VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE NGUYEN THI THANH XUAN ASSESSMENT OF AFLATOXIN B1 CONTAMINATION IN MAIZE AND AWARENESS OF AFLATOXINS IN SON LA PROVINCE Major : Food Science and Technology Code : 60540103 Supervisor : Dr Hu Suk Lee Dr Nguyen Thi Thanh Thuy AGRICULTURAL UNIVERSITY PRESS - 2017 DECLARATION This thesis contains no materials that have been accepted for the award of any other degrees or diploma in any educational institutions To the best of my knowledge, my paper includes no previously published work written by any person, except where indicated in references Hanoi, May 10th, 2017 Master candidate Nguyen Thi Thanh Xuan i ACKNOWLEDGEMENTS First of all, I am really grateful my supervisors Dr Hu Suk Lee from International Livestock Research Institute (ILRI) for his patience, enthusiasm, motivation and immense knowledge, and to Dr Nguyen Thi Thanh Thuy from Vietnam National University of Agriculture, who gave me the opportunity to carry out this thesis and always had a piece of advice ready regarding the practical aspect of my work Their guidance supported me during this research I wish to express my gratitude to Prof Nguyen Viet Hung, a Southeast Asia regional representative at ILRI in Vietnam, for giving me a profound insight into the research and sharing his valuable experience with me I always bear in mind the enthusiasm Prof Delia Grace and Dr Johanna Lindahl at ILRI in Nairobi I highly appreciate Ms Nguyen Le Thanh and Ms Le My Hanh for their help in the administrative aspect Eventually, I would like to thank to all the members at ILRI in Vietnam for giving me the opportunity to become temporarily a part of their team I owe my thanks to all staffs in Plant Protection Research Institute They helped me so much for conducting sampling, interview, data collection and laboratory analysis Many thanks particularly to the whole team, who went to Son La for their logistical support and suggestion I also express my admiration to all members in Son La who introduced me to the locals and supervised our team during sampling and interview process I am also very thankful to Son La residents who warmly welcomed our team in their homes and participated in the survey Last but not least, I want to convey my gratefulness to my family and friends for their unremitting support, patience and understanding Hanoi, May 10th, 2017 Master candidate Nguyen Thi Thanh Xuan ii LIST OF CONTENTS Declaration i Acknowledgements ii List of contents iii List of abbreviations v List of tables vii List of figures viii Thesis abstract ix Part Introduction 1.1 Introduction 1.2 Research objectives Part Literature review 2.1 Situation in the maize production in vietnam – case of Son La 2.1.1 Situation in the maize production in Vietnam 2.1.2 Study location - Son La province 2.2 Aflatoxins and their impacts on human and animal health 2.2.1 Aflatoxins 2.2.2 The impact of aflatoxins on human and animal health 2.3 Factors influencing aspergillus spp infection and aflatoxin development in maize 13 2.4 Situation in the aflatoxin contamination in maize 15 2.4.1 Situation in the aflatoxin contamination in the world 15 2.4.2 Situation in the aflatoxin contamination in Vietnam 16 2.5 Methods to detect aflatoxins 17 2.6 Regulations for aflatoxins in the world and Vietnam 18 2.6.1 Regulations for aflatoxins in the world 18 2.6.2 Regulations for aflatoxins in Vietnam 21 Part Materials and methods 24 3.1 Materials 24 3.1.1 Maize samples 24 3.1.2 The Sigma-Aldrich® Aflatoxin B1 Low Matrix ELISA kit 24 3.2 Methods 24 iii 3.2.1 Sample size calculation and selection 24 3.2.2 Sampling 25 3.2.3 Aflatoxin B1 analysis in Lab 25 3.2.4 Questionnaire survey 27 3.2.5 Survey data analysis 27 Part Results and discussions 30 4.1 Aflatoxin B1 contamination in maize in Son La 30 4.2 Awareness of aflatoxins in Son La 36 Part Conclusions and recommendations 46 5.1 Conclusions 46 5.2 Recommendations 46 References 47 Appendix 58 iv LIST OF ABBREVIATIONS Acronym Abbreviations A Aspergillus A flavus Aspergillus flavus A nomius Aspergillus nomius A parasiticus Aspergillus paraciticus AFB1 Aflatoxin B1 AFM1 Aflatoxin M1 AFs Aflatoxins CIT Citrinin DON Deoxynivalenol EC European Commission EFTA European Free Trade Association ELISA Enzyme Linked Immunosorbent Assay EU European Union F moniliforme Fusarium moniliforme FAO Food and Agriculture Organization of the United Nations FB1 Fumonisin B1 FDA Food and Drug Administration FUM Fumonisins GAPs Good agricultural practices GPS Global positioning system HBV Hepatitis B virus HCC Hepatocellular Carcinoma HPLC High-performance liquid chromatography IARC International Agency for Research on Cancer LOD Limit of detection v OD Optical density OTA Ochratoxin A PBS Phosphate-buffered saline PE Polyethylene ppb Part per billion TLC Thin-layer chromatography USA United States of America ZEA Zearalenone µg/kg Microgram per kilogram vi LIST OF TABLES Table 2.1 Area under maize, maize production and yield of districts in Son La in 2013 Table 2.2 The property of aflatoxins Table 2.3 Limits of aflatoxin growth and production by A flavus and A parasiticus 14 Table 2.4 The FDA action levels for aflatoxins in human food and livestock feed 20 Table 2.5 Vietnamese limits for aflatoxins in human food 22 Table 4.1 Awareness of moldy contamination in maize in Son La 30 Table 4.2 Prevalence of AFB1 contamination in maize in Son La 32 Table 4.3 Awareness of molds among 107 respondents in each category corresponding questions with answers 39 Table 4.5 Practices associated with consumption of maize, meat and milk 42 Table 4.6 Practices related to the medical examination 45 vii LIST OF FIGURES Figure 2.1 Agro-ecological regions in Vietnam Figure 2.2 The administrative map in Son La province Figure 2.3 Area under maize, maize production and yield in Son La province Figure 2.4 Chemical structure of aflatoxins Figure 3.1 The map shows sampling sites in Son La province 25 Figure 4.1 Difference in the prevalence of AFB1 contamination in maize 33 Figure 4.2 Socio- demographic characteristics of respondents 37 viii THESIS ABSTRACT Master candidate: Nguyen Thi Thanh Xuan Thesis title: Assessment of aflatoxin B1 contamination in maize and awareness of aflatoxins in Son La province Major:Food Technology Code: 60540103 Educational organization: Vietnam National University of Agriculture (VNUA) Research Objectives This research was conducted to evaluate the prevalence of aflatoxin B1 contamination in maize and residents’ awareness of aflatoxins in Son La province Materials and Methods Materials included maize samples and the Sigma-Aldrich® Aflatoxin B1 Low Matrix ELISA kit Maize samples were randomly collected at the end of the rainy season from 25 communes in districts in Son La with recording GPS location All maize samples were analysed for AFB1 contamination in Lab, using the Sigma-Aldrich® Aflatoxin B1 Low Matrix ELISA kit Besides, questionnaire survey was simultaneously carried out with maize collection using face-to-face interviews Eventually, all data were imported into Microsoft Excel 2010 and analyzed using STATA (version 14.0, StataCorp, College Station, TX, USA) ArcGIS version 10.4.1 ArcMap (ESRI, Redlands, CA, USA) was used to create the map Main findings and conclusions The prevalence of AFB1 contamination in maize in Son La was lower when harvested in the field; however, this prevalence was higher in the rainy season Noticeably, most people had adequate awareness of molds and their impacts on human and animal health, but they lack insight of aflatoxins and the impacts of aflatoxins, particularly ethnic minority groups In conclusion, the findings can conclude that maize in Son La is exposed to AFB1 to varying degree, and there may be a risk that maize production could contain AFB1 Therefore, we believe that this study produces evidence on potential risk to humans and animals in Son La and will inform science-based messages for reducing risk Addtionally, the findings underline the need to enhance public's awareness about aflatoxins and health risks associated with aflatoxins on human beings and animals ix Table 4.6 Practices related to the medical examination Characteristic Definition Porportion (%) Mean ($/year) Do you have a medical examination? Yes 55.1 No 44.9 How many times per year you have a medical examination? Rarely (2-3x/year) 88.7 77.8 Sometimes (1x/month) 10.4 98.8 Usually (1x/week) 0.9 12.5 Everyday 0.00 Has any member of your family been diagnosed with hepatitis? Yes 4.7 No 95.3 Has any member of your family been diagnosed with liver cancer? Yes 5.6 No 94.4 Consequently, aflatoxins contamination, particularly AFB1 not only pose serious health risks but also cause significant economic lost to farmers whether their maize might be refused or agreed for consumption Otherwise, AFB1 contamination in maize and awareness of aflatoxin are serious challenges to people in Son La as well as general public 45 PART CONCLUSIONS AND RECOMMENDATIONS 5.1 CONCLUSIONS The findings in this research can conclude that maize in Son La is exposed to AFB1 to varying degrees, and there may be a risk that maize production could contain AFB1 In addition, we found that the level of AFB1 is relatively high during the rainy season Thus, it is important to undertake control strategies and to distinguish the maize samples whether it is suitable for human consumptions and animal feed or not We believe that this study produces evidence on potential risk to humans and animals in Son La and will inform science-based messages for reducing risk People in Son La had adequately awareness of molds and their impacts on human and animal health, but they have lack of information and impacts of aflatoxins, particularly ethnic minority groups Therefore, the findings highlight the need to raise public's awareness about aflatoxins as well as conducting further investigation of health risks on humams and animals 5.2 RECOMMENDATIONS It is necessary to evaluate the association between AFB1 contamination and environmental risk factors (such as temperatures, relative humidity, cropping, agronomic practices or other potential risk factors) Additionally, the intervention programs may include timing of planting, avoiding drought and rodent/insect control for field management, washing, rapid and proper drying and cleaning for post-harvest (Hell, 1997) It is recommended that ethnic minority groups should be targeted for raising the public awareness of aflatoxin risks as well as introduction of control and prevention strategies Besides, a further dietary research 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Mar Apr May Percipitation (mm) 76.3 1.6 61.3 Jun Jul Agu Sept 75.1 42.6 359.8 429.5 258.9 266.2 Oct Nov 86 (Source: GSO, 2017) 58 Dec 44.9 101.2 APPENDIX 4: Prevalence and levels of AFB1 in maize in Son La in March, 2016 Districts (No sample) AFB1 level > ug/kg Prevalence (%) 95% CI AFB1 level > 20 ug/kg Prevalence (%) Mean 95% CI * Media n* Range (