ASSESSMENT OF QUALITY AND CONTAMINANTS IN COMMERCIALLY AVAILABLE MILK IN KARACHI AND ITS IMPACT ON HUMAN HEALTH BY RUBINA PERWEEN A thesis submitted to the Graduate Research Management Council In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY In Chemistry At Department of Chemistry, Federal Urdu University of Arts, Science and Technology, Karachi-75300, Pakistan 2014 i Federal Urdu University of Arts, Science and Technology, Gulshan-e-Iqbal Campus, Karachi, Pakistan Department of Chemistry Certification Certified that Rubina Perween enrolled for the research program M. Phil leading to Ph. D. She has fulfilled all criterion of Higher Education Commission (HEC) Islamabad for the degree of doctorate. This thesis entitled “Assessment of quality and contaminants in commercially available milk in Karachi and its impact on human health” is submitted by her is reveal for the partial fulfillment of the requirements for the Degree of Doctor of Philosophy. ______________________ ________________________ Prof. Dr. Qamar-ul Haque Dr. Humera Anwar (HEC Approved Supervisor) Supervisor Co- Supervisor ___________________________ __________________________ External Examiner Chairperson ii DEDICATION This thesis is dedicated to My Mother My Husband And to my Sisters For their unwavering support and understanding that kept I always focused on the goal iii ACKNOWLEDGEMENTS By the grace of Allah the Almighty and Benevolence of Prophet Hazrat Mohammad (PBUH), I have been able in carrying out and completing this Research work. I am indebted and extend sincere thanks to my Supervisor Prof. Dr. Qamar-ul Haque and Co- Supervisor Dr. Humera Anwar for their help in successful accomplishment of this task. I am also thankful to the chairperson of my department Prof. Dr. Iffat Mehmood for her generous cooperation. I really feel great pleasure and deep gratitude to Prof. Dr. Moinuddin Ahmed, Director of Laboratory of Dandrochronology and Plant Ecology (LDPE), Department of Botany Federal Urdu University for provision of working facilities and his student Dr. Kanwal Nazim, Dr. Mohammad Uzair Khan for their co-operation. I grateful to Prof. Dr. Abdul Hakim Sheikh, Dr. Mohammad Abid, Associate Professor and Dr Toqeer Ahmed Rao, Assistant Professor, Research Laboratory of Plant and Aerobiology, Department of Botany, Federal Urdu University for their help and guidance in the identification of mycobiota and their students Hina Zafar & Dr. Faisal Hussain for their assistance. I owe my special thanks to Prof. Dr. S. Shahid Shaukat for going through the Statistical Analysis and his generous intellectual encouragement. I obliged as well to Dr. Abbas Bhutto, senior research officer, Grain Quality Testing Laboratory (GQTL), Pakistan Agriculture Research Council (PARC), Karachi University and my colleague Dr. Kauser Yasmeen, Assistant Professor, Department of Chemistry, Federal Urdu University for their providing technical support in estimation of metals through Atomic Absorption Spectrometer. Thanks also Prof. Atia Naeem, Department of Microbiology, Federal Urdu University for her support in microbial assessment. Also, I am grateful to Dr. Mohammad Azam, Department of Geography, Federal Urdu University for providing District level map of Karachi city. iv The author grateful to Eng. Muhammad Yusuf, Senior scientific officer, Centralized Science Laboratories (CSL), University of Karachi to provide technical support in SEM-EDX spectroscopy. My cordial thanks are extended to my colleagues Mrs. Dr. Iffat Abul Tawab, Miss Asma Dilshad, Miss Nida Ali, and students of MSc. Final Mr. Zahoor, Mr. Kaleem, Mr. Ata Ullah, Miss. Sarwat, and Miss Sassi for their voluntary services in sample compilation. Some very special and joyful thanks are extended to all technical staff of Department of Chemistry and Department of Botany for their utmost help and support. I present my affable and deep gratitude to Dr. Zarina Ali (Chairperson Department of Botany) and Dr Alia Abbas Department of Botany, Federal Urdu University for their appreciation and moral support. Lastly, I also want to convey my deepest gratitude to all my honorable colleagues Prof. Shaheena Najmi, Prof. Rehana Iqbal (Ex-Chairpersons Department of Chemistry), Prof. Dr. Talat Tarique, Dr. Farah Kishwar, Anila Anwar and all friends for their encouragement and continued support. May, 2014 Rubina Perween v CONTENTS Title Certification Dedication Acknowledgements Content List of tables List of figures Abstract Khulasa Page no. Chapter 1: General Introduction_________________________________ 1-28 1.1 Overview……………………………………………………................. 1 1.2 Theme of study………………………………………………................... 4 1.3 Purpose of study……………………………………………….............. 4 1.4 Milk…………………………………………………….………............ 5 1.4.1Chemical Properties of Milk…………………………………. 5 1.4.1.1Fat…………………………………………………….. 5 1.4.1.2 Protein……………………………..……………….. 6 1.4.1.3 Lactose……………………………………………… 6 1.4.1.4 Mineral components………………………………. 6 1.4.1.5 Vitamins……………………………………............ 9 1.4.1.6 Enzymes……………………………………............ 9 1.4.1.7 Organic acids………………………………............... 9 vi 1.4.1.8 Hormone and hormone-like substances…….............. 10 1.4.2 Processing of fresh milk…………….………………………. 10 1.4.2.1 Standardization……………………………………. 11 1.4.2.2 Homogenization……………………………………. 11 1.4.2.3 Heat treatment……………………………………… 11 1.4.2.3.1 Pasteurization…………………………….. 11 1.4.2.3.2 Ultra heat treatment or ultra high 12 temperature (UHT)……………………… 1.4.2.3.3 Sterilization………………………................ 12 1.4.2.3.4 Packaging………………………………… 12 1.4.2.4 Toned milk…………………………………….......... 12 1.4.2.5 Skimmed milk……………………………………… 13 1.4.2.6 Milk analogues……………………………….......... 13 1.4.2.7 Some consequences of processed milk……………. 13 1.4.3 Milk allergy and milk borne illness…………………………. 13 1.5 Physico-chemical parameters examination………………………....... 14 1.5.1 Intrinsic factors …………………………………………….. 14 1.5.2 pH……………………………………………………………. 14 1.5.3 Electrical Conductivity (EC)………………………………… 14 1.5.4 Salinity……………………………………………….………. 15 1.5.5 Total Dissolved Solids (TDS)…………………………......... 15 1.5.6 Dissolved Oxygen (DO)………………………………........... 15 1.5.7 Specific Gravity……………………………………………… 15 vii 1.6 Assessment of Essential and Non-Essential Metals………………….. 15 1.6.1 Chromium (Cr)………………………………………………. 16 1.6.2 Manganese (Mn)…………………………….………………. 16 1.6.3 Iron (Fe)………………………………………………........... 17 1.6.4 Cobalt (Co)………………………………………………….. 17 1.6.5 Nickel (Ni)……………………………………………………. 18 1.6.6 Copper (Cu)…………………………………………………. 18 1.6.7 Zinc (Zn)………………………………………………........... 18 1.6.8 Cadmium (Cd)………………………………………………. 19 1.6.9 Lead (Pb)…………………………………………………….. 19 1.7 Microbial contamination investigation………………………………….. 23 1.7.1 Bacteria…………………………………………….………… 23 1.7.2 Fungi …………………………………….……..……………. 23 1.7.2.1 Alterneria………………………………….….…….. 24 1.7.2.2 Aspergillus……………………………….………….. 24 1.7.2.3 Drechslera…………………………………………… 25 1.7.2.4 Fussarium……………………………………………. 25 1.7.2.5 Mucor, Pythium, Penicillium & Rhizopus...……….. 27 Chapter 2: Facts Profile of Study Area____________________________ 29-38 2.1 Introduction…………………………………..………………….…….. 29 2.2 Topography and physical features…………………………………….. 29 2.3 Climate ……………………………………………..………………….. 30 2.4 Division of the city………….…………………………..…………….. 30 2.4.1 Karachi Central District….………………………………….. 31 2.4.2 Karachi East District……….……………………………….. 31 2.4.3 Karachi West District……….………………………………. 31 viii 2.4.4 Karachi South District……….………………………………. 32 2.4.5 Malir District………………….…………………………….. 32 2.5 Main dairy farms and cattle colonies of Karachi city…………………. 33 2.5.1 Landhi dairy colony (LDC) & surrounding…………………. 34 2.5.2 Bilal Dairy Colony (BDC) & surrounding…………………… 34 2.5.3 Surjani Dairy Colony (SDC) …………………………………. 35 2.5.4 Al-Momin Dairy Colony (ADC)………………………...……. 35 2.5.5 Nagori Dairy Colony (NDC)………………………………….. 35 2.5.6 Baldia, Lyari Diary Colony (BLDC) & Surrounding ……......... 35 2.5.7 Scattered Dairy Farms (SDF)………………………………….. 36 2.6 Main brands of processed milk …………..…………………………….. 36 2.7 Main brands of processed milk analogues …………………………....... 37 Chapter 3: Valuation of Fresh Milk______________________________ 39-98 3.1 Summary.…………………………………………………..…….............. 39 3.2. Introduction……………………………………………………..…….. 40 3.3. Methodology……………………………………………….……........ 42 3.3.1 Sampling…………………………..…………………...…….. 42 3.3.1.1 Sampling criteria………………………………........ 42 3.3.1.2 Sampling procedure………………………….………. 42 3.3.2 Physico-chemical Characteristics or intrinsic factors 43 Observations…………………………………………………. 3.3.3 Estimation of metals……………………….……………….. 43 3.3.4 Assessment of fungal contamination …………………...….. 43 3.3.5 Statistical Analysis………………………………………….. 44 3.4 Results and Discussion………………...…………………………..….. 45 3.5 Conclusion………………………………………………...……………. 97 ix Chapter 4: Quality assessment of processed milk and milk analogues__ 99-122 4.1Summary……………………………………………………………….. 99 4.2 Introduction………………………………………….…………………. 100 4.3 Materials and Methods…………………………………………………. 102 4.3.1Sampling…………………….………………………………… 102 4.3.2 Physical properties examination…………………….……….. 103 4.3.3Metal estimation……….………………………….………...... 103 4.3.4 Microbial analysis……………………………….….………… 103 4.3.5 Statistical analysis……………………………….….………… 104 4.4Results and Discussion………………………………………………….. 104 4.5Conclusion…………………………………………………….………… 122 Chapter 5: Elemental evaluation of Milk by SEM-EDX Technique___ 123-131 5.1Summary…………………….………………………………………...... 123 5.2 Introduction…………………………………………………………… 123 5.3 Materials and Methods…………………………………………………