehran, Iran. A total of 328 branded milk products and liquid milk samples were

collected and analyzed by ELISA method. The pasteurized liquid milk samples (128),

infant formula samples (20) and milk-based cereal weaning food samples (80) showed

that the incidence of contamination with AFM1 was 96.3%. The presence AFM1 of in

each group was 72.2 ± 23.5, 7.3 ± 3.9 and 16.8 ± 12.5 ng/ kg ranging between 31-113, 1-

14, and 3-35 ng/ kg respectively. In general the amount of AFM1 in 100 (78%) samples

of liquid milk and 24 (33%) samples of milk-based weaning food exceeded the maximum

tolerance limit accepted by EC, but in all of the infant formula samples it was lower than

the EC prescribed limit of 50 ng/ kg for AFM1 in milk and 25 ng/ kg for AFM1 in infant

milk products.

Decastelli et al. (2007) analyzed samples of raw cow’s milk and cattle feed from

the beginning of 2004 to the end of 2005 in Northern Italy. The presence of AFM1 in

milk and AFB1 in feed was higher than the maximum allowable limit in 1.7% of raw milk

samples and in 8.1% of feed samples. In 2005, the presence of these aflatoxins was

below the limits of EU regulations. In the analysis, an ELISA immunoassay was used as

screening test and the positive samples were confirmed by the HPLC analysis.

A study was made by Brukstiene et al. (2007) to determine the occurrence and

levels of AFM1 in the raw milk and dairy products produced in Lithuania. The obtained

results were compared with the maximum AFM1 tolerance limit for milk which is 50ng/

kg as determined by the regulation of EC. Raw milk samples were collected from forms

in various districts of Lithuania during 2004-2007 winter periods when cows are fed in

33

stables. Samples of the dairy products were analyzed during 2004-2006 by ELISA

immunochemical method. Nine raw milk samples out of 149 were fond contaminated

with AFM1. The concentration range determined was from 5.2 to 7.9 ng/ kg. After

testing 364 dairy product samples, it was found that only one sample of pasteurized milk

contained a 6 ng/ kg concentration of AFM1. In the remaining tested samples,

concentration of AFM1 was the ELISA determination level.

Atanda et al. (2007) undertook a survey to determine the AFM1 contamination of

milk and some locally produced dairy products in Abeokuta and Odeda local

governments of Ogun State, Nigeria. The samples of human and cow milk, yoghurt,

“wara”, ice-cream, and “nono” were collected randomly with in the local governments

and analyzed for AFM1 by using two dimensional TLC. Aflatoxin M1 contamination was

noticed in the range of 2.04 – 4.00 µg/ L in milk and ice-cream. Samples of human milk,

cow milk, and ice-cream showed high AFM1 concentration of 4.0, 2.04, and 2.23

respectively in abeokuta local government and AFM1 concentration of 4.0 µg/ L in cow

milk in Odeda local government. The study concluded that the AFB1 concentration in

feed which is transformed into AFM1 in milk should be reduced by good manufacturing

and good storage practices. Furthermore, there is need for stringent quality control during

processing and distribution of dairy products.

Tajkarimi et al. (2007) analyzed raw milk samples collected between April 2003

and February 2004 from milk tanks in one dairy plant in each of five regions in Iran.

Aflatoxin M1 analysis was made with validated HPLC method. Milk samples were

chosen with mean distances apart of 400 km, where there were different ecologies

(temperature, relative humidity etc.) and different agricultural products were used for

animal feeding. Twenty four to twenty five samples per season were analyzed for AFM1.

The overall mean of all samples was 0.041-0.065 µg/ L (95% confidence) and the

adjusted mean based on statistical modification was 0.039 µg/ L (61 samples had 0.000-

0.050 µg/ L, 29 samples were contaminated with 0.05-0.10 µg/ L, and remaining 8

samples had 0.10-0.39 µg/ L). All the samples were lower than Codex Alimentarius and

FDA standard for AFM1 (0.05 µg/ L). Levels of AFM1 were in winter and spring than in

summer and autumn seasons but the difference was not statistically significant (p> 0.07).

However, the level of AFM1 in milk from one region (Hamedan) was significantly lower

(p< 0.05) than in those of the other regions (Gorgan, Rasht, Shiraz, Tehran).

Ghiasian et al. (2007) carried out a survey on the occurrence of AFM1 in summer

and winter in raw milk samples from 93 traditional and industrial dairy farms of the

34

Hamedan district in order to address representative data on AFM1 in milk collected from

these regions. One nineteen samples (63.97%), out of 186 milk samples, were detected

contaminated with AFM1. The mean concentration of AFM1 in contaminated samples

was 43.4 ng/ L with the minimum and maximum levels of ≤ 10 anf 410 ng/ L

respectively. Fourteen (11.76%) contaminated samples had AFM1 concentration higher

than the maximum level specified in European Union regulations (50 ng/ L). The AFM1

contamination ratios of milk in summer and winter months were 56.5 and 71.7%

respectively.

Ayar et al. (2007) detected AFM1 contamination in 48 milk samples and AFB1

contamination in 48 feed samples. Different levels of aflatoxins were found in milk and

feed samples. Altogether, 20 raw milk samples (41.67%) and 15 feed samples (31.25%)

contained the aflatoxin levels above the legal limits established by the European

communities (EC) regulation and Turkish Food Codex. The study concluded that milk

and feed samples collected from this area constitute a potential risk for public health.

Oliveira and Ferraz (2007) analyzed 36 samples of pasteurized, ultra high-

temperature treated (UHT) and milk powder of goat traded in the city of Campinas, Brazil

for AFM1 contamination from October to December 2004 and March to May 2005.

Twenty five samples were found positive for AFM1 contamination at levels of 0.011-

0.161. The level of AFM1 contamination was below the tolerance limit of 0.50 µg/ L as

adopted for AFM1 in milk by Brazilian regulations. The mean levels of AFM1 in

pasteurized, UHT and milk powder of goat were 0.072 ± 0.048, 0.058 ± 0.044 and 0.056

± 0.031 µg/ L respectively. It was concluded that the incidence of AFM1 in goat milk

traded in Campinas is high, but at levels that probably leads to a non-significant human

exposure to AFM1 by consumption of goat milks.

Offiah and Adesiyun (2007) studied the occurrence of aflatoxins in peanuts, milk,

and animal feed in Trinidad, the West Indies. They determined the prevalence of AFB1 in

186 peanut products (140 peanut, 32 peanut butter, and 14 nut cakes) obtained from

supermarkets, road vendors, and sale outlets. They also determined the frequency of

aflatoxin M1 in 175 raw milk samples from milk collection centers and 37 pasteurized

milk samples obtained from supermarkets and sale outlets. The analytical technique used

for aflatoxins was that of radioimmunoassay method (Charm II Test) based on an indirect

competitive assay. Out of 175 raw milk samples, 13 (7.4%) were contaminated with

aflatoxin M1, while all the tested pasteurized milk samples were for aflatoxin M1

incidence.

35

Zinedine et al. (2007) surveyed 54 samples of pasteurized milk produced by five

different dairies from Morocco for the presence of aflatoxin M1 by using immunoaffinity

columns and liquid chromatography coupled to fluorescence detector. Confirmation of

AFM1 identity in positive samples was made by the formation of AFM1 hemi-acetal

derivative (AFM2a) after derivatization with TFA. The results showed that 88.8% of the

samples were contaminated with AFM1 and 7.4% were above the maximum level of 0.05

µg/ L set by the Moroccan and European regulations for AFM1 in liquid milk. The

contamination of AFM1 in milk of the five dairies was 100, 92.3, 90, 83.3, and 77.7%

respectively with AFM1 levels ranging from 0.001 to 0.117 µg/ L and the mean value of

AFM1 was 0.0186 µg/ L. According to the results of the study, the estimated daily intake

of AFM1 was 3.26 ng per person per day.

Gallo et al. (2006) reported a survey about in both raw and heat-treated milk from

Southern Italy from over the years from 2002 to 2005. Five hundreds and fifty two milk

samples of from cows, buffaloes, sheep, and goats were analyzed. Overall AFM1 was

detected in 248 samples (44.9%) from all the species. Most of these milk samples

contained AFM1 concentrations that were below the legal maximum residue level (MRL)

and only 33 samples (6.0%) were non-compliant. Bovine milk was fond the most

contaminated. The highest contamination levels of AFM1 were found during 2003 and

2005.

Unusan (2006) arranged a study having the purpose of determination of the levels

of AFM1 in UHT milk samples in central Anatolia, Turkey. The ELISA technique was

used to find out the occurrence of AFM1 in UHT milk samples. A total of 129 samples of

commercial UHT whole milk were analyzed and the mean value of AFM1 was found to

be 108.17 ng/ L. Sixty eight samples (53%) were below the limit of AFM1 in milk

permitted by EU and the remaining 61 (47%) were above the permitted limit. Four milk

samples exceeded the prescribed US regulations. The study concluded that the AFM1

levels in milk samples marketed in Antolia region appeared to be serious public health

problem at the moment. Dairy farmers must be educated by the government authorities

on potential health consequences of aflatoxins.

Diaz and Espitia (2006) conducted a study to establish the occurrence and levels

of contamination of AFM1 in retail milk from Bogota, Colombia. During 2004 and 2005,

241 samples were analyzed by HPLC coupled with fluorescence detector and with a prior

clean-up with immunoiaffinity columns. The analysis showed 69.2 and 79.4% AFM1

contamination in milk above 10 ng/ L, during 2004 and 2005 respectively. The range of

36

contamination was from 10.7 to 213.0 ng/ L in 2004 and from 10.6 to 288.9 ng/ L in2005.

In spite of the high incidence of AFM1 found in the analyzed milk samples, all samples

complied with current local regulations which allow AFM1 content in milk up to 400 ng/

L. The study emphasizes on the establishment of a permanent surveillance program for

milk consumed in Bogota in order restrict milk lots, containing AFM1 levels above the

regulatory level, entering the food chain.

Polychronaki et al. (2006) assessed the level and frequency of breast milk AFM1

as biomarker of maternal exposure. From May to September 2003, breast milk samples

were collected from a selected group of 388 lactating mothers of children attending the

New El-Qalyub Hospital, Qulyubiyah government, Egypt. After the extraction of

aflatoxin, AFM1 levels were assessed by HPLC with fluorescence detection. Aflatoxin

M1 contamination of breast milk was frequent (36%), albeit at moderate levels.

During 2002-2003, a total of 107 samples of raw, pasteueized, and UHT milk

commercialized in the cities of Sao Paulo and Marilia, Brazil, were analyzed for the

presence of AFM1 by Shundo and Sabino (2006). The samples were analyzed using

immunoaffinity columns for clean-up and TLC for determination. Aflatoxin M1 contents

were detected in 79 (73.8%) of milk samples ranging from < 0.02 to 0.26 µg/ L. The

parameters like recovery, repeatability, detection limit and quantification limit were

evaluated to in house optimization of this method. Based on spiking experiments, the

recovery values ranged from 85.83 to 73.86% at the levels of 0.010-0.50 µg/ L

respectively. The relative standard deviation for repeatability ranged from 7.73 to 2.08

µg/ L. LOQ was 0.02 µg/ L. The results of the analysis demonstrated a satisfactory

correlation when compared with HPLC. The study showed that immunoaffinity column

clean-up gave excellent results for recovery and sensibility.

Alborazi et al. (2006) evaluated AFM1 contamination in pasteurized milk samples

in Shiraz city, Iran. A total of 624 pasteurized milk samples were collected during April-

September 2003. Aflatoxin M1 was detected in 100% of the examined milk samples.

Aflatoxin M1 level in 17.8% of the samples was greater than the maximum tolerance limit

(50 ng/ L) accepted by EU. The study concluded that AFM1 contamination is a serious

problem for public health and to achieve a low level of AFM1 contamination in milk,

cows’ feed samples from various cowsheds must be evaluated routinely for aflatoxin and

kept away from fungal contamination as much as possible.

Deveci and Sezgin (2006) studied the effects of process stages on the AFM1

contents in skim milk powder which was produced from cow milk contaminated

37

artificially with AFM1 at two different levels, 1.5 and 3.5 µg/ L. Pasteurization,

concentration, and spray drying caused losses of about 16, 40, and 68% respectively in

content of the milk at 1.5 µg/ L level with AFM1, whereas losses of 12, 35, and 59

respectively were observed in the milk contaminated at the level 3.5 µg/ L of AFM1.

These losses were found to be significant statistically. After 3 and 6 months storage

periods the AFM1 content decreased by 2 and 5% respectively for the skim milk powder

produced from milk with 1.5 µg/ L AFM1, whereas the AFM1 content decreased by 2 and

5% respectively for the skim milk powder produced from milk with 3.5 µg/ L AFM1. For

3 and 6 months, changes in AFM1 content were found statistically insignificant (p > 0.05

and p > 0.01).

Bognanno et al. (2006) checked AFM1 in 240 samples of dairy ewes’ milk,

obtained from farms of Enna (Sicily, Italy) during the period of October-July 2000, by

HPLC using fluorimetric detector. The positive milk samples were for AFM1 were

confirmed by LC-MS. Aflatoxin M1 was found in 81% milk samples ranging from 2-108

ng/ L. Three samples were above the legal limit (50 ng/ L). The mean contamination of

AFM1 in milk samples obtained from ewes placed in stable was higher than that from

grazing ewes (35.27 vs 12.47 ng/ L). Samples collected in September-October showed

higher AFM1 contamination than those collected during other months (42.68 vs 10.55 ng/

L). The differences in AFM1 contamination are related to the administration of compound

feed. The present study concluded that according to the AFM1 contamination recorded in

ewe milk did not present a serious human health hazard. But the surveillance of AFM1

contamination should be more continuous and wide spread for ewe milk, because ewe

milk is exclusively used to produce cheese due to its higher protein content and AFM1 has

preferential binding to casein during coagulation of milk.

Jasutiene et al. (2006) evaluated how the technological factors of dairy product

processing affect the stability of AFM1. Milk powder was artificially contaminated with

AFM1 (0.31, 0.44, and 0.76 µg/ L) and it was dissolved in water to reconstitute milk (10%

w/v). This milk was pasteurized and fermented with three different starters (YC-180,

ABY-2, and CH-N-22) to pH 4.0 and pH 4.5. HPLC method with fluorescence detection

and with a prior clean-up step with immunoaffinity was used to determine AFM1 in milk,

pasteurized milk and fermented milk products. The excitation wavelength was set at 365

nm and emission wavelength was set at 435 nm. The study showed that three minutes

pasteurization at 95 °C had no significant effect on the AFM1 content in milk.

Fermentation with different starters to pH 4.0 and 5.0 produced significant effect on the

38

stability of AFM1 and on the average the AFM1 concentration in yoghurt and fermented

milk samples fell by 25%. It was found that the composition of starter and pH of the final

product had no statistically significant effect on AFM1 stability.

Gurbay et al. (2006) conducted a study to determine the levels of AFM1 in

commonly consumed milk samples in Ankara, capital city of Turkey. Aflatoxin M1

concentration in milk samples was determined by HPLC equipped with fluorescence

detector and the clean-up step was carried out with immunaffinity columns. The mean

recovery of the method was 117.9% and standard curves were linear in the range of 10-

200 ng/ L with correlation coefficient of 0.9998. The LOD was found to be 10 ng/ L. A

total of 27 samples were analyzed among which 24 were of UHT milk and 3 were of

daily pasteurized milk. Aflatoxin M1 was present in 59.3% samples, however only one

sample was contaminated at a level above the maximum permissible limit (50 ng/ L)

accepted by EU and Turkey.

The presence of AFM1 was determined by Kamkar (2005) in raw milk samples

from dairy plants of Sarab city of Iran during the year 2001. Total 111 milk samples were

analyzed and the presence of AFM1 was detected in 85 (76.6%) milk samples ranging

from 0.015 to 0.28 µg/ L. Level of AFM1 in 40% positive samples was higher than the

maximum tolerance limit (0.05 µg/ L) accepted by some European countries. The lowest

mean value of 0.024 µg/ L of AFM1 concentration was found in August and the highest

mean value of 0.024 µg/ L of AFM1 concentration was found in December. Aflatoxin M1

incidence level in January, February, April, and December was higher than the other

months. The mean contamination level of AFM1 was higher significantly (p< 0.01) in

autumn and winter samples than those of spring and summer. Furthermore no statistical

difference was found between AFM1 contents of spring and summer samples.

Sassahara et al. (2005) made a study aimed at to analyze the presence of

aflatoxins in foodstuffs (concentrated and roughage) destined for dairy cattle and in the

milk produced by these animals. Aflatoxin contamination of foodstuff happened mainly

in the feeds and silages did not present contamination of aflatoxins. Among the 42 milk

samples analyzed, 10 (24%) samples were found contaminated with AFM1 and 3 (7%)

samples were above the 0.5µg/ L limit.

Deveci and Sezgin (2005) made a study to determine AFM1 levels of skim milk

powders produced in Turkey. Analysis was made by HPLC along with immunoaffiniy

columns. Twenty one skim milk powder samples were collected from seven firms in four

different seasons (March-April-May, June-July-August, September-October-November,

39

and December-January-February). Aflatoxin M1 levels in the 21 samples were found in

the range of 0.0 to 0.705 µg/ kg. Two samples had the AFM1 levels (0.535 and 0.705 µg/

kg) which exceeded the Turkish Codex (0.5 µg/ kg). As a whole, 90.5% of the samples

did not exceed the maximum tolerance limit established by Turkish Codex. The study

showed that seasonal variations of AFM1 contents were statistically significant (p < 0.01)

and AFM1 contents of the samples collected in summer were lower than those of the

samples collected in the winter.

Kaniou-Grigoriadou et al. (2005) examined the presence of AFM1 in ewe’s milk

and the produced curd and Feta cheese by using ELISA. A total of 162 samples of Feta

cheese were obtained from traditional cheese-making plants of the West side of

Thessaloniki province, Greece. The levels of AFM1 in milk samples (highest value 18.2

ng/ L) were found far below the tolerance level. Higher levels of AFM1 were detected in

curd samples giving a mean enrichment factor of 4.9. The final ripened cheese was found

to be free of AFM1.

Sizoo and Van Egmond (2005) made a study the goal of which was to determine

the mass fractions of a number of analytes (AFM1, AFB1, ochratoxin A etc.) in the

duplicate diet samples collected by 123 participants in the spring and autumn 1994. For

the analysis of AFM1, AFB1, ochratoxin A, a method of analysis was developed that could

determine simultaneously these mycotoxins at very low levels. The method based on

chloroform extraction, liquid-liquid extraction, immunoaffinity cleanup and liquid

chromatography. The method was in-house validated and recoveries were found between

68-74% for AFM1 (at spiking levels of 30-120 ng/ kg, CV 7.6%), between 95-97% for

AFB1 (at spiking levels of 50-200 ng/ kg, CV 2.8%), and 75-84% for ochratoxin A (at

spiking levels of 150-600 ng/ kg, CV 4.3%). Limits of quantitation, defined as

signal/noise = 10, were found to be 24, 5, and 16 ng/ Kg in lyophilized material for

AFM1, AFB1, and ochratoxin A respectively. The method was used to analyze 123

samples of 24-hours diets and AFM1 was detected in 48% of the samples; the toxin

contents remained below the limit of quantitation in all samples. The 42% of the samples

showed AFB1 contamination and in 25% of the samples the toxin levels were above the

limit of quantitation. Ochratoxin A was detected in all the samples. The levels of the

toxins’ intakes were estimated from the analytical results. Intake levels of aflatoxins were

very low, while the mean intake of ochratoxin A was estimated to be 1.2 ng/ kg body

weight per day. This is well below the tolerable daily intake established by JECFA at 14

40

ng/ kg body weight per day. The study showed that the current dietary intake of

ochratoxin A in the Netherlands poses no appreciable health risk.

Oruc et al. (2005) determined AFM1 levels in 115 raw cow milk samples collected

from plain and mountain villages during March-April 2003 in Bursa province, Turkey, by

using ELISA technique. The mean AFM1 concentration was found 78.06 ± 6.34 ng/ kg

(0.00-212.40 ng/ kg) in milk samples plain villages and 71.72 ± 5.52 ng/ kg (12.30-

164.10 ng/ kg) in milk samples from mountain villages. The mean AFM1 concentration

in the plain villages’ milk samples was higher than those in those of mountain villages,

but the difference was not significant statistically. Overall, the incidence of in the

collected milk samples, regardless of the area differentiation, was found noticeably high

(99.13%) with approximately 60% of the samples exceeding the EU and Turkish

tolerance limit of 50 ng/ kg. The AFM1 concentration in 61.82% and 56.67% milk

samples from plain and mountain villages respectively were above the tolerance limit of

50 ng/ kg.

Celik et al. (2005) studied the contamination level of AFM1 in 85 pasteurized milk

samples which were consumed by the people of all ages including children. The

technique used for analysis was that of ELISA. Contamination of AFM1 was found in 75

(88.23%) samples, whereas 48 (64%) samples exceeded the legal level of AFM1 in milk

according to the Turkish Food Codex and Codex Alimentarius limit (50 ng/ kg or L). The

study showed serious risks for public health from milk consumption.

Rastogi et al. (2004) determined the occurrence of AFM1 contamination in Indian

infant products and liquid milk samples by using competitive ELISA technique. A total

of 187 samples comprising of infant milk food (18), infant formula (17), milk based

cereal weaning food (40), and liquid milk samples (12) were investigated and showed

87.3% contamination of AFM1. The infant milk products showed the higher range of

AFM1 contamination (65-1012 ng/ L) than that of liquid milk. Approximately 99% of the

contaminated samples exceeded the EU/ Codex Alimentarius recommended limit (50 ng/

L), whereas 9% contaminated samples the prescribed limit of US regulations (500 ng/ L).

The results suggested a need to introduce a safety limit for AFM1 levels in infant milk

products and liquid milk under the ‘ Prevention of Food Adulteration Act of India’ as

well as to describe the levels of AFB1 in dairy cattle feedstuffs so as to minimize the

health hazard risk from AFM1 contamination.

Carvajal et al. (2003) undertook a survey of AFM1 levels in milk as there was its

need due to high per capita milk consumption in Mexico. Quantification of AFM1 was

41

done in 580 samples by using HPLC. The AFM1 levels in the seven most consumed

brands from different regions were related with two processes (pasteurized and ultra-

pasteurized), different expiration dates, and different fat contents. Pasteurization and

ultra-pasteurization did not diminish AFM1 contamination. The milk with the lowest

contamination of AFM1 was a brand imported as powder and rehydrated in Mexico.

A study was undertaken by Abdulrazzaq et al. (2003) to determine whether breast

milk of mothers from the United Arab Emirates (UAE) contained aflatoxins. One

hundred and forty lactating mothers participated in the study, among these 55 were those

who had delivered premature infants and 85 were those who had full-term infants. Breast

milk samples were collected during regular feeding of infants in the special care baby unit

and postnatal wards using an electric breast pump and 10 mL milk was siphoned off into

a zinc free plastic container and AFM1 concentration was measured HPLC. Samples of

breast milk were collected between January 1999 and December 2000. Almost 66% of

the mothers were expatriates and 34% mothers were UAE nationals. The factors

considered were baby’s weight, postnatal age, sex, birth weight and gestational age.

Mothers’ nationality, age, and parity were also recorded. Overall, 92% of the breast milk

samples were found contaminated with AFM1. No significant correlation could be found

by univariate and multivariate regression analysis between AFM1 levels and the factors

studied. The study showed the need that public should be educated about hazards of

aflatoxin ingestion to reduce the presence of aflatoxin in breast milk.

Bonessi et al. (2003) conducted a study for observation of AFM1 in raw fresh or

pasteurized milk which is a part of commercial trade between members of the EU and

Emilia Romanga Region (Italy) in compliance with the institutional responsibilities of the

Veterinary Office Communitarian Exchange of the Emilia Romanga Region (Italy) and

collaboration of the Experimental Zoophylaxis Institute of Brescia. The results of the

study were favorable and none of the tested samples demonstrated AFM1 level beyond

acceptable and legal requirements.

Rodriguez-Velasco et al. (2003) used ELISA and HPLC methods for examination

of AFM1 in cow’s milk samples collected from dairy farms in the province of Leon,

Spain. Initially, the concentration of AFM1 in the milk extracts were estimated by

ELISA, with recovery rates of 74.6-109% for artificially contaminated milk at the levels

of 10-80 ng/ L. The samples, found contaminated with AFM1 at the levels above 10 ng/

L, were further quantified with HPLC. The mean recovery for HPLC was 89.3%. The

LOD was 10 ng/ L for both ELISA and HPLC. Aflatoxin M1 was confirmed in only 3.3%

42

of the samples and the concentrations in all these cases were lower than the maximum

limit applicable to these products pursuant to EU legislation. The validation of both of

the methods was done with reference material certified by the Community Bureau of

Reference.

The occurrence of AFM1 in the milk produced in an area of the Emilia region

(Italy) was surveyed from 1993 to 1998 by Pietri et al. (2003). A total of 332 milk

samples were collected from dairy farms delivering milk to factories for Parmigiano

Reggiano cheese production and AFM1 was detected in 95.5% of the samples. Twenty

eight samples (8.4%) exceeded the limit (50 ng/ kg) set by the Commission of the

European Communities (CEC) in 1998. The most contaminated samples (23 out of 28)

were found in the first two years of sampling, whereas in the later years there was a

general trend towards lower AFM1 levels.

Lopez et al. (2003) carried out a study in Argentina during winter to determine

AFM1 contamination degree of milk, as the exposure of infants to AFM1 is something to

worry about because milk is a main nutrient for children. The milk samples were

collected in winter and in the all cases AFM1 levels were lower than the recommended

limits. But, it is necessary to maintain control as AFM1 is a human carcinogen.

Garrido et al. (2003) determined the incidence of AFM1 and AFM2 in milk

samples (60 UHT and 79 pasteurized milk samples) collected from supermarkets in

Ribeirao Preto-SP, Brazil in 1999 and 2000. The analysis was done according to the

method 986.16 of AOAC International. Aflatoxin M2 could not be detected in any

sample, whereas AFM1 was detected in 29 (20.9%) samples ranging 50-240 ng/ L. The

results of the study showed that despite high incidence of AFM1 in commercial UHT and

pasteurized milk sold in Ribeirao Preto, the contamination levels of these toxins could not

be considered a serious public health problem according to MERCOSUR Technical

Regulations. Levels of AFM1 in 20.9% of the milk samples exceeded the concentration

of 50 ng/ L permitted by the EU.

Waliyar et al. (2003) made a study in which various food and feed samples

including groundnut seed, maize, sorghum, soybean cake, groundnut cake, poultry feed,

buffalo milk, cow milk, and milk powders were collected from farmers’ field, farmers’

stores, oil millers’ storage, traders’ storage, retail shops, and supermarkets. More than

2000 samples were analyzed by ELISA for aflatoxin contamination and most of the

samples contained high levels of aflatoxins with the exception of sorghum seeds.

Groundnut cake, which is the major cattle feed ingredient in the peri-urban area of

43

Hyderabad (Andhra Pradesh, India), showed aflatoxin contamination in more than 75%

samples at levels above 100 µg/ Kg leading to a high level of AFM1 in milk samples.

Strategies to reduce aflatoxin levels especially in groundnut with management

interventions at pre-harvest, harvest, and storage are necessary.

Roussi et al. (2002) determined AFM1 contamination in 298 milk samples by

immunoafinity column extraction and HPLC. In the first sampling, from December 1999

to May 2000, 114 samples of pasteurized, UHT, and concentrated milk were collected

from supermarkets, whereas 52 raw milk samples of cow, sheep, and goat were collected

from different milk producers all over Greece. In the second sampling, from December

2000 to May 2001, 54 samples of pasteurized milk, 23 samples of bulk-tank raw milk,

and 55 raw milk samples of cow, sheep, and goat were collected. In the first sampling,

the incidence rates of AFM1 contamination in pasteurized, UHT, concentrated, cow,

sheep and goat milk samples were 85.4, 82.3, 93.3, 73.3, 66.7, and 40.0% respectively.

Only one cow milk and two concentrated milk samples exceeded the EU limit of 250 ng/

L. In the second sampling, the incidence rates of AFM1 contamination in pasteurized,

bulk-tank, cow, sheep, and goat milk samples were 79.6, 78.3, 64.3, 73.3, and 66.7%

respectively. Only one cow and one sheep raw milk sample exceeded the limit of 50 ng/

L. The results proved that the current regulatory status about aflatoxins in Greece is

effective.

A study was carried out by Salem (2002) to investigate the natural occurrence of

total aflatoxins in feedstuffs and AFM1 in raw milk of dairy farms in Assiut province,

Egypt. A total of 82 feedstuff samples and 85 raw milk samples were collected from six

dairy farms and two factories producing concentrate feed in Assiut province. All the

samples were obtained between October 1999 and February 2000 and total aflatoxins in

feedstuffs and AFM1 in raw milk were analyzed by ELISA. All feedstuff samples were

found to be contained total aflatoxins in the range of 2 to 60 ng/ mL except six feed

ingredients which contained values lower than detection limit (1.75 ng/ g). The mean

value of total aflatoxins in all the feedstuff samples from all the investigated dairy farms

was 3.2 ± 1.66 ng/ g. The mean values of total aflatoxins in feedstuffs ranged from 2.0-

5.0 ng/ g in the individual farms. Four samples exceeded the Egyptian maximum level

(20 ng/ g). The highest value was found in cottonseed meal. Aflatoxin M1 was found in

50 (58.8%) of the investigated milk samples and AFM1 concentration ranged from ND –

15 ng/ L. The results revealed that AFM1 levels in the analyzed milk samples were below

the maximum tolerance limit of EU countries (50 ng AFM1/ L milk). Sixteen samples out

44

of the 50 positive samples exceeded the Swiss limit (10 ng/ L) which is the most

restrictive in the world.

Abdel-El-Fatha et al. (2002) conducted a study to determine the level of AFM1 in

camels’ milk collected from Sinai Governorate, Egypt, because the people living in this

area consume camels’ milk without any heat treatment. The collected milk samples (50)

were analyzed for AFM1 using TLC. Results revealed that 16% of the samples were

positive with a mean value of 0.65 ± 0.008 mg/ L. The concentration of AFM1 in positive

samples ranged from 0.4 to 0.9 mg/ L. The study concluded that the control measures to

safeguard consumers from AFM1 should be emphasized.

Abdel-El-Fatha (2002) determined the occurrence of aflatoxins in infants’ milk

powder samples (30) from different pharmacies in Kaliubia Governorate, Egypt. TLC

was used for the analysis of aflatoxins. The collected samples were analyzed and

screened for aflatoxins B1, B2, M1, and M2 and the data showed that the incidence of

aflatoxins B1, B2, M1, and M2 were 0.8, 0.6, 1.5, and 0.0 ppb respectively. The study

concluded that control measures to safeguard infant’s powder from exposure to aflatoxins

should be given emphasis.

Kamkar (2002) studied AFM1 contamination in commercial UHT milk in Tehran,

Iran. A total of 64 milk samples were analysed for the presence of AFM1 by using TLC

method. The data was analyzed statistically by applying one-way ANOVA. Fifty three

samples (82%) were found positive for AFM1 and 11 samples (17.4%) were negative.

Aflatoxin M1 concentration in the milk samples ranged from 69 to 387 ng/ L. All the

contaminated samples had AFM1 levels higher than the EU standard (50 ng/ L).

Martins and Martins (2000) determined contamination in 101 milk samples

collected from individual farms and supermarkets in Lisbon, Portugal, during June to

September 1999. Thirty one samples of raw milk were obtained from individual farms

and 70 UHT milk samples (18 whole milk, 22 semi-skimmed milk, and 30 skimmed milk

samples) were collected from supermarkets. Overall, incidence of aflatoxin M1 was very

high (83.2%) with 80.6% of raw milk and 84.2% of UHT milk. Among the raw milk

samples, 17 samples (54.8%) contained low levels (0.005-0.010 µg/ L), two samples

(6.5%) had levels of 0.011 and 0.020 µg/ L and six samples (19.3%) had levels between

0.021 and 0.050 µg/ L. Of the 70 samples of UHT milk, 10 samples (one whole milk,

two semi-skimmed milk, and seven skimmed milk samples) had levels below 0.005 µg/

L. Nine samples (two whole milk and seven skimmed milk samples) were contaminated

with AFM1 levels between 0.005 and 0.010 µg/ L. Twenty five samples (eight whole

45

milk, one semi-skimmed milk, and 16 skimmed milk samples) had AFM1 contamination

levels of 0.011-0.020 µg/ L. Six samples of whole milk and 18 samples of semi-skimmed

milk had levels of 0.021-0.050 µg/ L. One whole milk sample and one semi-skimmed

milk sample were contaminated at the levels of 0.059 and 0.061 µg/ L respectively and

exceeded the legal limit. The study concluded that, at the moment, the contamination of

milk with AFM1 does not appear to be a risk to public health, although the presence of

this toxin was detected in 83.2% of the samples analyzed.

Campiglio and Cerutti (1999) tested 360 samples of 23 raw materials for

aflatoxins B1, B2, G1, and G2. Eighty two samples of whole milk, skimmed milk,

powdered milk, and powdered whey were tested for AFM1. In raw materials, aflatoxins

were present in concentrations up to 40 ppb in a proportion of samples of nutmeg,

capsicum and paprika.

Ioannou-Kakouri et al. (1999) monitored aflatoxins B1, B2, G1, and G2 in locally

produced and imported foodstuffs (nuts, cereals, oily seeds, pulses) during 1992-1996.

The samples (peanuts/groundnuts, pistachios) which showed the total aflatoxins above the

Cyprus maximum level (10µg/ kg) lied between 0.7 and 6.9%. The highest incidence of

aflatoxin contamination was found in groundnut butter (56.7%) and the highest level of

AFM1 was detected in groundnuts (700 µg/ kg). Levels of AFM1 in raw and pasteurized

milk, analyzed in 1993, 1995, and 1996), were within both Cyprus tolerance limit (0.5 µg/

L) and the tolerance the tolerance limit adopted by of some European countries (0.05 µg/

L). Analysis of AFM1 was made by immunochemical methods with recoveries near 80%.

The results of the study indicated effectiveness of monitoring as well as the need for

constant surveillance and control. The control is meant for to prevent unfit products from

entering the Cyprus market and it includes sampling, retainment, analysis, and destruction

of foodstuff’ lots containing aflatoxin levels above tolerance limits.

Lopez et al. (1998) used TLC to detect AFM1 in 50 Argentinian milk samples

collected in autumn from commercial sources and from small dairy farms where cows

were manually milked. Aflatoxin M1 could not be detected in any of the samples tested.

Dhand et al. (1998) screened for aflatoxin samples of cow milk (15), buffalo milk (14),

paneer (27), khoa (28), and burfi (28) from Ludhiana, Indian Punjab. Aflatoxin M1 was

found to be present in 55 samples (7 cow milk, 5 buffalo milk, 8 paneer, 18 khoa, and 17

burfi samples). Seven samples (2 buffalo milk, 2 paneer, and 3 burfi samples) were found

to be contaminated with AFB1. Aflatoxin B2 and M2 were not detected in any of the

samples tested.

46

Choudhary et al. (1997) analyzed for AFM1 after collection of samples of raw

cow milk and raw buffalo milk from individual animals and from bulk supplies located in

and around Anand town, Gujarat, India and from the Gujarat Agriculture University

Farm. The samples of market milk supplied by three dairies in Anand town were

collected from retail outlets. The samples were analyzed for the presence of AFM1 by

HPLC. For raw cow milk, 30 (88.23%) of 34 individual samples and 31 (96.87%) of 32

of bulk samples were positive for AFM1. However on average, concentrations of AFM1

were lower in the bulk samples (mean 0.110 µg/ L) than in individual ones (mean 0.143

µg/ L). Similarly for raw cow milk, 84.21% of individual samples and 96.43% of bulk

samples were positive for AFM1. However on average, concentrations of AFM1 were

lower in the bulk samples (mean 0.074 µg/ L) than in individual samples (mean 0.076 µg/

L). Raw buffalo milk samples contained relatively lower AFM1 concentration as

compared to raw cow milk. All market raw milk samples, except one, were positive for

AFM1 but concentrations (ranging from 0.006-0.282 µg/ L) were below the action level

of 0.50 µg/ L which is laid down by Food Drug Administration. On the other hand,

83.3% of raw milk samples from individual cows exceeded this limit.

Domagala et al. (1997) collected 187 milk samples from 10 farms in the Krakow

Disrict of Poland and bulk milk samples from 5 collection points belonging to the

Krakow Dairy Cooperative during 1993-1994 and analyzed for AFM1 using ELISA.

Among the milk samples from the farm, 20% were positive for AFM1 with concentrations

ranging from 3.6 to 10.6 ng/ kg milk. From the 157 bulk milk samples, 37 (23.6%) were

positive for AFM1 and 25 samples had AFM1concentrations above 10 ng/ kg, whereas 12

had concentrations between 10 and 50 ng/ kg. None of the milk samples tested exceeded

the tolerance limit of 50 ng/ kg.

Meerarani et al. (1997) carried out HPLC analysis on 325 milk samples (buffalo

and cow milk) collected at random from milk vendors and farms during different months

of the year. Aflatoxin AFM1 was detected in 36 (11%) samples at concentration levels

ranging from 0.1 to 1.0 µg/ L. Three samples had AFM1 concentration above 0.5 µg/ L.

Saitanu (1997) examined for AFM1 270 samples of raw milk and commercial milk

products by using a radioimmunoassay. Except one sample of raw milk and eleven

samples of imported dry milk, AFM1 was found in all milk samples. In 48 (18%)

samples, including milk (17/67), pasteurized milk (20/63), UHT milk (7/60), sterilized

milk (3/60), and ‘pelleted’ milk (1/7), aflatoxin AFM1 contents were above 0.5 ppb. All

47

dried milk samples were negative for AFM1 except two samples with les than 0.1 ppb.

The results of the 5 milk samples positive for AFM1 were confirmed by HPLC.

Markaki and Melissari (1997) collected 81 samples of commercial pasteurized

milk collected from market in Athens, Greece and analysed for the presence of AFM1.

For the rapid and reliable determination of AFM1, a combination of a commercial ELISA

kit and a modified HPLC method was used. Initially the AFM1 concentrations in milk

extracts were estimated by ELISA. Samples containing AFM1 above 5.0 ng/ L, were

further quantified with HPLC. Aflatoxin M1 was derivatized to its hydroxylated product

aflatoxin M2a and then determined. The recovery of the HPLC method was nearly 100%.

Thirty two samples contained AFM1 at levels of 2.5-5.0 ng/ L and 31 samples contained

only traces of the toxin. None of the sample contained the toxin above 5.0 ng/ L. In 9

samples AFM1 could not be detected.

Oliveira et al. (1997) surveyed AFM1 in samples 300 of dried whole milk

consumed by infants at municipal schools and nurseries in Sao Paulo, Brazil. The

analyses were performed by using commercially available direct competitive ELISA kits.

Samples were reconstituted in water (1:8), centrifuged at 1630 × g for 15 minutes and

subjected directly to the assay without clean-up procedures. Results showed 33 (111%)

positive samples for AFM1 at levels of 0.10 to 1.00 ng/ mL with mean value of 0.27 ±

0.20 ng/ mL. By using the data on milk consumption patterns for 4-month old infants

who have highest intake of milk, a mean daily intake of 3.7 ng/ Kg per day was estimated.

Domagala and Kisza (1996) determined mycotoxin levels in 30 milk samples and

42 cattle fed samples from farms in Poland’s Krakow region, and also in 157 milk

samples taken at collection points for the Krakow Dairy Cooperative. Contents of

sterigmatocystin (ST), o-methyl serigmatocystin (OMST) and AFM1 were determined

using chromatography and ELISA. Analysis of feed samples included determination of

levels of ST, OMST, and AFB1, as well as toxin producing strains of Aspergillus species.

While milk samples form collection points were only analyzed for the presence of AFM1.

In case of farm milk samples, ST and OMST were not found in any sample, whereas

AFM1 was found in 4 samples when analysis was carried out by chromatography and in 6

samples when ELISA was used. In case of milk from collection points, 37 samples were

found to be contaminated with AFM1. The maximum AFM1 level was 25 ng/ kg. The

study concluded that while the levels of mycotoxins detected in feed and milk samples

were low, the risk that they present should not be disregarded, as they can easily increase

as a result of conditions. As a result of this study, it was recommended that systematic

48

testing for these contaminants should be carried out. Other recommended strategies

include raising quality requirements for both feeds and milk with respect to mycotoxin

levels. The development of sensitive and cheap analytical methods is also necessary.

Balata and Bhout (1996) analyzed for AFM1 contamination 24 samples of raw

milk collected from camels from North-Western coastal desert region (Mariout Research

Station and the Bourg El-Arab farm). Aflatoxin M1 contamination was detected in 25%

of the samples in the range of 0.30 to 0.85 µg/ L, with the mean value of 0.55 µg/ L.

Mitchell (1996) assayed for AFM1 a total of 258 samples of dried milk produced

in Northern Ireland, UK from April 1992 to October 1995. All the tested samples had

AFM1 concentrations below the advisory limit of 0.5 µg/ kg. In 42% of the samples,

concentrations were below 0.05 µg/ kg. In 1992 and 1995, 23.5% and 50% of samples

respectively had AFM1 concentrations below the LOD. In summer, AFM1 concentrations

were significantly lower than those in winter with 51% and 30% samples respectively

having concentration below the LOD. The results of the study demonstrated that the

Feeding Stuffs Regulations for controlling the levels of AFB1 in cattle feed are effective

in controlling AFM1 in milk and milk products.

Cow milk from suppliers belonging to the Arifo Milk Producers’ Cooperative,

Perugia, Italy, was collected in February and May 1995 and was analyzed for AFM1

contents by Rossi et al. (1996). The mean values for the sets of 61 and 62 samples were

approximately 10 and 13 ppt respectively. There were 11 and 8 negative samples, but

some samples showed high contamination with two farms consistently recording AFM1

contents above 80 ppt. Similarly testing of bulk ewe milk, from 42 suppliers in June,

1995 showed contents less than 5 ppt in 13 samples.

Fu (1996) evaluated the method for the detection of in milk and dried milk. The

two immunoaffinity columns used were (1) the Afla Test-P and (2) the AFLAPREP M.

By using (1), the average recoveries of AFM1 were 84.4 ± 7.0 and 94.2 ± 9.5% for the

milk spiked at 0.5 and 1.0 ppb respectively. The LOD was 0.1 ppb and the method was

easy to use and was suitable for multi-sample treatment. By using (2), the average

recoveries of AFM1 were 84.7 ± 2.7, 88.7 ± 1.9 and 89.2 ± 4.2% for the milk spiked at

0.1, 0.5 and 1.0 ppb respectively. The LOD was 0.05 ppb and the method was easy to

use. Twenty five samples of milk (sterilized, low fat and pasteurized) and 25 samples of

dried milk products (dried milk and infant formulae) were purchased from supermarkets

in Taipei, Taiwan, from July 1994 to June 1995 and tested for AFM1 by using (2).

49

Aflatoxin M1 was detected in three samples of milk at concentrations of 0.33, 0.12, 0.08

ppb. No AFM1 was detected in the tested dried milk products.

A survey of 79 milk samples collected by the UK dairy cooperative, Milk Marque,

from farms throughout England and Wales showed the presence of AFM1 (Anonymous,

1996). The limit of detection was 0.01 µg/ kg. Aflatoxin M1 was present in 8 of 40

winter samples and two of 39 summer samples ranging 0.01-0.04 µg/ kg and in 3 winter

samples ranging 0.05 to 0.10 µg/ kg. When the results were compared with previous

surveys, the maximum level of AFM1 in milk has decreased from 0.52 and 0.78 µg/ kg

when surveillance started in 1977 to 0.18 µg/ kg in 1988, 0.16 µg/ kg in 1989, and to 0.09

µg/ kg in the present survey. This decrease showed the effectiveness of Feeding-stuffs

Regulations in limiting levels of aflatoxins permitted in feed.

Diaz et al. (1995) determined occurrence of AFM1 in 100 samples of commercial

milk taken from a supermarket in Madrid, Spain, from January to June 1993. The

analytical method used for the determination of AFM1 was a dialysis diphasic membrane

procedure and high-performance TLC. Eighty six samples contained AFM1 levels below

0.01 µg/ L and 14 samples contained the toxin levels in the range of 0.02-0.04 µg/ L.

Thirty six dried milk samples of six different brands and 49 pasteurized milk

samples of four different brands were purchased from retail stores in Hermosillo, Mexico

and were analyzed for AFM1 content by Esqueda-Valle et al. (1995). Aflatoxin M1 was

isolated by antibody affinity columns and quantified by fluorometry. Recovery was 97%

at spike levels of 0.05-0.50 µg/ kg with LOD of 0.05 µg/ kg. The mean AFM1

concentrations ranged from 0.13 ± 0.01 to 0.02 ± 0.07 µg/ Kg in dried milk and from 0.13

± 0.07 to 0.27 ± 0.12 µg/ kg in pasteurized milk. The highest levels of AFM1 found in

dried and pasteurized milk samples were 0.33 and 0.49 µg/ kg respectively. The AFM1

levels in 76% of samples were below 0.2 µg/ kg and 24% samples had levels between 0.2

and 0.5 µg/ kg. None of the samples exceeded the maximum level of 0.5 µg/ kg

permitted by the FDA.

In the Gambia, West Africa, Zarba et al. (1992) initiated a study to explore the

relationship between dietary intake of aflatoxins during a one week period and a number

of aflatoxin biomarkers including aflatoxin metabolite excretion into breast milk. Five

lactating women were selected for this study and milk samples were collected by hand

expression once a day during days 3-7 for three women and during days 3-6 for the two

other women. Aflatoxin M1 was measured in all the breast milk samples by a preparative

monoclonal antibody immunoaffinity column/ HPLC method. Aflatoxin M1 was found in

50

milk samples of three of the women and the percentage of aflatoxin excreted as AFM1 in

milk ranged from 0.09 to 0.43%. The study indicated that this method can be used to

assess the levels of AFM1 in human milk and this can be used as a biomarker for exposure

of children to this carcinogen.

Fritz et al. (1977) described a method enabling a simultaneous identification and

determination of AFM1 and AFB1 in milk and dairy products by means of a self

registering fluorescence spectrophotometer with TLC accessory, directly from the plate.

The recovery rates of AFM1 and AFB1 in milk were about 83 and 82% respectively and

LOD was 0.1 µg/ kg. The recovery rates of AFM1 and AFB1 in milk powder were about

89 and 94% respectively and LOD was 0.5 µg/ kg. Of 24 analyzed samples of

commercial winter milk, four were found aflatoxin M1 positive, whereas AFB1 could not

be detected. In milk powder products, could not be detected but, AFB1 was found in one

sample.

2.4.2. Survey of AFM1 in Cheese

Dashti et al., (2009) analyzed a total of 321 milk samples (177 fresh, 105 long-

life, 27 powdered milk, and 12 human milk), 40 cheese samples and 84 feed samples for

the detection of AFM1 and total aflatoxin. The samples were collected from Kuwaiti

markets during January 2005- March 2007. The ELISA method was used and results

showed that all fresh milk samples, except one, were contaminated with AFM1 ranging

from 0.0049 to 0.0687 µg/ kg. Among the contaminated samples, 8 samples exceeded the

European Union regulatory limit. Among the long life samples, the ranges of AFM1 were

from below the detection limit to 0.0888 µg/ kg, with the four samples above the action

limit of EC. The powdered milk contained AFM1 ranging from 0.0020 to 0.0414 µg/ kg.

Among the human milk samples, only five were found contaminated AFM1 with levels

ranging 0.0088 to 0.0152 µg/ kg with a mean 0.0097 µg/ kg. The cheese samples showed

80% contamination with AFM1 with a range 0.0238- 0.452 and mean of 0.0876 µg/ kg

with one sample being above the regulatory limit (0.250 µg/ kg). The feed samples

showed 79.8% contamination with total aflatoxin.

Manetta et al. (2009) investigated the distribution of AFM1 in samples of whey,

curd, and a typical hard and long maturing cheese like Granda Padano (ripened for 12

months), produced with naturally contaminated milk in the range of 30-98 ng AFM1/ kg.

Determinations AFM1 of were carried out on 25 samples of each product by reverse-phase

51

HPLC and fluorescence detection with post-column derivatization, after a preliminary

C18-SPE clean-up. Results showed that, in comparison to milk, AFM1 concentration

levels increased both in curd (three fold) and long maturing cheese (four to five fold),

while AFM1 occurrence in whey decreased by 40%.

Ardic et al. (2009) undertook a study to determine the presence and levels of

AFM1 in Turkish white brined cheese consumed in the province of Erzurum, Turkey. A

total of 193 cheese samples were randomly collected from retail outlets and AFM1

determination was made by ELISA technique. Aflatoxin M1, at detectable level (0.05 µg/

kg), was fond in 82.4% of the samples. The AFM1 concentration ranged from 0.052 to

0.860 µg/ kg. The 26.4% samples exceed the legal limit of 0.250 µg/ kg established by

Turkish Food Codex. Widespread occurrence of AFM1 in Turkish white brined cheese

was considered to be possible hazard for public health especially for children.

Tekinsen and Eken (2008) performed a study for the analysis of AFM1 in 100

UHT milk and 132 kashar cheese samples. The samples were obtained from five big

cities: Istanbul, Izmir, Konya, Tekirdag, and Edrine. ELISA (competitive) technique was

used for the determination of AFM1. AFM1 was detected in 67% UHT milk samples and

in 82.6% kashar cheese samples. The incidence of AFM1 in positive UHT milk and

kashar cheese samples was in the range of 10 to 630 ng/ Kg and 50 to 690 ng/ kg

respectively. Aflatoxin M1 levels in 31 (31%) UHT milk samples and 36 (27.3%) kashar

cheese samples exceeded the maximum tolerable limit of EC and Turkish Food Codex.

Study showed the presence of high levels of AFM1 that that constitute a human health risk

in Turkey, therefore, milk and dairy products have to be controlled continuously for

presence of AFM1 contamination by the Turkish public health authorities.

Kamkar et al. (2008) determined the AFM1 concentration in curd and whey of

Iranian white cheese. The cheese milk samples were artificially contaminated with

aflatoxin M1 at six levels (0.25, 0.50, 0.75, 1.00, 1.25, 1.75 µg/ L). Iranian traditional

recipe was used to produce cheese. Aflatoxin M1 concentration in curd, whey, and cheese

was determined by HPLC using immunoaffinity column clean-up and fluorescence

detection. Aflatoxin M1 was found in curd, whey, and cheese in the concentrations of

0.43, 1.47, 1.57 µg/ L respectively. The aflatoxin M1 level found in curd and cheese was

found 3.12- and 3.65-fold more than that in whey that shows the affinity of AFM1 to the

protein fraction of milk.

Mohammadi et al. (2008) used a chemometric approach to minimize the AFM1

content of Iranian white brine cheese. The effects of various processing factors such as

52

renneting temperature (30-40 °C), cutting size (0.5-1.0 cm), stirring time (10-20 min),

press time (1-2 hours), curd size (64-256 cm3), and saturated brine pH (4.6-6) on AFM1

content of the cheese curds were explored. Increasing renneting time and press time, the

AFM1 content of the cheese samples decreased. Lowering of the saturated brine pH

reduced the AFM1 concentration in the cheese curds. The study showed that the optimum

processing conditions for minimization of AFM1 in cheese curds were: renneting

temperature 39.91 °C, cut size 0.51 cm, stirring time 17.71 min, press time 19.48 min,

curd size 73.27 cm3 and saturated brine pH 4.79.

Torkar and Vengust (2008) evaluated the level of AFM1 and microbiological

contamination with yeast and moulds in 60 samples of raw milk and 40 samples of curd,

soft salted or non-salted cheese and semi-hard cheese manufactured by small artisan

food-processing plants in Solvenia, collected in autumn and winter season. The results

obtained from ELISA method were confirmed with HPLC method. Four (10%) cheese

samples were found contaminated with aflatoxin M1 at the concentrations higher than 50

ng/ kg. One sample of fresh cheese, two samples of curd, and one sample of semi-hard

cheese contained 223 ng/ kg, 127 ng / kg, 51 ng/ kg, and 68 ng/ kg respectively. In one

sample of fresh cheese and in one sample of semi-hard cheese, aflatoxin M1 was found in

the concentrations of 39 and 25 ng / kg respectively. In the other 34 (85%) samples the

AFM1 concentration was lower than the limit of detection of the HPLC method (1.7 ng/

kg).

Tekinsen and Ucar (2008) studied 92 butter and 100 cream cheese samples for the

analysis of AFM1. The samples were obtained from retail outlets in the five big cities:

Istanbul, Izmir, Kayseri, Konya, and Tekirdag. The occurrence and concentration range

of AFM1 in the samples was investigated by competitive ELISA method. At the

detectable level of 10 ppt, all 100% of the butter samples and 99% of the cream cheese

samples contained AFM1. The concentration of AFM1 in butter samples and cream cheese

samples ranged from 10 to 7000 ng/ Kg and from 0 to 4100 ng/ kg respectively. The

aflatoxin M1levels in 28% of the butter and 18% of the cream cheese samples were above

the maximum tolerable limit of the Turkish Food Codex. The study showed the presence

of high aflatoxin level that constitutes a human health risk in Turkey. It was concluded

that the farmers and dairy companies need to be informed about the aflatoxin and

aflatoxicosis and the continuous monitoring of the aflatoxin level must be applied by the

Turkish public health authorities.

53

Virdis et al. (2008) reported the results of a two years survey on AFM1

contamination in goat milk and hard goat cheese produced in Sardinia, Italy. ELISA

commercial kit was used to measure the AFM1 content in 208 bulk-tank goat milk

samples. The samples obtained from 45 extensive and 7 intensive farms. Four samples

were collected during each lactating period. Aflatoxin M1 content was also studied in 41

hard goat cheese samples from 12 cheese factories. Aflatoxin M1 was detected in 36

(17.3%) bulk-tank goat milk samples at concentration of 5 to 40 ng/ L, while, in 172 it

was not detectable (< 5 ng/ L). The percentage of contaminated samples was higher in

intensive (71.4%) samples than in extensive (11.2%) ones. Aflatoxin M1 was detected in

4 (9.8%) out of 41 samples of ripened goat cheese at levels of 79.5 to 389 ng/ kg. These

contaminated samples were collected from the same cheese factory where the milk from

the intensive farms was used.

Deveci (2007) produced White Pickled cheese from cow’s milk contaminated

artificially with AFM1 at two different levels, 1.5 and 3.5 µg/ kg. The effects of process

stages on the AFM1 content were investigated. Pasteurization at 72 °C for two minutes

caused losses of AFM1 about 12% and 9% in milk contaminated with 1.5 µg/ kg AFM1

and 3.5 µg/ kg AFM1 respectively. These losses were statistically significant (p < 0.01).

It was found that after the cheese production, about 56% and 59% of total AFM1 remained

in cheese-curd, while about 32% of the total AFM1 transferred to the whey for both 1.5

µg/ kg AFM1 and 3.5 µg/ kg AFM1 contaminated milk. After the storage of three months

in brine, AFM1 content of cheeses produced from 1.5 µg/ kg AFM1 and 3.5 µg/ kg AFM1

contaminated milk decreased by 2.9% and 2.8% respectively. Changes in AFM1 content

in cheese samples were found statistically insignificant (p> 0.05 and p> 0.01) for three

months storage periods.

Colak (2007) made a study aimed at the determination of AFM1 levels in Turkish

White and Kashar cheeses which were produced with experimentally contaminated raw

milk. The distribution of AFM1 in White cheese during ripening was observed. Aflatoxin

M1 was added in concentrations of 0.25, 0.50, and 1.0 µg/ L of milk and then the cheeses

were produced according to their technologies. Whey, boiling water, cheese and brine

samples were checked for AFM1 residues. The quantification of AFM1 was done by

ELISA method. The toxin remained as 42.87% in White cheese samples. The change of

AFM1 concentration during White cheese ripening (0-90 days) was determined as the

average of 9.8%.

54

Oruc et al. (2007) investigated the distribution and stability of aflatoxin M1 in

Kashar cheese. Raw milk samples were spiked with AFM1 at 50, 250 and 750 ng/ L and

distribution of toxin in milk, cheese curd, whey, Kneading brine and cheese and its

stability during ripening was determined by HPLC. The concentration of aflatoxin M1 in

curds for each contamination level were 2.93, 3.19, and 3.37 times higher than those in

milk. After syneresis the percentage distribution of aflatoxin M1 was 40-46% in curds and

53-58% in whey, which indicated that relatively higher concentration of the toxin passed

to whey. By the kneading process, approximately 2-5% of AFM1 passed to kneading

brine. Over a sixty day storage period, there was no decrease in the concentration of

aflatoxin M1 suggesting that the toxin was stable during ripening.

Oruc et al. (2006) studied the distribution of between curd, whey, cheese and

pickle samples of Turkish white pickled cheese produced according to traditional

techniques and its stability during the ripening period. Cheeses were produced from the

milk artificially contaminated with AFM1 at the levels of 50, 250, and 750 ng/ L and

allowed to ripen for three months. Aflatoxin M1 determination was carried out with

HPLC with fluorescence detection employing immunoaffinity columns for clean-up.

During the synereses of the cheese a high concentration of AFM1 remained in curd and for

each trial the level was 3.6, 3.8, and 4.0 times higher than levels in milk. It has been

found in the study that only 2-4% of the initial spiking of AFM1 transferred into the brine

solution. During the ripening period of cheese, AFM1 levels remained constant

suggesting that AFM1 was quite stable during manufacturing and ripening.

Kamkar (2006) undertook a study to determine the presence and levels of AFM1

in cheese produced by different plants in the province of Tehran. A total of eighty cheese

samples were analyzed with TLC. Oh the 80 samples, 82.5% of the cheese samples were

found contaminated with AFM1. The range of AFM1 contamination level varied among

different months. Aflatoxin M1 concentration in May, August, November, February

samples ranged from 0.17 to1.30, 0.15 to 2.41, 0.16 to1.11 and 0.19 to 2.05 µg/ kg

respectively, while the mean values were 0.41, 0.35, 0.36, and 0.52 µg/ kg respectively.

The highest mean concentration of AFM1 was found in February samples (0.52 µg/ kg).

The lowest mean AFM1 concentration was found in August samples (0.35 µg/ kg).

According to statistical evaluation there was no significant difference (p > 0.05) between

the concentration of cheese samples taken in May and August with November and

February. It means that AFM1 contents in cheese samples taken in November were not

lower than those of cheese samples taken in May and February. Among the contaminated

55

samples, 66.6 % exceeded the maximum acceptable level (0.25 µg/ kg) that was accepted

by some of the countries such as Turkey. The study concluded the high occurrence of

AFM1 in cheese samples and was considered to be possible hazard for human health.

Baskaya et al. (2006) determined AFM1 levels in various cheese samples in

Istanbul, Turkey. A total of 363 cheese samples (131 white cheese, 132 processed cheese

and 100 kashar cheese samples) were brought to Istanbul Military food Control

Laboratory and were analyzed from 2oo2 to 2004. AFM1 levels quantification was

performed by competitive ELISA method. In 283 (77.96%) of 363 cheese samples,

AFM1 levels were found to be lower than 0.25 µg/ kg which is maximum acceptable limit

according to the Turkish standards, while in 80 (22.04%) samples the µg/ kg levels were

higher than the acceptable limit. The highest rate of AFM1 incidence (40.19%) was

determined in 2002 and the rates of AFM1 incidence 2003 and 2004 were 15.39% and

13.49% respectively. The results indicated high level of AFM1 incidence in cheese

samples which could be a potential risk for consumer health.

Kamber (2005) determined the and level of AFM1 and total mould counts in Cecil

cheese and Kars kasher cheese sold on the markets of Kars province, Turkey. A total of

60 cheese samples were purchased randomly from different shops in Kars, Eastern

Turkey. Competitive ELISA was used to determine the levels AFM1 of in cheese samples

and the concentration of AFM1 were found in the range of 51-115 ng/ kg. The mean

values of AFM1 concentration were 82.5 ng/ kg and 62.4 ng/ kg for Cecil cheese and Kars

kashar cheese respectively.

Aflatoxin M1 levels in 600 cheese samples (200 white, 200 kashar, and 200

processed cheese) were determined by Yaroglu et al. (2005). The cheese samples

werecollected from some provinces of Turkey and analyzed between January 2001 and

February 2002 in Bursa, Turkey. Competitive ELISA technique was used to determine

AFM1 levels and the highest concentration was 800 ng/ kg in kashar cheese. AFM1

contamination was detected in 30 (5%) samples (10 white, 12 kashar, and 8 processed

cheese samples). The AFM1 contamination levels in 6 (1%) samples exceeded the legal

limit of 250 ng/ kg established by the Turkish Food Codex.

The occurrence and the concentration range of AFM1 in white pickle and Van otlu

cheeses were investigated by Tekinsen and Tekinsen (2005) with fluorometric method

using immunoaffinity columns. Total 50 samples of white pickle cheese and 60 samples

of Van otlu (herb) obtained from retail outlets in Van and Hakkari, Turkey. Aflatoxin M1

was found in 86.7% samples of Van otlu cheese and 62% samples of white pickle cheese

56

at the detection level of 0.1 µg/ kg. The incidence of AFM1 ranged from 0.16 to 7.26 and

0.10 to 5.20 µg/ kg in Van otlu and white pickle cheese samples respectively. Forty

percent white pickle cheese and eighty percent Van otlu cheese samples exceeded the

maximum acceptable level of 0.25 µg AFM1 / kg cheese set by Turkish government. The

occurrence of the AFM1 in cheese samples indicated that the necessary precautions will

have to be taken to minimize the contamination of AFB1 in feed.

Aycicek et al. (2005) analyzed for AFM1, total aflatoxin, and AFB1 , 223 samples

of dairy products (49 cheese, 94 white cheese, 53 kashar cheese, 27 butter, 51 dehulled

hazelnut, and 40 cacao hazelnut samples) marketed in Ankara, Turkey during September

2002-September 2003. ELISA technique was used for analysis. The incidence of AFM1

contamination was 90.58% in the analyzed dairy products. Aflatoxin M1 levels in 19

(8.52%) out of 223 samples of dairy products were determined higher than the maximum

tolerance limit of AFM1 accepted by the Turkish Food Codex. Total aflatoxin levels in

only one of 51 dehulled hazelnut and one of 40 hazelet cacao cream exceeded the legal

limit. The study warranted for continuous surveillance programme to monitor regularly

the occurrence of aflatoxins in food and foodstuffs which are consumed by humans.

Elgerbi et al. (2004) analysed for the presence of AFM1 49 samples of raw cow’s

milk and 20 samples of fresh white soft cheese collected directly from local dairy

factories in the north-west of Libya. The HPLC method was used for toxin detection and

quantification. Thirty five (71.4%) of the 49 milk samples showed AFM1 contamination

between 0.03 and 3.13 ng/ mL. Five milk samples (free of AFM1) were artificially

contaminated with AFM1 at the levels of 0.01, 0.05, 0.1, 1.0, and 3.0 ng/ mL and the

analysis showed the average recoveries of 66.85, 72.41, 83.29, 97.94, and 98.25% with

coefficients of variation of 3.77, 4.11, 1.57, 1.29, and 0.54% respectively. Fifteen (75%)

of 20 white soft cheese samples showed AFM1 contamination between 0.11 and 0.52 ng/

g of cheese. Four cheese samples (free of AFM1) were artificially contaminated with

AFM1 at the levels of 0.1, 0.5, 1.0, and 3.0 ng/ g and the analysis showed the average

recoveries of 63.23, 78.14, 83.29, and 88.68% with coefficients of variation of 1.53, 9.90,

4.87, and 3.79% respectively. AFM1 concentrations were lower in cheese samples as

compared to milk samples.

A study was undertaken by Sarimehmetoglu et al. (2004) to determine the

presence of levels of AFM1 in cheeses consumed in the province of Ankara. A total of

400 cheese samples containing 100 samples of each of white, kashar, tulum, and

processed cheeses were studied. The cheese samples were purchased randomly from

57

different markets and were analyzed by competitive ELISA technique. Three hundred

and twenty seven cheese samples (81.75%), consisting of white (82%), tulum (81%),

kashar (85%), and processed cheese (79%) samples, were found to contain AFM1 at

different levels. One hundred and ten cheese samples (27.5%) were found to have AFM1

levels that exceed the legal limits of 250ng/ kg established by the Turkish Food Codex.

The samples exceeding the Turkish safety limits were 27% of white cheese, 24% of tulum

cheese, 34% of kashar cheese, and 25% of processed cheese.

Finoli and Vecchio (2003) analyzed samples, from sheep and dairy farms or

markets in western Sicily, of feedstuffs (15), milk (40), and cheese (30) for their

respective contents of AFB1 and AFM1 to evidence any possible indirect mycotoxin

contamination risk to the consumer. Analysis was performed with HPLC and

fluorescence detection after immunoaffionity column extraction and cleanup. Aflatoxin

M1 was detected in 30% of the milk samples at levels ranging from 4 to 23 ng/ L and 13%

of the cheeses from 21 to 101 ng/ kg. The levels of aflatoxin B1 in the feedstuff samples

ranged from < 10 to 769 ng/ kg. None of the contaminated samples exceeded the legal

limits set down by the EU for milk (50 ng/ L) and for feed (5g/ kg) or that in force in the

Netherlands for cheese (200 ng/ kg).

Gunsen and Buyukyoruk (2003) made a study in which 125 samples of

commercially available vacuum packed fresh kashar cheese randomly collected between

October 1999 and December 2000 were analyzed for Coliform bacteria, Escherichia coli,

Salmonella, Staphylococcus aureus, moulds, yeasts and levels of AFM1. Twenty eight

(32.55%) of 86 samples exceeded the Turkish kashar limits for AFM1.

Gunsen and Buyukyoruk (2002) determined by ELISA aflatoxin B1 in 25 cacao

hazelnut cream and 15 dried apricot samples and AFM1 in 130 cheese samples (35 full

fatty Turkish white cheeses, 35 fresh kashars, 25 old kashars, 20 Gravyer cheeses, and 15

cream cheeses), randomly collected traditional retail markets with insufficient chilling

facilities in Bursa, Turkey during 2001. Mean AFB1 / AFM1 concentrations in the cacao

hazelnut cream, dried apricot, and cheese were 1076.5 ± 194.4, 1441.3 ± 331.9 and 142.2

± 18.7 ng/ Kg respectively. The percentage of the cheese samples which exceeded the

Turkish AFM1 tolerance limit of 250 ng/ kg was 15.45%.

Aycicek et al. (2002) studied the occurrence of AFM1 in 183 white cheese and

butter samples in Istanbul, Turkey in 2001. The study showed the high incidence AFM1

of in white cheese (65%) and butter (81%). The study marked the importance that should

be given to routine analysis of these products for contamination of AFM1. Lopez et al.

58

(2001) carried out a study to find out the distribution of AFM1 in both whey and cheese

when cheese was manufactured on small-scale using artificially AFM1 contaminated milk

as raw material. The AFM1 was added in concentrations that varied from 1.7 to 2.0 µg/ L

of milk. The production of cheese was home-made and the concentration of AFM1 was

determined in both whey and cheese using ELISA. The greatest proportion (60%) was

detected in whey, while 40% AFM1 remained in cheese.

Oruc and Sonal (2001) determined aflatoxin M1 by ELISA in 57 cheese and 10

milk samples collected from supermarkets and street milkmen in Bursa province, Turkey.

The highest concentration of aflatoxin M1 was found 810.0 ng/ kg in full fatty white

cheese. The incidence of aflatoxin M1 was higher in cheese (89.47%) than that of milk

(10%). The concentration of aflatoxin M1 in 7 (12.28%) out of 57 cheese samples

exceeded the Turkish tolerance limit of AFM1 (250ng/ kg). None of the milk samples

exceeded EU/ Turkish tolerance limit (50ng/ L).

Govaris et al. (2001) produced Telemes cheeses using milk that was artificially

contaminated with AFM1 at levels of 0.05 and 0.10 µg/ L. The cheeses were produced in

two cheese-making trials and were allowed to ripen for two months and stored for an

additional 4 months to stimulate commercial production of Telemes cheese. The

concentration of AFM1 in whey, curd, brine, and the produced cheeses were determined

at intervals by HPLC and fluoremetric detection coupled with immunoaffinity column

extraction. The concentrations of AFM1 in the produced curds were 3.9 and 4.4 times

higher than those in milk, whereas concentrations in whey were lower than those in curd

and milk. Aflatoxin M1 was present in cheese at higher concentrations at the beginning

than at the end of the ripening/ storage period and it declined to concentrations 2.7 and

3.4 times than those initially present in milk by the end of the sixth month storage.

Concentrations of AFM1 in brine were low and increased by the end of the ripening/

storage period but only a portion of the amounts of AFM1 lost from cheese was found in

the brine. The results revealed that Telemes cheeses produced from milk containing

AFM1 at a concentration close to either the maximum limit of 0.05 µg/ L set by EU or at

double of this value, will contain AFM1 at a level that is much lower or slightly higher

respectively than the maximum acceptable level of 0.25 µg of AFM1/ kg of cheese set by

some countries.

Bakirci (2001) studied the levels of AFM1 in raw milk samples obtained from the

dairy plants of Agricultural Faculty of Yuzuncu Yil University. The fat and carry-over of

AFM1 in milk products manufactured by the same dairy plant were also studied.

59

Aflatoxin M1 was found in 79 (87.77%) of the milk samples examined and 35 (44.30%)

of the positive samples were higher than the maximum tolerance limit (0.05 ng/ mL)

accepted by Turkey and some other countries. Statistical analysis showed that there was

significant difference (p < 0.05) between the mean concentrations of AFM1 of milk

samples taken from March to April and March to May. Ninety three samples were

investigated to determine the fate and carry-over of AFM1 in milk products. The results

showed no statistical differences between AFM1 contents of bulk milk, pasteurized milk,

skimmed milk, yoghurt, buttermilk, and whey. Aflatoxin M1 contents of white pickled

cheese and kashar cheese, were higher than those of bulk milk samples, whereas those of

cream and butter samples were lower.

Prado et al. (2001) determined AFM1 in soft and parmesan cheese by

immunoaffinity column and HPLC. The samples were collected in Belo Horizonte city,

Brazil. A purified extract was obtained by extraction with dichloromethane followed by

washing with n-methane and immunoaffinity clean-up. The quantification of AFM1 was

made by HPLC using fluorescence detector. Aflatoxin M1 was detected in all the

analyzed samples of soft cheese brand, the concentration ranging between 0.02 and 0.54

ng/ g with mean level of 0.15 ng/ g. In grated parmesan cheese, AFM1was detected in 13

(93%) of 14 samples analyzed and the concentrations ranged between 0.04 and 0.30 ng/ g

with the mean level of 0.14 ng/ g.

Finoli and Vecchio (1997) analyzed for AFM1 80 samples of goat milk and 25

samples of goat milk cheese from the same suppliers collected from dairy farms

Lombardy, Italy during March-October 1996. The analyses were carried out by HPLC

with fluorescence detection after clean-up with immunoaffinity columns. Aflatoxin M1

was detected in 30% of samples from farms in the range of 3 to 37 ng/ L for milk and 19

to 160 ng/ L for cheeses. Aflatoxin M1 was detected in 11 commercial cheese samples in

the rage of 18 to 200 ng/ kg.

A total of 223 samples of Grana Padano cheese manufactured in 4 years (1991-

1994) by dairies in all provinces of the Po valley, Italy were checked for AFM1 by Pietri

et al. (1997). Grated cheese was extracted with chloroform and the defatted extract was

purified by an immunoaffinity column. Then AFM1 was determined by HPLC with

fluorescence detection. Only one sample exceeded the maximum tolerance limit (250 ng/

kg) set by some European countries. Only 15 (6.7%) samples had AFM1 concentrations

in the range of 100-250 ng/ Kg, whereas most of the samples (91%) were in the range 5-

100 ng/ kg. Mean AFM1 contamination levels for 1992 and 1994 were significantly

60

higher (p < 0.05) than those for 1993 and 1991. No significant difference of

contamination was observed among provinces or dairies of origin. It was concluded that

the situation regarding AFM1 contamination can be considered fairly satisfactory.

Barrios et al. (1996) analyzed, for the presence of AFM1, 35 samples of

commercial cheeses: 9 fresh, 9 semi-cured or semi-ripened and 17 ripened made with

different types of milk (cow, ewe, goat, and mixtures of milk of various species)

produced in the south of Spain. The method used for analysis was that of HPLC. In 16

(45.71%) of the 35 cheese samples, the presence of AFM1 was detected ranging in

concentration from 20 to 200 ng/ g of cheese. In the positive samples, the mean levels of

AFM1 were 105.33 ng/ g in ripened cheese, 73.80 ng/ g in semi-ripened cheeses, and

42.60 ng/ g in fresh cheeses.

A total of 22 samples of Spanish cheeses (12 samples of Manchego cheese with

visible signs of mould spoilage and 10 of a Blue cheese, Valdeon) were analyzed for the

presence of mycotoxins (AFB1and AFM1, sterigmatocystin, patulin, penicillic acid and

mycophenolic acid in Manchego cheese, and mycophenolic acid and roquefortine in

Valdeon cheese) by Lopez-Diaz et al. (1996). In addition, 24 Penicillium and Aspergillus

strains isolated from the samples were assessed for their ability to produce mycotoxins.

Four of the Manchego cheese samples were found to contain mycophenolic acid and one

of the Valdeon cheese samples contained roquefortine. No other mycotoxin could be

detected. One Aspergillus strain isolated from Manchego cheese had the ability to

produce aflatoxin M1. On the other hand, 7 of 9 Penicillium (P. roquefortii) strains

isolted from Valdeon cheese were able to produce roquefortine, with one strain also

producing mycophenolic acid.

Dragacci and Fremy (1996) used immuno-affinity columns to determine the

AFM1 content of cheeses with good recoveries. The analysis of cheeses in France in

1990-1995 showed that the occurrence was not very frequent. With the exception of

samples from 1989-1990, when AFB1 contaminated maize meals were used to

supplement feeds, a small portion of cheese samples contained AFM1 above the maximum

acceptable level (0.200 µg/ kg).

Dagoglu et al. (1995) investigated the presence of in 75 cheese samples by using

ELISA. Fifty samples (herb cheeses) were collected from Van and 25 were collected

from Istanbul, Turkey. Overall, 45.2% of samples contained AFM1. The highest level of

AFM1 was found in white cheese at a concentration of 0.51 ppb and the lowest was found

in herb cheese at concentration of 0.06 ppb.

61

Jahn and Rothe (1995) analyzed milk and milk products originating in Saxony,

Germany for the presence of residues and contaminants (e.g. organochlorine pesticides,

poly chlorinated biphenyls, and aflatoxins etc.) in 1994 and 1995 on behalf of Saxon

Agriculture Institute, under the ‘healthy eating’ project. Two eighty one samples of raw

milk, market milk, whipping cream, butter, cheese, quarg, and dried skim milk were

tested and it was concluded that concentrations of residues and contaminants were similar

to those for other areas of Germany.

Martins and Martins (1995) studied the presence of aflatoxins B1, B2, G1, G2, and

M1, ochratoxin A, sterigmatocystin and patulin using TLC in 182 samples of eight

commercial varieties of cheese (three types of Flamengo, Casteloes, Camembert, two

low-fat cheeses and Emmental) from five areas of Portugal from 1990 to 1993.

Mycotoxins were not detected in any of the samples studied despite the high sensitivity

and reproducibility of the method used.

Piva et al. (1988) checked 313 samples of imported liquid milk (225 from FR

Germany and 88 from France) and 159 samples of imported cheese (34 from FR

Germany, 82 from France and 43 from the Netherlands) for AFM1 in 1984. The number

of positive samples was small for both German and French milks i. e., 13.8% and 12.5%

respectively. The contamination levels were very low with maximum value of 23 ng/ L.

In case of cheeses, AFM1 was detected in 19.5, 26.5, and 53.5% of the French, German,

and Dutch samples respectively. Only two French samples of cheese exceeded 250 ng/

kg, the limit set by Swiss law. In 1985 two surveys for AFM1 contamination were carried

out on 276 milk samples mostly obtained from individual farms and on 416 cheese

samples taken from all parts of the country. As regards the milk, 70 samples (25.3%)

contained AFM1 but generally at very low levels. Only 7 samples (2.5%) exceeded 50 ng/

L. In case of cheese, 130 samples (31.3%) contained AFM1 but here again only 9

samples (2.2%) exceeded 250 ng/ L. The study showed that there was no significant in

AFM1 levels in Italian, German, and French cheese samples but these were significantly

lower (p < 0.01) than in Dutch samples.

Fremy and Roiland (1979) made several classic cheeses of Camembert from raw

milk spiked with AFM1. Three aflatoxin levels of 7, 5, and 3 µg/ L were used. Aflatoxin

M1 was recovered 35.6, 47.1, and 57.7% in the respective curds and 64.4, 52.9, and

42.3% in the respective whey. During the storage, contents of different cheeses decreased

25, 55, and 75% respectively. Similar results were obtained in milk contaminated with

AFM1 C14.

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2.4.3. Survey of AFM1 in Yoghurt

Maqbool et al. (2009) used a highly sensitive competitive ELISA method to

determine AFM1 in milk and dairy samples randomly collected from the city of

Faisalabad, Pakistan. Riedel-de-Haen, aflatoxin M1 ELISA kit (Art-No. 45169) was used

for analysis. A total of 21, 10, and 10 random single fresh milk (1L), yoghurt (1 kg), and

butter (1 kg) samples respectively collected from different areas, in the city of Faisalabad,

such dairy farms, sale points, bazaars, and markets. Duplicate analysis of the total 41

samples showed AFM1 contamination levels below the EC maximum permissible limit

(50 ng/ L). The maximum levels of AFM1 concentration found in milk, yoghurt and

butter were 40, 13, and 7.4 ng/ L respectively. Despite these results, possibility of

exceeding the limit cannot be excluded and regular monitoring is necessary.

Gurbay et al. (2006) arranged a study to investigate the levels AFM1 of in dairy

products due to its importance potential human health hazard. Aflatoxin M1 Levels were

determined by a competitive ELISA kit. Eleven cheese samples were found

contaminated AFM1 with ranging from 78.2 to 188.44 ng/ kg, while 32 0f the 40 yoghurt

samples had AFM1 levels from 61.61 to 365.64 ng/ kg. The study showed the importance

of continuous surveillance of commonly consumed cheese and yoghurt samples for AFM1

contamination in Turkey.

Lin et al. (2004) conducted a survey to probe the incidence of AFM1

contamination in dairy products in Taiwan. AFM1 contamination was investigated in 44

samples of fresh milk, 45 samples of milk powder, and 24 samples of drinking yoghurt

collected from supermarkets, convenience stores, and drug stores located in 23 counties

of Taiwan from June to August 2002. The analytical method used was that of HPLC for

quantification of AFM1 using immonoaffinity columns for extraction step. The detection

limits for fresh milk (pasteurzed milk), milk powder and drinking yoghurt were 0.002,

0.02 and 0.005 ng/ mL respectively. The recoveries of AFM1 from fresh milk at spiked

levels of 0.5 and 0.05 ng/ mL were 83.3 ± 2.9 and 89.5 ± 2.9 % respectively. The

recoveries of AFM1 from milk powder at spiked levels of 5 and 0.5 ng/ mL were 86.0 ±

1.9 and 88.7 ± 1.9 % respectively. The recoveries of AFM1 from drinking yoghurt at

spiked levels of 0.5 and 0.05 ng/ mL were 99.9 ± 1.4 and 94.8 ± 3.2 % respectively.

AFM1 contamination could not be detected in the powder milk samples. Forty fresh milk

samples showed the contamination in the range of 0.002-0.083 ng/ mL. Aflatoxin M1was

detected in three drinking yoghurt samples at the levels of 0.007, 0.009, and 0.044 ng/

63

mL. According to the food sanitary standard regulation in Taiwan, the action level of

AFM1 is 0.5 ng/ mL for fresh milk and 5 ng/ mL for milk powder. All the samples (113)

collected in this survey met the regulation requirements.

Martins and Martins (2004) analyzed for the presence of AFM1 in 96 samples of

commercial yoghurts (48 natural yoghurts and 48 yoghurts with pieces of strawberries)

that were produced in Portugal. Immunoaffinity column extraction and HPLC technique

was used for the analysis. The LOD was 10 ng/ kg and the recoveries of AFM1 from the

samples spiked at the levels of 10.0, 50.0, 100.0, and 150.0 ng/ kg were 88.0%, 91.0%,

93.0%, and 99.0% respectively. Aflatoxin M1 was detected in 18 yoghurt samples

(18.8%) ranging from 19 to 98 ng/ kg, while 78 yoghurt samples (81.2%) did not reveal

the presence of AFM1. Two samples (4.2%), out of 48 natural yoghurt samples, were

found contaminated with AFM1 containing 43 and 45 ng/ kg of AFM1. Sixteen samples

(33.3%), out of 48 yoghurt samples with pieces of strawberries, were contaminated with

AFM1 ranging 19 to 98 ng/ kg. The contamination level in six samples (12.5%) was

found to be low ranging from 19 to 35 ng/ kg, in four samples (8.3%) was found in the

range of 36 to 50 ng/ kg and in two samples (4.2%) was found with the levels of 51 and

65 ng/ kg. The contamination level in four samples (8.3%) was found to be high ranging

from 90 to 98 ng/ kg.

Sarimehmetoglu et al. (2003) analyzed AFM1 by ELISA procedure in 132

samples of yoghurt collected from different markets in Ankara Turkey during February to

July 2002. Aflatoxin M1 was detected in 49 (37.12%) yoghurt samples in the range of 50-

800 ng/ kg. The AFM1 contamination levels were above the legal limits established by

the Turkish Food Codex. The study concluded the AFM1 contamination in yoghurt as a

serious human health hazard and measures should be undertaken to avoid AFM1

contamination.

Bahout and Moustafa (2003) collected eighty random samples of plain and

flavoured yoghurt from supermarkets around Zagazig city in Egypt, during the summer of

2002 and tested for yeasts, moulds, and aflatoxins. Aflatoxin M1 residue was detected in

two (5%) plain yoghurt samples, at the levels of 0.20 and 0.30 µg/ kg. One sample of

fruit-flavoured sample showed AFM1 contamination at the level of 0.42 µg/ kg.

Aflatoxin B1 was not detected in any of the plain yoghurt samples, but it was present in

two (5%) samples of fruit-flavoured yoghurt at levels of 0.32 and 0.50 µg/ kg.

Govaris et al. (2002) studied the distribution and stability of AFM1 during

production and storage of yoghurt. Cow’s milk was artificially contaminated with AFM1

64

at levels of 0.050 and 0.100 g/ L and fermented to yoghurt to reach pHs 4.0 and 4.6.

Yoghurt fermented to pH 4.6 was also used to prepare strained yoghurt. Yoghurts were

stored for 4 weeks at 4 °C. Analysis of AFM1 in milk, yoghurt, strained yoghurt, and

yoghurt was carried out using immunoaffinity column extraction and HPLC coupled with

fluorescence detector. A significant decrease (p< 0.01) was observed in AFM1 levels

compared with those initially added to milk. The growth of culture lactic acid bacteria

was found not to be affected in the AFM1 contaminated yoghurts, with the exception of

streptococcus thermophilus that showed significantly (p < 0.01) lower increase in the

yoghurt containing the toxin at high concentration. After fermentation, AFM1 was

significantly lower (p< 0.01) in yoghurts with pH 4.0 than in yoghurts with pH 4.6 at both

the contamination levels. During the storage in refrigerator, AFM1 was rather more stable

in yoghurts with pH 4.6 than with pH 4.0. The percentage loss of the initial amount of

AFM1 in milk was estimated 13% and 22% by the end of the fermentation, 16% and 34%

by the end of storage for yoghurts with pHs 4.6 and 4.0 respectively. The percentage

distribution ratio of the initial toxin (AFM1 in the yoghurt) in strained yoghurt/ yoghurt

whey was about 90/ 10 and 87/ 13 for the lower and the higher contamination levels

respectively.

Srivastava et al. (2001) analyzed, as part of the programme on environmental

contaminants in food stuffs in Kuwait, 54 samples of fresh full cream, skimmed milk,

yoghurt, and infant formula were analyzed for by HPLC following clean-up with

immunoaffinity columns. The contamination of aflatoxin M1 was found in 28% samples.

Galvano et al. (2001) collected, during 1996, 161 samples of milk, 92 samples of

dry milk for infant formula, and 120 samples of yoghurt from supermarkets and drug

stores in four big Italian cities and checked for AFM1 by immunoaffinity extraction and

HPLC. Aflatoxin M1 was detected in 125 (78%) of milk samples ranging from < 1 to 23.5

ng/ L with the mean level of 6.28 ng/ L. In case of dry milk samples, AFM1 was detected

in 49 (53%) samples ranging from < 1 to 79.6 ng/ kg with the mean level of 32.2 ng/ kg.

While, in case of yoghurt samples, AFM1 was detected in 73 (61%) samples ranging from

< 1 to 32.1 ng/ kg with the mean level of 9.06 ng/ kg. Altogether, only in four dry milk

samples AFM1 concentration was above the legal limit established by EC in 1999.

Kim et al. (2000) investigated the occurrence of AFM1 in pasteurized milk and

dairy products by using direct competitive ELISA and HPLC. By ELISA, the recoveries

of AFM1 from the samples spiked at levels between 5 and 500 pg/ mL were 88.0-106.5%

for pasteurized milk and 84.0-94.0% for yoghurt. By HPLC, the recoveries were 103-

65

120% and 87.0-93.0% for pasteurized milk and yoghurt respectively. The LOD was 2 pg/

mL by ELISA and 10 pg/ mL by HPLC. A total of 180 samples were collected in Seoul,

Korea and analyzed. The incidence of AFM1 in pasteurized milk, infant formula,

powdered milk and yoghurt was 76, 85, 75, and 83% respectively mean concentrations of

18, 46, 200, and 29 pg/ g respectively when determined by ELISA. The results obtained

by ELISA were closely related to those by HPLC for AFM1.

Galvano et al. (1998), during 1995, conducted a study in which 159 samples of

milk, 97 samples of dried milk for infant formula, and 114 samples of yoghurt were

collected from supermarkets and shops in 4 Italian cities and were analysed for by

immunoaffinity extraction and HPLC. Aflatoxin M1 was detected in 136 (86%) of the

milk samples in the range < 1 to 108.5 ng/ L with mean 21.77 ng/ L. Among the dried

milk samples, AFM1 was detected in 81 (84%) of the samples in the range of < 1 to 101.3

ng/ kg with mean 21.77 ng/ kg. Aflatoxin M1 was detected in 91 (80%) of yoghurt

samples in the range of <1 to 496.5 ng/ L with mean 18.08 ng/ L. Only 5 samples (2

milk, 2 dried milk, and 1 yoghurt samples) contained AFM1 levels exceeding the Swiss

legal limits. Aflatoxin M1 levels were 4 times higher in milk and yoghurt samples

collected during November to April than those collected during May to October. The

study concluded that during 1995, despite the widespread occurrence of AFM1, the mean

contamination levels in milk products sold in Italy were not a serious human health

hazard.

Galvano et al. (1996) reviewed critically data from literature since 1980 on the

occurrence of AFM1 in human and animal milk, infant formula, dried milk, cheese and

yoghurt. Moreover, the influence of storage and processing of milk and milk products on

the occurrence and stability of AFM1 was reviewed. It was concluded that: (1) attempts

should be made to harmonize already existing regulatory limits for aflatoxins in foods and

feed; (2) to avoid uncertainty in actual practice, further investigations should verify the

influence of milk storage and processing on AFM1 occurrence; (3) the occurrence of

AFM1 in animal milks and milk products is widespread, however, contamination levels do

not appear to be a serious health hazard; (4) monitoring programmes should be made

extensive and frequent; and (5) in tropical and subtropical countries, especially in African

countries, particular attention should be devoted to monitoring milk and milk products as

well as feed. Finally the review concluded that extensive and periodic surveys on the

occurrence of aflatoxins and their metabolites in human milk should be performed, since

there is risk of a serious health hazard to mother, fetus, or infants.

66

De Sylos et al. (1996) analyzed for AFM1, a total of 152 samples of pasteurized

milk, powdered milk, cheese and yoghurt, collected from groceries and supermarkets in

Campinas, Brazil, during 1989-1990 by using The AOAC method (visual quantification)

980.21. Fifty two pasteurized milk samples were also analyzed in 1992 by using HPLC

method. Aflatoxin M1 was not detected in the samples studied during 1989-1990. Four

milk sample of the 1992 batch were found contaminated with AFM1 in the range of 73-

370 ng/ L. Except for the sample containing 370 ng/ L, which would also have been

positive using the TLC method, the study concluded that the detection of AFM1 in 1992

reflected the higher sensitivity of the HPLC method and not a greater occurrence of the

toxin.

Stoloff et al. (1981), in the year 1979, conducted a survey of manufactured dairy

products (992 samples of nonfat dry milk, vanilla ice cream, yoghurt, Cheddar cheese,

and cottage cheese) for AFM1 contamination. One sample of cottage cheese had

detectable aflatoxin equivalent to 0.08 ng/ mL in the milk from which the product was

made. The samples were collected by Food and Drug District inspectors from randomly

selected establishments at three times throughout the year. The study concluded that in a

“normal” year should not be in manufactured dairy product in the United States at a level

in excess of that from milk with 0.1 ng AFM1/ mL.

Polzhofer (1977) investigated, during the period from September 1972 till

December 1974, 260 samples of milk, 41 of milk powder, 54 of yoghurt, 80 of fresh

cheese, 65 of Camembert cheese, 77 of hard cheese, and 134 of processed cheese for their

contents of aflatoxin B1, B2, G1, G2 , and M1. Only AFM1 could be detected in all

products. During the period of investigation, the average values of AFM1 concentration

were 0.07, 0.50, 0.20, 0.23, 0.43, 0.26, and 0.31 µg/ kg for milk, milk powder, yoghurt,

fresh cheese, hard cheese, processed cheese, and Camembert cheese respectively. The

maximum values of AFM1 concentration found were 0.33, 2.0, 0.47, 0.51, 0.73, 1.3, and

0.55 µg/ kg for milk, milk powder, yoghurt, fresh cheese, Camembert cheese, hard cheese

and processed cheese respectively.

67

3. MATERIALS AND METHODS

3.1. Materials and Instruments

3.1.1. Milk Samples

In this study, a total of 817 milk samples were analyzed. The 168 samples of raw

milk were randomly obtained from several collaborating dairies in 14 districts of the

Punjab, during the year 2005. The samples were collected in the middle of every month.

Samples were kept in freezer in case of delayed analysis. A total of 480 fresh milk

samples, comprising of 360 buffalo milk samples and 120 cow milk samples, were

procured by directly approaching the milking sites between January 2007 and March

2007. Area for milk collection was divided into three categories namely, urban, semi-

urban, and rural. The milk samples were stored in freezer compartment inside a

refrigerator until these were analyzed for AFM1. The milk samples were placed in a

cooler with icepacks during transportation. The samples of buffalo milk (55), cow milk

(40), goat milk (30), sheep milk (24), and camel milk (20) were obtained during the first

half of the year 2007 from the area of Faisalabad district in the Punjab province of

Pakistan. The milk samples were either analyzed immediately or stored in freezer in case

of delayed analysis.

3.1.2. Samples of Cheese and Yoghurt

The samples of cheese and yoghurt were collected from Faisalabad city during

2008. Eighty samples of cheese and eighty samples of yoghurt were analyzed.

3.1.3. Feed Samples

The total 260 samples of dairy feedstuff were analyzed for the contamination of

AFB1 which comprised of 60 concentrate feed, 80 cotton-seed cake, 36 wheat bran, 24

bread pieces, 36 wheat straw, and 24 paddy straw samples. Samples of concentrate feed

and other feedstuffs were collected in the start of 2007 and in the start of 2008 from the

areas of district Faisalabad and district Lahore in Pakistan.

68

3.1.4. Chemicals and Standards

All the reagents used were of Analar Grade. Acetonitrile (HPLC grade) of Sigma-

Aldrich (Steinheim, Germany) was used. The immunoaffinity columns AflaM1TM HPLC

were obtained from VICAM (Watertown, MA, USA). The MycoSep® 226 AflaZon+

(push-through format) columns were obtained from Romer Labs, Inc. (Stylemaster Drive

Union, MO 63084-1156 USA). The water used during analysis was double distilled with

water distillation system (Bibby, UK, model W14S). Alternatively, water was double

distilled with Millipore water purification system (Bedford, MA, USA). Standards of

AFM1 (10 µg/mL in acetonitrile) and AFB1 (10 µg/mL in acetonitrile) were purchased

from Supelco (Bellifonte, PA, USA) and stored with care in freezer. Working standard

solutions of 0.1 µg/mL were prepared from stock standard solutions and were stored in a

tightly Stoppered (closed) vial below 4oC.

3.1.5. Instruments

(a) Fluorometer, Vicam series-4 model (VICAM, USA),

(b) HPLC system of Agilent 1100 series (Agilent, USA)

(c) HPLC system of Shimadzu, model LC-10 (Shimadzu, Japan)

(d) Millipore water purification system (Bedford, MA, USA)

(e) Bibby water distillation system, model W14S (J.Bibby Science Products Ltd.,

UK)

(f) Gerber butyrometer (Gerber, Switzerland)

(g) Digester for protein analysis, Model DK 6 (Velp Scientifica, Italy)

(h) Distillation unit for protein analysis, Model UDK 126 D (Velp Scientifica, Italy)

(i) ELISA reader, model A3 (DAS Inc., Italy)

(j) Vacuum manifold, SPE-10 ( J.T. Baker Inc. USA)

(k) Water Bath-Shaker (GFL, Germany)

(l) Mini shaker/Vortex mixer (Thermolyne, USA)

(m) Micropipettes (Eppendorf AG, Hamburg, Germany)

(n) Oven (Memmert, Germany)

(o) Centrifuge machine for Gerber method (locally made, Pakistan)

(p) Centrifuge (Gallenkamp, UK)

(q) Nitrogen generator (CLAIND, Italy)

(r) Grinding mill (Retsch KG, Germany)

69

(s) Refrigerator (NATIONAL, Pakistan)

(t) Water bath, model WB 14 (Memmert, Germany)

(u) Fume hood (Fisher Scientific Co., USA)

(v) Analytical balance (Sartorius, German)

(w) Sonicator (OGAWA SEIKI Co., Japan)

3.2. Methods

3.2.1. Determination of Aflatoxin M1 with Fluorometer

The analysis was carried out with Fluorometer along with the use of affinity

chromatography columns for clean-up step according to the method described by Hansen

(1990). Before analysis sample was brought to room temperature. To remove cream

from the milk sample, it was centrifuged at 2000×g for 10 minutes. The 10 mL sample of

skim milk was passed through AflaTest affinity column of the VICAM, USA. These

affinity columns contain antibodies to aflatoxin. The column was then washed twice

with 10 mL portions of 10% methanol and the aflatoxin M1 was eluted from the affinity

column by passing 1.0 mL of 80% methanol. All the sample eluate (1.0 mL) was

collected in a glass cuvette.

The concentration of aflatoxin M1 was measured in a fluorometer, Vicam series-4

model with the option of 360 nm excitation filter and 440 nm emission filter. The results

were recorded using digital Fluorometer readout with automatic printing device.

3.2.2. Determination of Aflatoxin M1 by HPLC

The method used for determination of AFM1 was the AOAC Official Method

2000.08 (AOAC Official Method 2000.08, 2005).

3.2.2.1. Extraction Procedure

After warming at about 37oC in water bath, liquid milk was centrifuged at 2000×g

to separate fat layer and then filtered. The prepared test portion of 50 mL was transferred

into syringe barrel attached with immonoaffinity column (IAC) and passed at slow steady

flow rate of 2-3 mL/ min. The washing of column was done with 20 mL water and then it

was blown to dryness and afterwards aflatoxin M1 was eluted with 4 mL pure acetonitrile

by allowing it to be in contact with the column at least 60 seconds. The eluate was

70

evaporated to dryness using gentle stream of nitrogen and at the time of LC determination

it was diluted with the mobile phase.

3.2.2.2. LC Determination with Fluorescence Detection

The HPLC system of Agilent 1100 series (Agilent, USA), equipped with an auto

sampler LAS G1313A and a fluorescence detector FLD G1321A with excitation and

emission wavelength of 365nm and 435nm respectively, was used for AFM1

determination. The ZORBAX Eclipse XDB-C18 (Octadecyl silane chemically bonded to

porous silica) column (Agilent, USA), 4.6×150 mm with particle size 5 µm in diameter,

was used. Acetonitrile in ratio of 25% with 75% water was used as mobile phase. The

flow rate was 0.8 mL/min. Calibration curve was determined using a series of calibration

solutions of AFM1 in acetonitrile with concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0

µg/ L. The retention time for aflatoxin M1 was 6.1-6.5 min.

3.2.2.3. Calculations

Calculations were made according to the following equation:

Wm = Wa × (Vf/Vi) × (1/Vs)

Where Wm = amount of AFM1 in the test sample in µg/L; Wa = amount of AFM1

corresponding to area of AFM1 peak of the test extract (ng); Vf = the final volume of re-

dissolved eluate (µL); Vi = volume of injected eluate (µL); Vs = volume of test portion

(milk) passing through the column (mL).

3.2.3. Determination of Aflatoxin B1 by HPLC

For the determination of AFB1, the AOAC Official Method 994.08 was used with

small modifications (AOAC Official Method 994.08, 2000).

3.2.3.1 Extraction and Clean-up Procedure

A test portion of 50.0g and 100mL extraction solvent (850mL acetonitrile with

150mL deionized water) was taken in 250mL Erlenmeyer flask and placed in a shaker for

1 hour at high speed. After filtration, 8mL extract was taken with pipette in 10mL glass

tube. MycoSep® column (rubber flange end) was pushed slowly into the tube. As

column was pushed into the tube, extract was forced through frit, through 1-way valve,

and through packing material and was collected in column reservoir. The purified extract

71

(2mL) was transferred quantitatively from top of column to screw cap vial (derivatization

vial) and was evaporated under nitrogen.

3.2.3.2. Aflatoxin Derivatization

After adding n-hexane (200µL) in the derivatization vial to re-dissolve aflatoxin,

50µL of trifluoroacetic acid was added and it was mixed on vortex mixer for 30 seconds.

After five minutes, 1.95mL of deionized water: acetonitrile (9:1) mixture was added and

again mixed on vortex mixer for 30 seconds. Layers were allowed to separate and

aqueous layer (lower layer) containing aflatoxins was removed, filtered through 0.45µm

syringe filter and then injected onto LC column.

3.2.3.3. LC Determination with Fluorescence Detection

The high-performance liquid chromatography equipment (LC-10, Shimadzu,

Japan), comprising liquid pump LC-10AS, column oven CTO-10A, system controller

SCL-10A, fluorescence detector RF-530, communication bus module CBM-101, and data

acquisition software class LC-10A was used for the present study. The excitation

wavelength of 365nm and emission wavelength of 435nm was set during analysis. The

stainless steel column Discovery® C18 of Supelco (Bellifonte, PA, USA) with dimensions

of 25cm×4.6mm (id) and with particle size of 5 µm diameter was used. The mobile phase

(acetonitrile: methanol: deionized water in the ratio of 20:20:60) was degassed with

sonicator before use. The flow rate was 1.0 mL/ min. Calibration curve was determined

using a series of calibration solutions of AFB1 in acetonitrile with concentrations of 0.5,

1.5, 2.5, 5.0, 10.0, and 15.0 µg/ L. The retention time for aflatoxin B1 was 5.36 min.


Aflatoxin B1 peak was identified in derivatized extract chromatogram by

comparing its retention time with corresponding peak in the standard chromatogram. The

quantity of the aflatoxin B1, C, was determined in the derivatized extract (injected) from

the respective standard curves. The concentration of aflatoxin B1 was calculated in test

sample as follows:

Aflatoxins B1 ng/g = C/W

W = 50g × (2mL/ 200mL) × (0.02mL/ 2mL) = 0.005g

72

Where W = equivalent weight of test portion (in 20µL) injected into LC; C = ng aflatoxin

(in 20µL) injected into LC.

3.2.4. Determination of Aflatoxin M1 in Cheese and Yoghurt by

ELISA

The samples cheese and yoghurt were analyzed for AFM1 using the competitive

ELISA procedure as described by the protocol provided with RIDASCREEN® ELISA kit

(RIDASCREEN® Aflatoxin M1 30/15, 2007).

3.2.4.1. Sample Preparation

Cheese (2.0g) samples were triturated and yoghurt samples (2.0g) were directly

analyzed. Extraction was completed with 40 mL dichloromethane by shaking for 15

minutes. The suspension was filtered and 10 mL of the extract was evaporated at 60°C

under weak N2-stream. The oily residue was re-dissolved in 0.5 mL methanol, 0.5 mL

PBS buffer and 1 mL n-heptane. After mixing thoroughly, it was centrifuged for 15

minutes at 2700 × g. The upper heptane layer was removed completely. From the lower

methanolic-aqueous phase, 100µL was taken and diluted with 400 mL buffer 1 and

100µL of it was used per well in the test.

3.2.4.2. Test Procedure

The standard solutions (100µL) and prepared samples (100µL) were added into

the microtiter well placed in the microwell holder. Gentle mixing was accomplished by

shaking the plate manually and incubated for 30 minutes at room temperature (20-25°C)

in the dark. The liquid was poured out of the wells and microwell holder was tapped

vigorously upside down against adsorbent paper to ensure complete removal of liquid

from the wells. The wells were washed by adding 250 µL washing buffer in each well

and poured out the liquid again. Washing step was repeated for two times. Then 100 µL

of the diluted enzyme conjugate was added and mixed gently by shaking the plate

manually and incubated for 15 minutes at room temperature in the dark. After incubation

the wells were washed again. The 100 µl of substrate/chromogen was added and mixed

gently by shaking the plate manually and incubated for 15 minutes at room temperature in

the dark. Now stop solution (100µL) was added in each well. Mixing was by shaking the

73

plate manually. The absorbance was measured photometrically at 450 nm against an air

blank with in 15 minutes after the addition of stop solution.


The following formula was used to measure the % absorbance.

(Absorbance of standard or sample / absorbance of zero standard) × 100 = % absorbance

The zero standard is made equal to 100 % and absorbance values are taken in

percentages. A calibration curve is obtained by plotting %absorbance values for the

standards against the aflatoxin M1 concentration (µg/L). The concentration of AFM1 in

samples was calculated from the calibration curve.

3.2.5. Determination of Milk Fat Percentage

The Gerber method was used for the determination of fat percentage in milk

samples (Egan et al., 1981). According to this method, fat determination in milk is

performed with butyrometer. The 10 mL H2SO4 (95-98%) is taken into a Gerber

butyrometer (Gerber, Switzerland). The 10.94 mL of mixed milk is carefully taken with

specific pipette (Gerber, Switzerland) on to the side of the butyrometer to form a layer on

H2SO4. One mL of amyl alcohol (fat free) is added on to the layer of milk. The

butyrometer is sealed with special metal key and rubber stopper. The sample and

sulphuric acid are mixed by rolling the tube in hand at an angle of 45o. The butyrometer

is inverted once or twice to complete the mixing in the graduated stem and then

centrifuged at 1100 rpm for 4 minutes. The butyrometer (stopper downward) is incubated

in a water bath (Model WB 14, Memmert, Germany) at 65o C for at least 3 minutes and

fat% is read off directly from the scale.

3.2.6. Determination of Milk Protein

The Kjeldahl method was applied for the determination of milk protein in milk

samples using Block Digester/ Steam Distillation procedure according to the AOAC

official method 991.20 (AOAC Official Method 991.20, 1995).

3.2.6.1. Test Portion Preparation

The 6.75g K2SO4 and 0.75g CuSO4·5H2O catalyst (Total =7.5g) is added in the

digestion tube. More or less 1.0g of the test portion is weighed in the digestion tube after

74

warming at 38±1oC and mixing. Then 10 mL of H2SO4 is added. Tube is closed with

stopper and held for digestion. A blank (all reagents except test portion) is digested and

distilled each day.

3.2.6.2 Digestion

The digestion tubes are placed in the digester attached with the venting tubes to

remove the acid vapors. Initially the digester (Model DK 6, Velp Scientifica, Italy) is set

at low temperature (180oC-230oC) to control foaming. Digestion is carried out until white

fumes develop. The temperature is increased up to 420oC and digestion is continued.

Normal digestion temperature is 420oC and lower temperatures increase digestion time.

Digestion is completed in approximately 45 minutes at 420oC. After digestion when

contents are clear (clear with light blue-green color), boiling is continued for at least one

hour with total digestion time from 1.75-2.5 hours. The digestion tubes are removed from

the Block digester and then allowed to cool.

3.2.6.3. Distillation

The solution of NaOH (40%) is placed in alkali tank of distillation unit (Model

UDK 126 D, Velp Scientifica, Italy). Graduated Erlenmeyer flask (500 mL), containing

25 mL H3BO3 (4%) solution, is placed on receiving platform, with tube from condenser

extending below surface of H3BO3 solution. The digestion tube is attached to distillation

unit and then 50 mL sodium hydroxide solution is added. The steam-distillation is

started and continued until almost 150 mL distillate is collected. Receiving flask is

removed, few drops of indicator (methyl red/ bromocresol green) are added and then it is

titrated against 0.10 M HCl solution until first trace of pink coloration.


Result is calculated by applying the following equation.

Nitrogen % = 1.4007 × (Vs- Vb) × M / W

Where Vs and Vb = mL HCl titrant used for test portion and blank respectively; M =

molarity of HCl solution; and W = test portion weight in gram. Multiply percent protein

by factor 6.38 to calculate percent protein.

75

3.2.7. Statistical Analysis

The data was analyzed by applying SPSS (statistical package for social sciences,

version 11.5) and mean and standard deviation of all variables (AFM1, AFB1, fat%, and

protein %) were calculated. The arithmetic mean is the most commonly used type of

average. It is usually referred to as simply the average or the mean. It is obtained by

dividing the sum of all the observations by the total number of observations. It is denoted by

" X ".

For ungrouped data

n

X =

nx...+x + x + x = X n321 ∑

Another term used in Statistics is variance. The Variance is defined as the

arithmetic mean of the squared deviations of the observations measured from their mean.

The population variance is denoted by σ2 where as sample variance is denoted by S2 and

defined as

For ungrouped data

sampleFor n

)x - (x = S

population For N

) - (x =

22

22

∑

∑ µσ

Alternative formula

∑∑

∑∑

n

X -

nX = S

N

X -

NX =

222

222σ

The positive square root of the variance is called Standard Deviation. It is

denoted by σ (S for sample). The standard deviation is an absolute measure of dispersion

(Steel et al., 1997).

Analysis of Variance (ANOVA) gives the comparison among the mean values of

the results. ANOVA technique was applied for various variables (AFM1 concentration

and AFB1 concentration) using one-way/simple ANOVA. Mathematically, ANOVA and

Regression are very similar. Both analyses require an interval scale and normally

distributed dependent variable. When the independent variable is a nominal or ordinal

scale variable, we use ANOVA. When the independent variable is an interval scale

variable, we use regression. When there are more than one independent variables, and some

76

of the variables are nominal scale and some are interval scale type variables, we use either

ANOVA or Logistic regression. If we use ANOVA, the interval scale variables are called

"covariates".

To determine the relationship between different variables (concentrations of

AFB1/ AFM1 and corresponding areas of the peaks in the chromatogram/ %absorbance),

regression analysis/curve fitting was performed. The regression shows the dependency of

one variable (dependent) on the other variable (independent). The details of regression

are given in the following lines. If there are two variables in a sample, peak area (y-axis)

and concentration (x-axis) and both are continuous variables, their values can plotted to

observe whether they form a pattern. The plot shows a regression line and indicates that

how the area of peak can be influenced by the concentration. The Fig. 2 shows the

regression line.

Fig. 2: The

regression line.

The simplest

type of linear

regression or linear

equation is: y = a +

bx. Sometimes it is

referred to as f(x) = a

+ bx. In the linear

equation, “a” represents the intercept of the line on the y-axis, “b” represents the slope of

the line relative to the x-axis, i. e. b = tan θ as shown in the Fig. 3.

Fig. 3: The linear equation.

77

The most commonly used criteria to describe the relation between independent and

dependent variables, is the Coefficient of Determination (r2 or R2).

Coefficient of Determination is calculated as:

r2 = Σ(ŷ - µy)2/Σ(y - µy)2

= bSxy / Syy

= Regression Sum Square/Total Sum Square

= Explained Variation / Total Variation

r2 = S2xy / Sxx . Syy from (1) bSxy / Syy (b = Sxy/Sxx)

In fact, it can be proved that

R = Cov (x, y) / σx . σy

Where, Cov (x, y) stands for the covariance of the two variables x and y. It can be

expressed as Sxy / n, where n is the number of cases. Moreover, σx and σy are the standard

deviations of x and y.

The highest value of r2 is 1 and the highest value shows the most perfect

relationship between the variables. Closer the value of r2 to 1, more perfect the relationship

between the variables.

Correlation analysis gives the relationship between the variables. Correlation

analysis was performed to find out the correlation between AFM1 concentration in milk

and fat% of the milk. Correlation analysis was also performed to find out the correlation

between AFM1 concentration in milk and protein% of the milk. The Coefficient of

Correlation is calculated by using the Pearson Product Moment r.

r = Σ xy – Σx Σy/ Σx2 – (Σx)2/ n × Σ y2 – (Σy)2/ n

The value range of the coefficient of correlation is -1 to 1, where "0" means no

correlation, "+1" means perfectly positively correlated and "-1" means perfectly negatively

correlated (Steel et al., 1997). Anything ‘r’ value above "0.5" is considered to be high.

Anything from 0.2 to below 0.5 is referred as "medium", whereas anything below 0.2 will

be considered as low correlation.

78

4. RESULTS AND DISCUSSIONS

Milk, a balanced diet provided by nature, is of great worth for human beings.

Dairy products including milk are important sources of animal protein, vitamins, and

essential fatty acids for infants and young adults (Jensen and Nielsen, 1982). Due to the

common occurrence and harmful effects of aflatoxin contamination, there is a need for

detection and quantification of aflatoxin M1 in milk and its products.

4.1. Aflatoxin M1 Contamination in Raw Milk- A

General Survey

In the first phase of study, a survey was conducted regarding the contamination of

AFM1 in the Punjab province of Pakistan. Concentration of AFM1 was determined by

Fluorometer with a prior clean-up step with immunoaffinity columns. Immunoaffinity

columns have been successfully used in the analysis of aflatoxins in food and feed during

the last few years (Scott and Trucksess 1997). Many researchers have used IAC in

combination with HPLC (Gurbay et al., 2006; Tuinstra et al., 1993) for the analysis of

aflatoxins. The IAC in combination with Fluorometer was applied by Chiavaro et al.

(2005), for determination of aflatoxin B1 and aflatoxin M1 in pig liver.

A total of 168 milk samples were analyzed to evaluate the contamination level of

aflatoxin M1 in the raw milk. Contamination of AFM1 was detected in the raw milk.

Aflatoxin M1 was found to be present in all of the examined milk samples obtained from

14 districts of the Punjab province. The Table 2 gives the distribution of AFM1 in milk

samples by district-wise and month-wise in the Punjab during the year 2005. Graphically

the distribution of AFM1 in milk has been shown in Fig. 4. The graph shows that there is

no variation in the concentration of AFM1 in milk according to districts. Table 3 gives

minimum, maximum, mean and standard deviation by month-wise. The 162 samples

(96.4%) had contamination less than 0.5 µg AFM1/ L milk which is US tolerance limit for

AFM1 in milk. Only one sample (0.6%) contained 0.5µg/ L of AFM1 in milk, while the

remaining 5 samples (3.0%) contained more than o.5 µg/ L of AFM1 in milk.

In general, regardless of the samples which are below and at the borderline limit

(0.5 µg/ L), 3.0% of the samples had concentration of AFM1 which exceeded the

79

prescribed limit of US regulations i.e. 0.5µg/ L. Almost 99.4% of the contaminated

samples exceeded the European community / Codex Alimentarius recommended limit.

The European Community and Codex Alimentarius Commission prescribe that the

maximum level of AFM1 in liquid milk and dried or processed milk products should not

exceed 0.05µg/ L or 0.05µg/ kg (Codex Alimentarious Commission, 2001; Creppy,

2002). This limit has been established following the ALARA (as low as reasonable

achievable) principle. In Austria and Switzerland the maximum level is further reduced

to the level of 0.01 µg/ kg for infant food commodities. Although US regulations

prescribe 10 times higher limits of AFM1, even then 3% Pakistani milk samples exceeded

the maximum limit.

The results of the present study were compared with those of other studies made

in Morocco, Iran, and Italy. Fifty four samples of pasteurized milk produced by five

different dairies from Morocco were surveyed for the presence of AFM1 and 88.8% of the

samples were contaminated with AFM1; 7.4% being above the maximum level of 0.05

µg/ L set by the Moroccan and European regulations for AFM1 in liquid milk (Zinedine et

al., 2007). In Iran, of the 111 samples, 85 (76.6%) were found contaminated with AFM1

in concentration between 0.015 and 0.28 µg/ L (Kamkar, 2005). The 40 milk samples

were analyzed in Italy and AFM1 was detected in 30% of milk samples at levels ranging

from 0.004 to 0.023 µg/ L. None of the contaminated samples exceeded the legal limit of

0.05 µg/ L set down by the European Union for milk (Finoli and Vecchio, 2003).

Seasonal effect influences concentration of aflatoxin M1 in milk. Table 4 gives

the distribution of AFM1 in milk by district-wise and season-wise. Graphically district-

wise and season-wise distribution of AFM1 in milk has been shown in Fig.5. The Fig. 5

shows that in almost all the districts the concentration of aflatoxin M1 in raw milk was

lower in summer season and maximum in winter season. ANOVA indicates significant

(p < 0.01) difference in AFM1 concentration among seasons. Fig. 6 depicts a significant

variation in the concentration of AFM1 in milk among all the four seasons. AFM1

concentrations are the highest in winter and the lowest in summer. Fig. 7 shows that the

minimum concentration of AFM1 in milk was observed during summer. Many authors

also reported higher concentration of AFM1 in cold seasons as compared to hot seasons

(Rossi et al., 1996; Blanco et al., 1988), the reason being in winters mostly milking

animals are fed with compound feeds and thus concentration of aflatoxin B1 increases

which in turn enhances AFM1 concentration in milk. Moreover temperature and moisture

contents also affect the presence of aflatoxin B1 in feeds.

80

Table 2: Distribution of aflatoxin M1 (µg/ L) in raw milk samples by month-wise and district-wise in the Punjab.

Districts Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Mean SD SE

Lahore 0.55 0.46 0.46 0.45 0.28 0.39 0.39 0.36 0.36 0.45 0.47 0.47 0.42 0.059 0.017

Sheikhupura 0.44 0.38 0.46 0.32 0.32 0.32 0.16 0.19 0.28 0.27 0.34 0.37 0.32 0.081 0.023

Hafizabad 0.39 0.41 0.31 0.31 0.31 0.28 0.36 0.22 0.23 0.45 0.45 0.41 0.34 0.078 0.023

Jhang 0.46 0.46 0.25 0.39 0.26 0.35 0.34 0.13 0.17 0.28 0.30 0.41 0.32 0.095 0.028

T.T.Singh 0.70 0.49 0.46 0.36 0.36 0.34 0.34 0.20 0.34 0.34 0.38 0.38 0.39 0.071 0.021

Okara 0.48 0.46 0.46 0.34 0.34 0.34 0.42 0.01 0.40 0.28 0.40 0.35 0.36 0.119 0.034

Sahiwal 0.49 0.49 0.38 0.38 0.39 0.38 0.38 0.19 0.35 0.30 0.39 0.45 0.38 0.074 0.021

Khanewal 0.49 0.44 0.33 0.40 0.33 0.28 0.28 0.23 0.29 0.45 0.41 0.45 0.37 0.076 0.022

Pakpatan 0.66 0.49 0.33 0.33 0.24 0.49 0.34 0.19 0.44 0.30 0.41 0.44 0.39 0.095 0.027

Vehari 0.46 0.57 0.49 0.50 0.35 0.27 0.41 0.14 0.32 0.32 0.41 0.46 0.39 0.116 0.034

Bahawalnagar 0.49 0.49 0.44 0.45 0.28 0.30 0.30 0.42 0.40 0.40 0.47 0.37 0.40 0.069 0.020

Mazafargarh 0.40 0.49 0.39 0.42 0.37 0.42 0.37 0.12 0.38 0.41 0.45 0.37 0.38 0.090 0.026

Layya 0.44 0.41 0.43 0.46 0.36 0.38 0.32 0.16 0.30 0.31 0.46 0.25 0.36 0.089 0.026

Lodhran 0.59 0.49 0.47 0.46 0.33 0.37 0.20 0.22 0.33 0.27 0.30 0.46 0.37 0.099 0.029

Mean0.50

30.46

60.40

40.39

80.32

30.35

10.32

90.19

90.32

80.34

50.40

30.40

3

SD0.09

20.04

70.07

40.06

10.04

40.06

10.07

50.09

90.07

10.07

10.05

70.06

0

81

SE0.02

50.01

30.02

00.01

60.01

20.01

60.02

00.02

70.01

90.01

90.01

50.01

6

82

Table 3: Distribution of aflatoxin M1 (µg/ L) in raw milk samples by month-wise.

Months Samples Minimum Maximum Mean SD SEJanuary 14 0.39 0.70 0.503 0.092 0.025February 14 0.38 0.57 0.466 0.047 0.013March 14 0.25 0.47 0.404 0.074 0.020April 14 0.31 0.50 0.398 0.061 0.016May 14 0.28 0.39 0.323 0.044 0.012June 14 0.27 0.49 0.351 0.061 0.016July 14 0.16 0.39 0.329 0.075 0.020August 14 0.01 0.42 o.199 0.099 0.027September 14 0.17 0.40 0.328 0.071 0.019October 14 0.27 0.45 0.345 0.071 0.019November 14 0.30 0.47 0.403 0.057 0.015December 14 0.25 0.47 0.403 0.060 0.016

83

Figure 2: Districtwise distribution of fresh milk samples (aflatoxin M1 concentration)

0

0.1

0.2

0.3

0.4

0.5

0.6

Laho

re

Sheikh

upur

a

Hafiza

bad

Jhan

g

T.T.S

ingh

Oka

ra

Sahiw

al

Khane

wal

Pakpa

tan

Vehar

i

Bahaw

alnag

ar

Maz

afar

garh

Layy

a

Lodh

ran

District

Afl

ato

xin

M1

co

nce

ntr

ait

on

(µ

g/L

)

Fig. 4: District-wise distribution of AFM1 in raw milk samples.

84

Table 4: Distribution of aflatoxin M1 (µg/ L) in raw milk by district-wise and season-wise.

Districts Winter Spring Summer Autumn Mean SD SELahore 0.49 0.46 0.36 0.41 0.42 0.071 0.020Sheikhupura 0.38 0.39 0.25 0.28 0.32 0.089 0.026Hafizabad 0.42 0.31 0.29 0.34 0.34 0.079 0.023Jhang 0.41 0.32 0.27 0.23 0.32 0.106 0.030T.T.Singh 0.49 0.41 0.31 0.34 0.39 0.120 0.035Okara 0.42 0.40 0.28 0.34 0.36 0.125 0.036Sahiwal 0.46 0.38 0.34 0.33 0.38 0.081 0.023Khanewal 0.45 0.37 0.28 0.37 0.37 0.085 0.025Pakpatan 0.50 0.33 0.32 0.37 0.39 0.127 0.037Vehari 0.48 0.50 0.29 0.32 0.39 0.118 0.034Bahawalnagar 0.46 0.45 0.33 0.40 0.40 0.074 0.021Mazafargarh 0.43 0.41 0.32 0.40 0.38 0.090 0.026Layya 0.39 0.45 0.31 0.31 0.36 0.092 0.027Lodran 0.46 0.47 0.28 0.30 0.37 0.120 0.035Mean 0.44 0.40 0.30 0.34SD 0.078 0.066 0.093 0.071SE 0.010 0.013 0.012 0.013

85

Figure 4: Distribution by season and

districtwise of fresh milk samples (aflatoxin M1 concentration)

0

0.1

0.2

0.3

0.4

0.5

0.6

Laho

re

Sheikh

upur

a

Hafiza

bad

Jhan

g

T.T.S

ingh

Oka

ra

Sahiw

al

Khane

wal

Pakpa

tan

Vehar

i

Bahaw

alnag

ar

Maz

afar

garh

Layy

a

Lodh

ran

Distrcit

Afl

ato

xin

M1

con

cen

trai

ton

(µ

g/L

)Winter

Spring

Summer

Autumn

Fig. 5: Distribution of AFM1 season-wise and district-wise in raw milk samples.

86

Figure 1: Seasonwise distribution of fresh milk samples (aflatoxin M1 concentration)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Winter Spring Summer Autumn

Seasons

Afla

toxi

n M

1 co

nce

ntr

aito

n (µ

g/L

)

Fig. 6: Season-wise distribution of AFM1 in raw milk samples.

87

Figure 1: Monthwise distribution of fresh milk

samples (aflatoxin M1 concentration)

0

0.1

0.2

0.3

0.4

0.5

0.6

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Month

Afl

ato

xin

M1

co

nc

en

tra

ito

n (

µg

/L)

`

Fig. 7: Month-wise distribution of AFM1 in raw milk samples.

Aspergillus flavus and Aspergilllus parasiticus easily grow in feeds having

moisture from 13% to 18% and environmental moisture from 50% to 60%, furthermore,

they can produce toxin (Jay, 1992). Another reason of low AFM1 level in summer may

be attributed to out-pasturing of milking cattle. Out-pasturing of milking cattle is an

effective factor for low level of AFM1 concentration in milk (Kamkar, 2005). Fig. 8

shows an attractive scene of buffaloes grazing on an island in the Ravi river near Lahore

city, Pakistan. In the present study the lowest AFM1 concentration has also been noted in

summer, which is found to be 0.01 µg/ L. Similar results were stated by other researchers

too (Applebaum et al., 1982; Blance and Karleskind, 1981) and they found that low

aflatoxin M1 level or no toxin production was obtained in summer season.

88

This is the first ever systematic study on aflatoxin M1 in Pakistan. According to

the results obtained from this study, the situation calls for continuous monitoring

aflatoxin M1 concentration in milk and milk products. Almost 99.4% of the analyzed

samples crossed the EU tolerance limit; there is urgent need to create awareness among

the people about the high concentration of AFM1 which is health hazard.

Picture 1: Buffaloes grazing at island in the Ravi river near Lahore, Pakistan.

(Courtesy daily The News August 30, 2007)

89

4.2. Aflatoxin M1 Contamination Variation with Respect

to Localities and with Respect to Herd-Size of

Cattle

Depending on the local conditions and traditions, different feed regimen is used in

different areas like urban and rural and feed plays main role in milk contamination with

AFM1. The present study showed the levels of aflatoxin M1 in raw milk of buffaloes and

cows from different localities of urban, semi-urban, and rural in the central areas of the

Punjab province of Pakistan.

High-performance liquid chromatography (HPLC) method was used for analysis

in this study. Fig. 8 shows the peaks of AFM1 in standard and sample. HPLC is a

commonly used technique in latest aflatoxin determination (Nachtmann et al., 2007; Van

Egmond and Dragacci, 2001). Methodology for determination of aflatoxin M1 in milk

has been greatly improved in recent years with the application of immunoaffinity columns

(IAC) which provide a combination of extraction and clean-up stages. With the advent of

immunoaffinity columns, AOAC Official Method 2000.08 has come into force. This

method is meant for aflatoxin M1 in milk using IAC by liquid chromatography with Final

Action 2004. The previous AOAC liquid chromatographic method for aflatoxin M1 and

M2 in fluid milk was AOAC Official Method 986.16 with Final Action 1990 (AOAC

Official Method 986.16, 2000).

The clean-up step was carried out with immunoaffinity columns in this study for

determination of AFM1 along with HPLC. Fig. 9 shows the standard curve. The standard

curve was linear (y = 0.312 + 6.578x, y = area, x = amount) from 0.05µg/ L to 10µg/ L

AFM1 concentration. The coefficient of determination (R2) was 0.9997. The limit of

detection (LOD) was defined as the lowest limit of the analyte which could be detected

by the instrument. The LOD was determined as 0.004 µg AFM1/ L of milk.

Concentration of AFM1 in raw milk of buffaloes from urban, semi-urban, and

rural areas is given in Table 5, Table 6, and Table 7 respectively. Whereas concentration

of AFM1 in raw milk of cows from urban, semi-urban, and rural areas is given in Table 8,

Table 9, and Table 10 respectively. Fig. 10 shows area-wise comparison of AFM1 in milk

of buffaloes and cows. According to the statistical analysis of the data, there exists

difference in the concentration of AFM1 in the raw milk of buffaloes (F= 27.615, p<

0.05), and cows (F= 4.617, p<0.05) from the three areas urban, semi-urban and rural. The

90

higher concentration of AFM1 is present in urban and semi-urban area samples and lower

one is present in rural area samples, the reason being that the feeding practices are varied

in these localities. In urban and semi-urban areas there is less availability of green fodder

and there is excessive use of concentrated feed, cottonseed cake, corn, soybean, wheat

straw, paddy straw, and wheat bran. All these commodities are vulnerable to the attack of

moulds and there is a high possibility of presence of AFB1 in these commodities (Dutton

and Kinsey, 1996). The contamination of aflatoxin happens mainly in the feeds

(Sassahara et al., (2005). On the other hand, in rural areas there is abundance of green

fodder and it is commonly used as a feed of cattle by all and sundry. There is less use of

supplements like concentrated feed and oil seed cakes in these areas but wheat straw is

used along with green fodder. In some areas, there is also a common practice of grazing

which is another factor for less concentration of AFM1 in milk samples from rural areas.

91

Fig. 8: Comparison of HPLC chromatograms: (A) standard (B) sample, for AFM1 in

milk.

92

Aflatoxin M1

A

B

Aflatoxin M1

Fig. 9: Calibration curve of standard solutions of AFM1 by HPLC analysis.

Tale 5: Concentration of aflatoxin M1 (µg/ L) in the contaminated raw milk samples of

urban buffaloes.

Concentration of AFM1 (µg/ L)0.0990 0.1030 0.0218 0.0047 0.0209 0.01800.1321 0.0824 0.1074 0.0041 0.0261 0.01650.0792 0.0318 0.0895 0.0708 0.0508 0.01760.0660 0.0286 0.0043 0.0849 0.0172 0.01090.0878 0.0215 0.0290 0.0044 0.0636 0.01310.1047 0.0172 0.0174 0.0042 0.0848 ×0.0785 0.0043 0.0145 0.0726 0.0344 ×0.0628 0.0034 0.0062 0.0043 0.0954 ×0.0063 0.1343 0.0048 0.1210 0.0348 ×0.1545 0.0035 0.1062 0.0047 0.0174 ×0.1373 0.1791 0.1416 0.0605 0.0258 ×0.0058 0.0045 0.0046 0.0908 0.0206 ×

y = 0.312 + 6.578x

Regression Standardized Predicted Values of AFM1

2.01.51.0.50.0-.5-1.0

AR

EA

70

60

50

40

30

20

10

0

-10

93

Table 6: Concentration of aflatoxin M1 (µg/ L) in the contaminated raw milk samples of

semi-urban buffaloes.

Concentration of AFM1 (µg/ L)0.0741 0.1432 0.0249 0.0043 0.07040.0988 0.0659 0.2513 0.0041 0.09380.2387 0.0878 0.0332 0.0044 0.04800.0593 0.0439 0.3770 0.0255 0.04900.0667 0.0527 0.5027 0.0042 0.04850.0889 0.1860 0.3016 0.0047 0.04950.0533 0.2232 0.0311 0.0340 0.02590.0444 0.0199 0.0207 0.0062 ×0.1790 0.2790 0.0415 0.0046 ×0.0494 0.0166 0.0249 0.0170 ×0.1193 0.3720 0.0046 0.0204 ×


rural buffaloes.

Concentration of AFM1 (µg/ L)0.0045 0.0113 0.0326 0.00690.0313 0.0151 0.0435 0.00460.0391 0.0226 0.0388 0.02080.0056 0.0166 0.0498 0.00470.0522 0.0207 0.0042 0.00480.0075 0.0276 0.0044 0.02320.0329 0.0311 0.0043 0.02910.0064 0.0139 0.0058 0.02400.0447 0.0174 0.0040 ×0.0263 0.0232 0.0052 ×0.0090 0.0261 0.0059 ×


urban cows.

Concentration of AFM1 (µg/ L)0.0306 0.0133 0.1114 0.0741 0.01460.0275 0.0148 0.0823 0.1237 0.49710.0344 0.4825 0.1234 0.0926 0.24400.8183 0.0167 0.1392 0.4454 0.04480.0290 0.0150 0.0152 × ×

94


semi-urban cows.

Concentration of AFM1 (µg/ L)0.0084 0.0028 0.0386 0.0429 0.5106 0.02730.0094 0.4538 0.0045 0.0118 0.0483 0.02150.4083 0.0022 0.0050 0.0105 0.0336 ×0.0075 0.0029 0.0057 0.0095 0.0217 ×

Table 10: Concentration of aflatoxin M1 (µg/ L) in the contaminated raw milk samples

of rural Cows.

Concentration of AFM1 (µg/ L)0.0125 0.0089 0.0075 0.0094 0.00760.0062 0.0078 0.0126 0.0057 0.00510.0104 0.0065 0.0084 0.0092 ×0.0069 0.0151 0.0108 0.0046 ×

Fig. 1. Area-wise comparison of aflatoxin M1 in milk of buffaloes and cows

54.16

42.5

30.8

57.555

45

0

10

20

30

40

50

60

70

Urban Semi-urban Rural

Pe

rce

nta

ge

of m

ilk s

am

ple

s

con

tam

ina

ted

with

afla

toxi

n M

1

Buffaloes

Cows

Fig. 10: Area-wise comparison of aflatoxin M1 in milk of buffaloes and cows.

During the survey of AFM1 contamination in buffalo milk by keeping in view the

herd-size of cattle, there was found variation in AFM1 concentration in milk according to

herd-size of cattle in the area under study. Rearing of dairy animals is a source of income

95

for some people in this area and people adapt it as an occupation. For some people

milking animals are the only source of their livelihood. People rear dairy animals

according to their financial capacity and according to their resources. Therefore the

number of milking animals varies from one dairy to the other. Three categories were

under investigation in the present study. Category I was of small herd-size comprising of

animals up to five. Category II was that of medium herd-size comprising of animals from

six to ten. Category III was that of large herd-size comprising of animals above ten.

Furthermore, all these categories were studied in urban, semi-urban, and rural areas.

Table 11, Table 12, and Table 13 give the concentration of AFM1 in raw milk of urban

buffalo in small herd-size, medium herd-size, and large herd-size respectively. Table 14,

Table 15, and Table 16 give the concentration of AFM1 in raw milk of semi-urban buffalo

in small herd-size, medium herd-size, and large herd-size respectively. Table 17, table

18, and table 19 give the concentration of AFM1 in raw milk of rural buffalo in small

herd-size, medium herd-size, and large herd-size respectively. Table 20 gives a

composite result of AFM1 level in buffalo milk and cow milk in urban, semi-urban, and

rural areas along with variation of AFM1 concentration in buffalo milk with respect to

herd-size.


urban buffaloes belonging to the small herd-size category.

Concentration of AFM1 (µg/ L)0.0990 0.1321 0.0792 0.06600.0878 0.1047 0.0785 0.06280.1545 0.1373 0.1030 0.08240.0318 0.0286 0.0215 0.01720.0043 0.0044 0.0063 0.00580.0180 0.0165 × ×


urban buffaloes belonging to the medium herd-size category.

Concentration of AFM1 (µg/ L)0.1343 0.1791 0.1074 0.08950.0290 0.0174 0.0218 0.01450.0043 0.0045 0.0045 0.00480.1062 0.1416 0.0708 0.08490.0176 0.0109 0.0131 0.00470.0062 0.0046 0.0041 ×

96


urban buffaloes belonging to the large herd-size category.

Concentration of AFM1 (µg/ L)0.1210 0.0605 0.0908 0.07260.0042 0.0047 0.0044 0.00430.0508 0.0848 0.0954 0.06360.0261 0.0209 0.0174 0.03480.0344 0.0258 0.0172 0.0206


of semi-urban buffaloes belonging to the small herd-size category.

Concentration of AFM1 (µg/ L)0.0741 0.0988 0.0494 0.05930.0667 0.0889 0.0533 0.04440.2387 0.1193 0.1432 0.17900.0704 0.0938 0.0480 ×0.0485 0.0495 0.0490 ×


of semi-urban buffaloes belonging to the medium herd-size category.

Concentration of AFM1 (µg/ L)0.0659 0.0878 0.0527 0.04390.1860 0.2232 0.2790 0.37200.0199 0.0166 0.0332 0.02490.2513 0.3770 0.5027 0.30160.0311 0.0207 0.0415 0.02490.0259 × × ×

97


of semi-urban buffaloes belonging to the large herd-size category.

Concentration of AFM1 (µg/ L)0.0042 0.0041 0.0043 0.00460.0046 0.0047 0.0044 0.00620.0340 0.0255 0.0170 0.0204


of rural buffaloes belonging to the small herd-size category.

Concentration of AFM1 (µg/ L)0.0045 0.0056 0.0090 0.01130.0075 0.0064 0.0313 0.03910.0522 0.0329 0.0263 0.01510.0291 0.0232 × ×


of rural buffaloes belonging to the medium herd-size category.

Concentration of AFM1 (µg/ L)0.0166 0.0207 0.0276 0.01390.0174 0.0232 0.0388 0.04980.0311 0.0261 0.0326 0.04350.0240 × × ×


of rural buffaloes belonging to the large herd-size category.

Concentration of AFM1 (µg/ L)0.0044 0.0042 0.0058 0.00410.0052 0.0059 0.0069 ×0.0208 0.0046 0.0048 ×

98

Table 20: Aflatoxin M1 (AFM1) level (µg/ L) in buffalo milk and cow milk in urban,

semi-urban and rural areas along with variation of aflatoxin M1 concentration in buffalo

milk with respect to herd-size.

Species Area Herd-size Total Contaminated Mean ±SD

Samples Samples (%age)

Buffalo Urban Small 40 22 (55%) 0.038±0.053

Buffalo Urban Medium 40 23(57.5%) 0.027±0.047

Buffalo Urban Large 40 20 (50%) 0.021±0.033

Buffalo Semi-urban Small 40 18 (45%) 0.039±0.087

Buffalo Semi-urban Medium 40 21 (52.5%) 0.075±0.130

Buffalo Semi-urban Large 40 12 (30%) 0.003±0.076

Buffalo Rural Small 40 14 (35%) 0.007±0.013

Buffalo Rural Medium 40 13 (32.5%) 0.009±0.015

Buffalo Rural Large 40 10 (25%) 0.002±0.004

Cow Urban Nil 40 23(57.5%) 0.087±0.174

Cow Semi-urban Nil 40 22(55%) 0.042±0.120

Cow Rural Nil 40 18(45%) 0.004±0.005

The result of statistical analysis applied to the data of AFM1 concentration in milk

samples of buffalo appeared to be significant with respect to herd-size (F= 6.631, p=

0.001). Milk samples in case of small herd-size (1-5 cattle) and medium herd-size (6-10

cattle) showed higher concentration than that of large herd-size (more than 10 cattle).

Most probably people belonging to the categories of small and medium herds use more

concentrated feed and other supplements to get high yield of milk as in many cases milk

is the only source of income for them and they try to acquire more and more milk to cater

their needs. Therefore to reduce the level of AFM1 in milk, it is imperative to educate

people belonging to these categories about the menace of aflatoxin contamination.

99

4.3. Aflatoxin M1 Contamination in Milk of Different

Species

There is an abundant production of milk in Pakistan and the major sources of milk

are five species of mammals namely buffalo, cow, goat, sheep, and camel. Currently in

Pakistan, there are 29.56 million cows, 27.33 million buffaloes, 53.79 million goats,

26.49 million sheep, and 0.92 million camels (Pakistan Livestock Census, 2006).

Pakistan is blessed with high yielding genetic dairy animals such as Nilli-Ravi buffalo,

Sahiwal cow, Kajli sheep and Beetle goat. Even without application of scientific tools,

dairy farmers are producing more than 33 million tons of milk per annum (Tipu et al.,

2007). A number of studies have been undertaken to determine AFM1 in cow milk (Gallo

et al., 2008; Rodriguez-Velsco et al., 2003; Choudhary et al., 1998). Buffalo milk AFM1

contamination has been shown by Garg et al. (2004). Occurrence of aflatoxin M1 in goat

milk was confirmed by Virdis et al. (2008) and by Oliveira and Ferraz (2007). Many

studies declared contamination of AFM1 in sheep milk (Bognanno et al., 2006; Benedetti

et al., 2005; Battacone et al., 2005). The study of Abdel-El-Fatah et al. (2002) has shown

the contamination of camel milk with AFM1.

Due to the common occurrence and harmful effects of aflatoxin contamination,

there is a need for detection and quantification of aflatoxin M1 in milk. The present study

has been designed in this perspective. HPLC method combined with clean-up step with

immunoaffinity columns was used for AFM1 detection in the study. Fig.11 shows the

calibration curve. The standard curve was linear (y= -1.520 + 17.579x, y = area, x =

amount) from 0.05 µg/ L to 10 µg/ L AFM1 concentration. The coefficient of

determination (R2) was 0.9998. Fig. 12 shows the peaks of AFM1 in standard and sample.

100

Fig. 11: Calibration curve of standard solutions of AFM1 with concentrations of 0.05,

0.1, 0.5, 1.0, 5.0 and 10.0 µg/ L by HPLC analysis.

y = -1.520 +17.579xR2 = 0.9998

Regression Standardized Predicted Values of AFM1

2.01.51.0.50.0-.5-1.0

Are

a

200

100

0

-100

101

Fig. 12: Comparison of HPLC chromatograms: (A) standard (B) sample, for AFM1 in

milk of five species.

102

A

B

Aflatoxin M1

A

Aflatoxin M1

B

Considering the contamination of AFM1, total 169 milk samples were analyzed

and these were taken from five species made up of 55, 40, 30, 24, and 20 samples of

buffaloes, cows, goats, sheep, and camels respectively. Table 21 gives the concentration

of AFM1 in buffalo milk, cow milk, goat milk, sheep milk, and camel milk. It was

observed that 15.8% of contaminated buffalo milk samples and 20% of contaminated cow

milk samples were above the EU action level of 0.05 µg/ L for AFM1 (European

Commission, 2006). However, none of the contaminated milk sample from goat milk and

sheep milk exceeded the EU action level for AFM1. No contamination of AFM1 in the

analyzed samples of camel milk was detected. In present study the contamination level of

AFM1 in raw milk samples was found to be low as compared to the results of earlier

studies in the same area (Hussain and Anwar, 2008; Hussain et al., 2008).

Table 21: Concentration of aflatoxin M1 (µg/ L) in the contaminated milk samples of

five species.

Sample No. Buffalo milk

Cow milk Goat milk Sheep milk Camel milk

12345678910111213141516171819

0.02110.03230.02350.03410.01950.02300.02370.03180.03340.00570.03000.04100.12000.02600.02010.07210.04060.04530.0845

0.03900.02410.03340.07290.08700.01520.02540.03720.02830.02100.02220.06770.02840.02150.0543

0.01590.01450.00660.00790.00890.0136

0.00530.00770.01360.0125

Not detected in all the

tested samples

According to the statistical analysis of the data, there exists a variation in the

concentration of AFM1 (F = 4.952, p = 0.001) in the raw milk of the five concerned

species. In cow milk the percentage of AFM1 contamination is the highest whereas in

103

camel milk the percentage of AFM1 contamination is zero. The AFM1 contamination

level of goat milk and sheep milk is also well below the EU action level. The reason for

this seems to be that the species namely camel, goat, and sheep are mainly fed by grazing

and this practice is effective to reduce the level of AFM1 concentration in milk.

Moreover, cotton-seed cake, corn, and concentrate feed are not used for these species in

this area and these feed commodities are the major sources of aflatoxin contamination.

The milk of sheep and goat is safe with respect to AFM1 contamination, whereas the

camel milk is the safest regarding the contamination of AFM1 in this area.

Fat% in buffalo milk, cow milk, goat milk, sheep milk, and camel milk is given in

Table 22, Table 23, Table 24, Table 25, and Table 26 respectively. Protein% in buffalo

milk, cow milk, goat milk, sheep milk, and camel milk is given in Table 27, Table 28,

Table 29, Table 30, and Table 31 respectively.

Table 22: Fat% in buffalo milk samples.

Fat% in buffalo milk samples7.7 6.5 7.5 7.1 8.5 7.5 5.3 7.5 7.3 7.0 8.27.3 6.2 10.0 6.8 7.5 8.2 7.3 3.5 6.2 7.0 7.47.1 6.6 5.1 6.7 5.8 6.0 5.7 4.5 11.0 5.8 6.16.8 6.5 7.2 6.2 7.3 7.6 4.2 6.7 7.4 8.5 7.67.7 7.7 7.5 8.2 8.1 8.3 8.1 6.4 7.9 7.8 7.6

Table 23: Fat% in cow milk samples.

Fat% in cow milk samples2.3 6.0 4.5 2.2 2.5 6.1 3.5 3.3 2.7 2.65.8 4.6 5.1 5.3 3.6 4.2 3.8 5.2 3.5 3.23.7 4.3 4.3 4.2 3.1 4.0 3.9 3.0 4.1 2.93.8 3.4 3.2 4.3 4.4 3.6 4.1 4.2 3.4 3.2

104

Table 24: Fat% in goat milk samples.

Fat% in goat milk samples5.3 5.1 5.4 5.5 4.0 5.0 3.6 3.8 3.5 3.74.1 3.9 3.5 3.6 3.7 3.1 3.3 3.4 3.3 4.23.8 3.7 3.9 3.6 3.2 3.4 3.9 4.1 3.6 3.7

Table 25: Fat% in sheep milk samples.

Fat% in sheep milk samples7.2 5.5 8.0 8.7 8.8 8.3 8.8 8.57.9 7.7 7.6 7.8 7.9 8.9 8.2 7.68.4 8.5 8.6 8.8 8.7 7.8 8.1 8.7

Table 26: Fat% in camel milk samples.

Fat% in camel milk samples4.4 3.4 3.5 3.3 3.3 3.63 3.13.5 3.8 3.4 3.2 3.1 4.0 3.72.5 2.3 2.2 2.3 3.2 3.1 ×

Table 27: Protein% in buffalo milk samples.

Protein% in buffalo milk samples4.9 4.7 3.8 5.1 4.7 3.6 4.83.2 4.2 4.5 4.9 4.3 3.8 4.84.3 4.2 3.3 5.1 5.5 5.6 4.83.7 4.9 5.3 4.9 4.1 4.5 5.34.6 5.1 5.3 5.1 4.7 4.4 5.04.2 5.3 2.7 4.5 4.8 4.6 4.74.3 4.3 6.0 3.8 4.9 3.0 5.04.7 4.1 4.8 3.4 5.2 4.6 ×

Table 28: Protein% in cow milk samples.

105

Protein% in cow milk samples3.6 4.6 4.5 4.3 3.8 3.6 4.63.7 4.3 4.2 4.4 3.6 4.1 3.42.9 3.1 2.8 2.9 3.1 2.7 3.23.2 3.0 3.4 3.4 2.7 3.3 3.53.5 3.3 2.3 2.9 3.3 3.6 ×3.0 2.9 3.2 3.4 4.1 3.1 ×

Table 29: Protein% in goat milk samples.

Protein% in goat milk samples3.9 4.0 5.5 5.0 4.1 3.8 3.53.3 3.6 3.8 3.6 3.7 3.9 4.23.9 4.0 3.8 3.4 3.6 4.3 3.83.2 3.9 3.4 3.1 3.6 × ×4.0 3.0 3.5 3.6 × × ×

Table 30: Protein% in sheep milk samples.

Protein% in sheep milk samples5.4 5.7 7.2 5.5 5.3 5.4 5.25.8 5.7 5.6 5.3 5.9 5.1 5.55.1 5.1 5.2 5.7 5.8 6.0 5.45.6 5.6 6.1 × × × ×

Table 31: Protein% in camel milk samples.

Protein% in camel milk samples3.4 3.5 3.2 3.3 3.4 3.1 3.03.6 3.7 3.8 4.0 3.9 3.2 3.53.4 3.6 3.7 3.8 3.5 3.6 ×

106

Table 32: Composite result of AFM1 (µg/ L) contamination, fat%, and protein% in milk

of the five species.

Species→ Buffalo Cows Goats Sheep Camel

Total samples 55 40 30 24 20

Contaminated

samples 19 15 6 4 Nil

AFM1 Mean±SD 0.013±0.024 0.014±0.022 0.002±0.005 0.002±0.004 ND

Fat% Mean±SD 7.0±1.26 3.8±0.95 3.9±0.66 8.1±0.73 3.2±0.60

Protein% Mean±SD 4.5±0.67 3.4±0.57 3.8±0.50 5.5±0.44 3.5±0.28

107

Fig. 13: Comparison of AFM1 contamination%, fat%, and protein% in milk of the five

species.

Table 32 gives a summary of results of AFM1 contamination, fat%, and protein%

in milk of the five species. Fig. 13 gives comparison of AFM1 contamination%, fat%,

and protein% in milk of the five species. The detail of AFM1 contamination, fat%, and

protein% in buffalo milk, cow milk, goat milk, sheep milk, and camel milk is given in

Table 33, Table 34, Table 35, Table 36, and Table 37 respectively. Table 38 gives the

detailed correlation analysis for all species. It was observed that there was no correlation

between AFM1 concentration and fat % for all species. Similarly no correlation was

found between AFM1 concentration and protein%.

7

3.8 3.9

8.1

3.24.5

3.4 3.85.5

3.5

34.5

37.5

20

16.7

11.1

0

5

10

15

20

25

30

35

40

Buffalo Cow Goat Sheep Camel

Fat %

Protein %

AFM1 contamination %

108

Table 33: Aflatoxin M1 (µg/ L) contamination, fat%, and protein% in buffalo milk.

Sample Fat% Protein% AFM1

(µg/ L)Sample Fat% Protein% AFM1

(µg/ L)123456789101112131415161718192021222324252627282930

7.76.57.57.18.57.55.37.57.37.08.27.36.210.06.87.58.27.33.56.27.07.47.16.65.16.75.86.05.74.5

4.94.73.85.14.73.64.84.24.13.84.63.24.24.54.94.33.84.84.32.73.43.04.34.23.35.15.55.64.84.7

0.0211NDND

0.0323NDNDND

0.0235ND

0.0341NDNDND

0.0195NDND

0.0230NDNDNDND

0.0237NDNDND

0.0318NDNDNDND

313233343536373839404142434445464.74.849505152535455

11.05.86.16.86.57.26.27.37.64.26.77.48.57.67.77.77.58.28.18.38.16.47.97.87.6

6.04.84.63.74.95.34.94.14.55.35.34.84.94.74.65.15.35.14.74.45.04.34.55.25.0

0.0334NDNDNDNDND

0.0057NDNDND

0.0300ND

0.0510ND

0.12000.0260

ND0.02010.07210.0406

ND0.0453

ND0.0845

ND

109

Table 34: Aflatoxin M1 (µg/ L) contamination, fat%, and protein% in cow milk.

Sample Fat % Protein % AFM1(µg/ L)12345678910111213141516171819202122232425262728293031323334353637383940

2.36.04.52.22.56.13.53.32.72.65.84.65.15.33.64.23.85.23.53.23.74.34.34.23.14.03.93.04.12.93.83.43.24.34.43.64.14.23.43.2

3.64.64.54.33.83.64.63.52.33.33.74.34.24.43.64.13.43.03.24.12.93.12.82.93.12.73.23.32.93.63.23.03.43.42.73.33.52.93.43.1

0.0390ND

0.0241NDNDNDND

0.0334ND

0.0729ND

0.087NDNDNDNDND

0.0152ND

0.0254ND

0.03720.0283

NDNDND

0.0210NDND

0.0222NDNDND

0.06770.0284

ND0.0215

ND0.0543

ND

110

Table 35: Aflatoxin M1 (µg/ L) contamination, fat%, and protein% in goat milk.

Sample Fat% Protein% AFM1(µg/ L)123456789101112131415161718192021222324252627282930

5.35.15.45.54.05.03.63.83.53.74.13.93.53.63.73.13.33.43.34.23.83.73.93.63.23.43.94.13.63.7

3.94.05.55.04.13.83.53.23.03.13.33.63.83.63.73.94.24.03.43.63.94.03.83.43.64.33.83.93.53.6

ND0.0159

ND0.0145

NDNDNDND

0.00660.0079

NDNDND

0.0089NDNDNDNDNDNDND

0.0136NDNDNDNDNDNDNDND

111

Table 36: Aflatoxin M1 (µg/ L) contamination, fat%, and protein% in sheep milk.

Sample Fat% Protein% AFM1(µg/ L)123456789101112131415161718192021222324

7.25.58.08.78.88.38.88.57.97.77.67.87.98.98.27.68.48.58.68.88.77.88.18.7

5.45.77.25.55.35.45.25.65.85.75.65.35.95.15.55.65.15.15.25.75.86.05.46.1

0.0053NDNDNDND

0.0077NDNDNDNDNDND

0.0136NDNDND

0.0125NDNDNDNDNDNDND

112

Table 37: Aflatoxin M1 (µg/ L) contamination, fat%, and protein% in camel milk.

Sample Fat% Protein% AFM1(µg/L)123456789101112131415161718

4.43.43.53.33.33.63.13.53.83.43.23.14.03.72.52.32.22.3

3.43.53.23.33.43.13.03.63.73.84.03.93.23.53.43.63.73.8

NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND

113

Table 38: Pearson correlation analysis of aflatoxin M1 (µg/ L) concentration, fat%, and

protein% in the milk of five species.

Species Variables Correlation values Status of correlation

Buffalo Fat% - AFM1

Protein%- AFM1

Fat% - Protein%

0.330(0.013)0.146

(0.286)0.130

(0.410)

Non-significant

Non-significant

Non-significant

Cow Fat% - AFM1

Protein%- AFM1

Fat% - Protein%

-0.075(0.064)0.075

(0.645)0.209

(0.196)

Non-significant

Non-significant

Non-significant

Goat Fat% - AFM1

Protein%- AFM1

Fat% - Protein%

0.328(0.077)0.150

(0.428)0.548

(0.002)

Non-significant

Non-significant

Significant

Sheep Fat% - AFM1

Protein%- AFM1

Fat% - Protein%

-0.047(0.826)-0.109(0.612)-0.195(0.361)

Non-significant

Non-significant

Non-significant

Camel Fat% - AFM1

Protein%- AFM1

Fat% - Protein%

N.A.

N.A.

-0.136(0.201)

N.A.

N.A.

Non-significant

114

4.4. Aflatoxin M1 Contamination in Milk Products

Milk is the fluid which is secreted by mammary glands of female mammals and it

contains all the nutrients necessary to sustain life and it is a major nutrient for humans

especially children. But it becomes hazardous when it contains contaminants like

aflatoxin M1 above the permissible level. The normal milk processes like pasteurization

have no significant effect on the AFM1 content in milk and AFM1 prevails in milk

products. However, fermentation produced significant effect on the stability of AFM1.

On the average, the concentration of AFM1 in yoghurt and fermented milk samples fell by

25% (Jasutiene et al., 2006). A number of countries have declared legal limits for AFM1

in milk and milk products. In some European countries, the limit for AFM1in milk has

been set 0.05 µg/ L and for AFM1 in cheese at 0.25 µg/ L (Manetta, et al., 2005).

However, there is no maximum tolerance level or such guidelines available for this

contaminant in Pakistan. Research in this area is among the major needs of the country.

Among the various milk products cheese and yoghurt are important ones. In

Pakistan the number of milk processing industries producing cheese and yoghurt is

limited. These industries are situated only in the Punjab province. The annual production

of cheese and yoghurt is 1164 and 10088 metric tons respectively (Agricultural Census

Organization, 2008). In the present study cheese and yoghurt samples, purchased from

local market, were analyzed for AFM1 contamination. Cheese is prepared from curdled

milk by removing whey and by curd ripening in the presence of special microflora, for

example, Lactococcus lactis is used as starter culture for Cheddar cheese. The process of

cheese formation consists of milk preparation, setting, molding, pressing, and ripening.

Yoghurt is prepared by bacteria culture. Yoghurt culture consists of thermophilic lactic

bacteria; Streptococcus thermophilus and Lactobacillus bulgaricus which live together

symbiotically (Belitz et al., 2004).

For AFM1 assay in milk and milk products, thin layer chromatography

(Van Egmond et al., 1978) and high-performance liquid chromatography (Dragacci et al.,

2001; Mortimer et al., 1987) are widely used. Through advances in biotechnology,

immunochemical procedures have been developed for the assay of AFM1, which are

based on the affinities of the monoclonal or polyclonal antibodies for aflatoxin. Three

types of immunochemical methods used for AFM1 analysis are: enzyme-linked

immunosorbent assay (Lee, 1996), radioimmunoassay (Saitanu, 1997), and

immunoaffinity column assay (Sharman et al., 1989; Dragacci & Fremy, 1996). Among

115

these, enzyme-linked immunosorbent assay method is simple, robust, and cost-effective.

It permits analysis of up to 200 samples per day. Enzyme-linked immunosorbent assay

(ELISA) is in frequent use for the assay of AFM1 (Domagala et al., 1997; Oliveira et al.,

1997). The reliability of ELISA in AFM1 determination was confirmed by Rosi et al.

(2007) by analyzing 1500 milk samples and results from 600 of them were compared with

those of the HPLC reference method. ELISA and HPLC assays of spiked samples gave

same precision (coefficient of variation, recovery, and regression coefficient R2 values are

0.9-8%, 96.8-108%, and 0.993 respectively)

The calibration graph was obtained from the standards provided along with the

ELISA kit having the concentration of AFM1 0.00 - 0.08 µg/ L in milk. Standard graphs

of ELISA analysis are shown in Fig. 14 and Fig. 15 for cheese and yoghurt respectively.

According to the protocol leaflet provided with the ELISA kit, detection limit is 0.005 µg/

L, recovery rate is 95% for spiking of AFM1 (0.01 µg/ L to 0.08 µg/ L) in fatty milk, and

specificity of the ELISA kit is 100% for Aflatoxin M1 (RIDASCREEN® Aflatoxin M1

30/15, 2007).

y = -968.66x + 95.857

R2 = 0.9932

0

20

40

60

80

100

120

0 0.05 0.1AFM1 concentration

(microgram/litre)

%A

bs

ora

nc

e

Fig 14: Standard curve from ELISA analysis for cheese.

116

y = -1104.1x + 94.857

R2 = 0.9907

0

20

40

60

80

100

120

0 0.02 0.04 0.06 0.08 0.1

AFM1 Concentration (microgram/Kg)

% A

bsor

banc

e

Fig. 15: Standard curve from ELISA analysis for yoghurt.

Total 160 samples of dairy products were analyzed for the contamination of AFM1

which comprised of 80 samples of cheese and yoghurt each. The Table 39 and Table 40

give the details of contamination of AFM1 in cheese and yoghurt samples respectively.

The study revealed that overall 87% samples of cheese and 70% of yoghurt samples were

found contaminated with AFM1

Table 39: Concentration of aflatoxin M1 (µg/ kg) in the contaminated cheese samples.

Concentration of AFM1 (µg/ kg) in cheese samples0.036 0.058 0.049 0.048 0.047 0.046 0.0370.011 0.041 0.039 0.028 0.031 0.053 0.0400.033 0.036 0.051 0.029 0.030 0.032 0.0420.034 0.035 0.021 0.055 0.057 0.066 0.0560.061 0.060 0.059 0.064 0.069 0.055 0.0660.062 0.060 0.063 0.044 0.058 0.064 0.0270.028 0.039 0.043 0.045 0.024 0.025 0.0740.072 0.075 0.054 0.073 0.075 0.074 0.0060.049 0.048 0.050 0.065 0.068 0.029 0.0700.071 0.067 0.065 0.054 0.052 0.041 0.038

117

Table 40: Concentration of aflatoxin M1 (µg/ kg) in the contaminated yoghurt samples.

Concentration of AFM1 (µg/ kg) in yoghurt samples0.018 0.033 0.055 0.022 0.044 0.025 0.038 0.0400.036 0.039 0.031 0.032 0.034 0.020 0.026 0.0370.041 0.029 0.027 0.007 0.006 0.073 0.006 0.0060.072 0.011 0.010 0.009 0.014 0.070 0.069 0.0650.064 0.057 0.067 0.062 0.060 0.059 0.035 0.0120.043 0.026 0.023 0.024 0.028 0.035 0.030 0.0350.021 0.019 0.017 0.042 0.045 0.047 0.049 0.050

Table 41: Aflatoxin M1 (AFM1) contamination (µg/ kg) in cheese and yoghurt.

Commidity→ Cheese Yoghurt

Total samples 80 80

Contaminated samples 70 56

AFM1 contamination% 87.5 70

AFM1 (µg/ kg) mean 0.049 0.036

Std. Deviation 0.016 0.019

Minimum (µg/ kg) 0.006 0.006

Maximum (µg/ kg) 0.075 0.073

There is higher percentage of contamination in the cheese samples as compared to

yoghurt samples. The details of AFM1 contamination in cheese and yoghurt samples are

given in the Table 41. According to the statistical analysis of the data, there exists

difference in the concentration of AFM1 in cheese and yoghurt samples (F= 18.218, p<

0.05). The mean value of contamination level of AFM1 is 0.049 µg/ kg (SD = 0.016) in

cheese samples and 0.036 µg/ kg (SD = 0.019) in yoghurt samples.

The results of AFM1 contamination in cheese and yoghurt were compared with the

studies of other researchers in different countries showing AFM1 contamination in dairy

products. Due to detrimental effect of AFM1 on human health, extensive research on

AFM1 is in process at this time. Large number of surveys are undertaken every year to

know the presence and levels of AFM1 in dairy products in different countries. Elgerbi et

118

al. (2004) analyzed 20 samples of fresh white soft cheese for the presence of AFM1, after

collecting directly from local dairy factories in the north-west of Libya. Out of 20, fifteen

samples (75%) showed the presence of AFM1 in concentration between 0.11 and 0.52 µg/

kg of cheese. Tekinsen and Tekinsen (2005) investigated the occurrence and

concentration range of AFM1 in 60 samples of Van otlu cheese and 50 samples of white

pickle cheese obtained from retail outlets in Van and Hakkari, Turkey. Aflatoxin M1

ranged from 0.16 to 7.26 µg/ kg and 0.10 to 5.20 µg/ kg in Van otlu and white pickle

cheese samples respectively. Two hundred and twenty three samples of dairy products

including 196 cheese samples, marketed in Ankara, Turkey during Sep. 2002 and Sep.

2003 were analyzed for AFM1. The incidence of AFM1 contamination was found 90.58%

(Aycicek et al. 2005). Commonly consumed cheese and yoghurt samples were randomly

collected from supermarkets in Ankara, Turkey and AFM1 contamination was determined

by Gurbay, et al. (2006). Aflatoxin M1 was detected in 11 cheese sample out of 39

samples in the range of .078 µg/ kg to 0.188 µg/ kg. Thirty two out of 40 yoghurt

samples had AFM1 levels between 0.061 and 0.365 µg/ kg. Aflatoxin M1 contamination

was determined by Yapar et al. (2008) in 105 samples of cheese produced in north eastern

Turkey. Seventy five cheese samples (71.42%) were found contaminated with AFM1,

whereas the level of AFM1 in 40 cheese samples (30.08%) was found to exceed the limit

(0.25 µg/ kg) allowed by Turkish Food Codex. Dashti et al. (2009) analyzed a total of 40

cheese samples for the contamination of AFM1, which were collected randomly during

January 2005-March 2007 from Kuwait markets. Results showed 80% contamination

with AFM1 in cheese samples with a range 0.023-0.452 and mean of 0.087 µg/ kg. One

sample was above the regulatory limit (0.25 µg/ kg). The AFM1 contamination in milk

and milk products results from aflatoxin B1 contaminated feedstuffs ingested by lactating

animals. The first step to prevent the transfer of aflatoxins to humans is to control the

feed hygiene and there is need for stringent quality control during processing and

distribution of dairy products and this can be achieved by adopting good manufacturing

practices.

119

4.5. Aflatoxin B1 Contamination in Dairy Feed

Feed of lactating animals has substantial role in the occurrence of AFM1 in milk.

A number of commodities are used as feedstuffs to meet the productive requirements of

dairy animals in addition to the fodder which is mainly required for maintenance

purposes. Aflatoxins are present in feed commodities. The feedstuffs may be divided

into two types i.e., balanced and non-balanced. The balanced feed includes concentrated

feed which may simply be called as concentrate. The non-balanced feedstuffs include

cottonseed cake, wheat bran, maize cake, and bread etc. Moreover, wheat straw and

paddy straw are also used as dry roughages along with fodder. The ingredients of

concentrate are cereals (wheat, maize, rice, millet, barley, oat, and sorghum), wheat bran

(bye-product of flour mills), rice bran and broken-rice (bye-product of rice Sheller),

cottonseed cake (bye-product of oil expeller), cottonseed meal (bye-product of oil solvent

extraction plant), maize gluten meal, sunflower meal, soybean meal, and canola meal etc.

Most of the feedstuffs and concentrate ingredients, under favorable conditions of

temperature and moisture, are prone to the attack of fungi which results in AFB1

contamination (Tanaka et al., 2007; Ammida et al., 2006; Hwang and Lee, 2006; Gao et

al., 2005).

The present study has been designed to explore the AFB1 contamination in

different commodities used as feedstuffs for dairy animals in Pakistan, by using high-

performance liquid chromatography with fluorescence detection. Methods including

enzyme-linked immunosorbent assay (ELISA), thin layer chromatography (TLC), high-

performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry

(LC-MS) have been developed to analyze aflatoxins (Bacaloni et al., 2008; Sodhi and

Kapur Ghai, 2007; Var et al., 2007). High-performance liquid chromatography with

fluorescence detection has been increasingly used in the determination of aflatoxins due

to its accuracy, precision, and ease in automation. HPLC has wide range applications in

analytical chemistry. For analyzing aflatoxins HPLC is a preferred method which may be

used in normal-phase mode or reversed-phase mode. Normal-phase chromatography uses

polar (water or triethyleneglycol) bonded silica surface as stationary phase and a non-

polar mobile phase such as hexane or iso-propylether, whereas reversed-phase

chromatography employs a non-polar stationary phase, mostly a hydrocarbon, and a polar

120

mobile phase such as water, methanol, or acetonitrile (Skoog et al., 2003). For the

analysis of aflatoxins, most of the HPLC columns contain C8 or C18 bonded silica and

the mobile phase may be binary or ternary mixture of polar solvents. The most

commonly used solvent mixture includes deionized water, acetonitrile, and methanol.

The different steps involved in aflatoxin analysis are extraction, clean-up, and

quantitation. In present study the clean-up step was accomplished with the use of

MycoSep® columns. MycoSep® column shortens the time-consuming process of clean-

up. These contain a combination of adsorbents for complex commodities and are

applicable to grains, silages, feed, and food.

The original HPLC chromatograms are shown in Fig. 16. The standard curve was

obtained from AFB1 standards of concentrations from 0.5µg/ L to 15µg/ L and was found

linear (y = -157.13 + 476.08x, y = area, x = amount of AFB1). The linear standard curve

is shown in Fig. 17. The coefficient of determination (R2) was 0.9923. The LOD was

determined as 0.5µg AFB1/ kg of feed. The validation of the method was done by spiking

feed samples with standard AFB1 solution. The spiking was made at the level of 8 µg/

kg, 10 µg/ kg and 15 µg/ kg and recovery was found to be 101%, 92% and 88.6%

respectively.

Concentration of AFB1 in samples of cottonseed cake, concentrate feed, wheat

bran, bread and paddy straw is given in Table 42, Table 43, Table 44, Table 45, and Table

46 respectively. Total 260 samples of feedstuffs were analyzed for the contamination of

AFB1 which comprised of 80 cottonseed cake, 60 concentrate, 36 wheat bran, 24 bread,

24 paddy straw and 36 wheat straw samples. The contamination percentage in cottonseed

cake, concentrate feed, wheat bran, wheat bread, and paddy straw samples is 100%, 91%,

55%, 33%, and 33% respectively. All these feedstuffs are vulnerable to the attack of

moulds. The Fig. 17 shows the growth of moulds on bread. When AFB1 contamination

was compared in old and new samples of feedstuffs, its percentage was higher in old

samples as compared to the new ones. None of the 16 samples of wheat straw was found

contaminated with AFB1. Table 47 gives the detail of contamination of AFB1 in different

commodities. The study revealed that overall 65.7% samples of different feedstuffs were

found contaminated with AFB1. The aflatoxin contaminated feedstuffs ultimately cause

AFM1 contamination in milk.

121

Fig. 16: HPLC chromatograms: (A) Standard (B) Sample, for aflatoxin B1.

122

Fig. 17: The linear standard curve of AFB1 standards with concentrations from 0.5µg/ L

to 15µg/ L.

Table 42: Concentration of aflatoxin B1 (µg/ kg) in the contaminated cottonseed cake

samples.

Concentration of AFB1 (µg/ kg) in cottonseed cake samples

232 230 218 616 204 202 262 260 257 255198 196 822 220 199 192 178 177 276 474284 282 230 228 224 223 230 228 263 861233 631 258 257 261 259 15 18 235 1214 13 11 19 16 17 220 410 216 220310 315 202 204 180 185 228 228 225 248243 215 404 250 246 238 232 235 225 190195 215 208 430 226 315 330 179 175 231

Regression standard predicted values of AFB1

2.01.51.0.50.0-.5-1.0

AR

EA

8000

7000

6000

5000

4000

3000

2000

1000

0

y=-157.13 + 476.08x

R2 = 0.9923

123

Table 43: Concentration of aflatoxin B1 (µg/ kg) in the contaminated concentrate

samples.

Concentration of AFB1 (µg/ kg) in concentrate samples

7 6 172 171 3 213 214 4 291 190 365164 309 188 337 186 172 171 484 183 404 203200 202 240 235 135 130 150 165 155 160 180175 145 148 211 12 216 218 14 18 15 166230 137 215 185 178 191 168 244 134 142 143

Table 44: Concentration of aflatoxin B1 (µg/ kg) in the contaminated wheat bran

samples.

Concentration of AFB1 (µg/ kg) in wheat bran samples

65 64 66 63 132 131 123 121 133 132130 128 10 8 14 160 55 140 145 148

Table 45: Concentration of aflatoxin B1 (µg/ kg) in bread samples.

Concentration of AFB1 (µg/ kg) in wheat bran samples

27 16 11 1418 24 37 39

Table 46: Concentration of aflatoxin B1 (µg/ kg) in the contaminated paddy straw

samples.

Concentration of AFB1 (µg / kg) in paddy straw samples

45 61 12 1713 46 58 47

124

Table 47: Aflatoxin B1 (AFB1) level (µg/ kg) in different feedstuffs.

Sample category Total samples Contaminated samples Mean SD

Concentrate 60 55 176.25 96.24

Cottonseed cake 80 80 242.04 146.71

Wheat bran 36 20 98.40 49.87

Bread 24 8 23.24 10.47

Paddy straw 24 8 37.38 20.27

Wheat straw 36 Nil ND ND

Picture 2: The growth of moulds on bread.

The results of AFB1 contamination in feedstuffs were compared with the studies

of other researchers in different countries showing aflatoxin contamination of feeds and

feedstuffs. In Korea, Han et al. (2006) confirmed the contamination of only one feed

sample out of 249 total samples, at the level of 11µg/ kg, by using HPLC. Martins et al.

(2007) reported a survey on the occurrence of AFB1 in dairy cow’s feed over ten years

125

(1995-2004) in Portugal. The levels of AFB1 above the maximum limit established in

Portugal (5 µg/ kg) for dairy feed were observed in 62 (6.2%) samples with levels ranging

from 5.1 to 74 µg/ kg. A two years survey was conducted by Binder et al. (2007) on

1507 samples sourced from European and Mediterranean countries and 1291 samples

originating from the Asian-Pacific region for the occurrence of mycotoxins in

commodities, feeds, and feed ingredients. Aflatoxin B1 was detected in 54 maize samples

(out of 311 with max. level of 457 µg/ kg), 3 maize gluten meal samples (out of 37 with

max. level of 45 µg/ kg), 3 soybean meal samples (out of 122 with max. level of 45 µg/

kg), 3 rice-bran samples (out of 27 with max. level of 11 µg/ kg), 8 peanut meal samples

(out of 9 with max. level of 381), and 109 finished feed samples (out of 536 with max.

level of 330 µg/ kg). In present study 59.6% feedstuff samples exceeded the US

maximum AFB1 tolerance limit (20 µg/ kg), while 65.7% samples were above the EU

action level for AFB1 (2 µg/ kg).

There is the highest percentage of contamination and level of contamination in

samples of cottonseed cake. According to the statistical analysis of the data, there exists a

difference in the concentration of AFB1 in different feedstuffs (F= 14.709, p< 0.05).

Thus, contamination level varies in different commodities. Cottonseed cake contains the

highest level of AFB1 and almost 80% dairy farmers use cottonseed cake as feedstuff

which should be discouraged in order to check AFB1 contamination. AFB1 contamination

was higher in old samples as compared to the fresh ones. Some old samples did not show

contamination of AFB1 which led to the deduction that if storage conditions were suitable

then there were less chances of AFB1 contamination.

4.6. CONCLUSIONS

In conclusion, this study revealed the contamination of AFM1 in milk and milk

products and AFB1 contamination in dairy feed commodities. The main results may be

summarized as following:

1. According to the overall study of AFM1 contamination in raw milk in fourteen

districts of the Punjab province of Pakistan, 99.4% samples showed AFM1

concentration higher than the EU tolerance limit, while 3% samples were higher

in AFM1 contamination than the US tolerance limit.

2. The AFM1 contamination in milk during winter season was found to be the

highest as compared to the AFM1 contamination in other seasons.

126

3. The percentage of AFM1 contamination in milk and level of contamination was

higher in the milk from urban and semi-urban areas as compared to that from rural

areas.

4. The AFM1 contamination was minimal in milk from rural areas.

5. Higher AFM1 contamination was observed in milk from buffaloes belonging to

small herd-size and medium herd-size as compared to AFM1 contamination in

milk from buffaloes belonging to large herd-size.

6. During the study of species of milking animals with respect to AFM1

contamination in milk, buffalo milk and cow milk showed higher AFM1

contamination in milk as compared to the AFM1 contamination in milk of goats,

sheep, and camels.

7. The AFM1 contamination in milk from goats and sheep was found insignificant

and was much below the US as well as EU tolerance limits. Thus the goat milk

and sheep milk from the area under consideration was found safe regarding AFM1

contamination.

8. The AFM1 contamination in camel could not be detected. Thus the camel milk

from the area studied was found to be the safest with respect to AFM1

contamination.

9. Due to the AFB1 contamination, dairy feed plays a major role in the AFM1

contamination in milk. The AFB1 contamination in feed commodities was

investigated and it was concluded that cottonseed cake and concentrate feed were

heavily contaminated with AFB1. The other feed commodities like wheat bran,

bread and straw were also found contaminated with AFB1.

10. Precautions must be taken in the storage of feed commodities. Low moisture

content, low temperature and low humidity conditions should be maintained

during storage because these depress the fungus growth and thus eliminate

aflatoxin contamination.

4.6.1. Implications of Study Results and Elimination of AFM1

Contamination in Milk and Milk Products

Pakistan is an agricultural country and its products should not be limited to the

local market, rather these should be trade oriented products and should be competent in

the international market. The ever changing world has been transferred into a global

127

village where the principle, survival of the fittest is strictly followed. Therefore, to enjoy

any meaningful share in the world trade, the products must meet established international

standards. Pakistan has accepted WTO rules and arrangements should be made for

improvement of quality of its agricultural products so that these might be according to the

international standards as well as safe for the consumers.

The AFM1 is cytotoxic, as shown by the results of in vitro studies in human

hepatocytes, and its acute toxicity in several species is similar to its parent compound

AFB1. According to the studies of carcinogenicity, AFM1 was about one-tenth as potent

as AFB1. The genotoxicity of AFM1 in vitro was found to be similar to that of AFB1 in

some test systems and between one-half and one-sixth of that of AFB1 in others. The

AFM1 contamination of milk and milk products has been found in the area under study

(Maqbool, et al., 2009; Hussain and Anwar, 2008; Hussain et al., 2008). The results of

the present study point to the need for measures to be taken to reduce and control the

AFM1 contamination in milk and milk products. As per results, contamination in buffalo

milk and cow milk is higher and these two species are the main source of milk in this

area. The milk of other species i.e., goat, sheep, and camel is used at limited level in this

area and furthermore the milk from these three species has been declared safe from the

AFM1 contamination point of view (Hussain et al., 2009). So attention should be mainly

focused on the reduction of AFM1 contamination in buffalo milk and cow milk.

Approximately 0.3-6.2% of AFB1 in animal feed is transformed to AFM1

in milk. The best way of controlling AFM1 in milk and milk products is to reduce the

amount of AFB1 in the feed of dairy animals. The concentration AFB1 in dairy feed can

be reduced by good manufacturing practice and good storage practices. Those feedstuffs

should be discarded which are apparently attacked by fungus. Long standing storage of

commodities used as dairy feed should be discouraged. Fresh feedstuffs for milking

animals should be used because during storage fungal growth and mycotoxin

contamination is suspected. In case of storage, aeration is recommended which helps to

maintain low humidity and is responsible for low moisture content; and thus chances of

fungal growth and toxin production are reduced.

As during the study, the cottonseed cake and concentrate feed have been found

heavily contaminated with AFB1, therefore, the use of these two commodities must be

controlled. If the use of these commodities is inevitable, these must be blended with the

commodities which are free from AFB1 contamination, so that the final feed product must

not contain the toxin exceeding the tolerance level.

128

Many studies have proved the effectiveness of toxin binders, used in feed, on the

reduction of carry-over of AFB1 to AFM1 in milk (Diaz, et al., 2004; Galvano et al.,

1996). Now-a-days a number of toxin binders for aflatoxins are available commercially

in Pakistan. A few examples are: Myco-AD (Special Nutrients Inc., USA), Mycofix®

Plus 3.0 (Biomin®, Austria), Mycotox® (Ceva Animal Health, France,). Proper use of

toxin binders is recommended for the feed of milking animals to reduce and control the

AFM1 contamination in milk and milk products. The correct dosage of most toxin

adsorbents in order to be effective is 5 to 20 kg per ton (1000 kg), depending on the level

of contamination.

During the study of AFM1 contamination with respect to herd-size and locality-

wise, the main contamination was observed in medium size herds and in the localities of

urban and semi-urban. Thus the people rearing medium-size herds must be focused. The

areas of urban and semi-urban must get attention. The people rearing milking animals are

not much educated. These people should be informed about the menace of aflatoxin

contamination. This is an era of media and media plays an important role in educating

people and to keep them well informed. Media should be properly used to get the people

informed about aflatoxin contamination and its consequences. Both print media and

electronic media should be utilized for this purpose. NGOs may play their role in this

regard.

Presently the Food Rules and specifications of Pakistan Standards and Quality

Control Authority (PSQCA) make no mention of maximum tolerance limits of aflatoxins

in food and feedstuffs (Pakistan Standard: 3189-1992, 1992; Pakistan Standard: 364-

1991, 1991; Pakistan Standard: 363-1982, 1982; Manual of Food Laws in Pakistan,

2004). In the light of this study, there is need for establishing food legislations governing

maximum tolerance limit of AFB1 in feed commodities for dairy animals and maximum

tolerance limit of AFM1 in milk and milk products in the country. The Government body

which deals and governs the standards in Pakistan is PSQCA. It may adopt US maximum

tolerance limit of 0.5 µg AFM1/ L of milk. This limit is both adequate for the protection

of consumers’ health and reasonably achievable in the country. It will also not restrict

adequate supply of milk for human use.

Analysis of aflatoxins at µg/ L or kg level needs high tech. laboratories equipped

with highly sophisticated instrumentation. Adequate number of laboratories must be

established for proper analysis of aflatoxins in different foods and feed commodities and

129

also for certification purposes, as required by the international trade. There should be

continuous surveillance programs in the country to monitor the occurrence of aflatoxins

regularly in milk and milk products.

130

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155

APPENDIX

LIST OF PUBLICATIONS

Hussain, I. & Anwar, J. (2008). A study on contamination of aflatoxin M1 in raw milk in

the Punjab province of Pakistan. Food Control 19 (4), 393-395.

Hussain, I., Anwar, J., Munawar, M. A., & Asi, M. R. (2008) Variation of levels of

aflatoxin M1 in raw milk from different localities in the central areas of Punjab,

Pakistan. Food Control 19 (12), 1126-1129.

Hussain, I., Anwar, J., Asi, M. R. Munawar, M. A., & Kashif, M. (2009) Aflatoxin M1

contamination in milk from five dairy species in Pakistan. Food Control xxx (2009),

in press.

156

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