Tài liệu Assessment of aflatoxin b1 contamination in maize and awareness of aflatoxins in son la province

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 : 1. Dr. Hu Suk Lee 2. 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 1. Introduction ........................................................................................................ 1 1.1. Introduction ........................................................................................................ 1 1.2. Research objectives............................................................................................ 2 Part 2. Literature review ............................................................................................... 3 2.1. Situation in the maize production in vietnam – case of Son La ........................ 3 2.1.1. Situation in the maize production in Vietnam .................................................... 3 2.1.2. Study location - Son La province ....................................................................... 4 2.2. Aflatoxins and their impacts on human and animal health ................................ 8 2.2.1. Aflatoxins ........................................................................................................... 8 2.2.2. The impact of aflatoxins on human and animal health ...................................... 9 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 3. 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 4. 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 5. 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 ......................................................................................................... 7 Table 2.2. The property of aflatoxins............................................................................ 8 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 .............................................................. 3 Figure 2.2. The administrative map in Son La province .................................................. 4 Figure 2.3. Area under maize, maize production and yield in Son La province .............. 6 Figure 2.4. Chemical structure of aflatoxins .................................................................... 9 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 5 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 PART 1. INTRODUCTION 1.1. INTRODUCTION Vietnam has an extremely advantageous geographical location in Southeast Asia and Asia. It is located in both tropical and sub-tropical areas. Thus, its climate features a tropical monsoon climate with high temperature and humidity which is a favorable condition for fungal growth, especially mycotoxins produced from Aspergillus spp. (CAST, 2003). Among mycotoxins, aflatoxins, particularly aflatoxin B1 (AFB1) produced primarily by two species of ubiquitous fungus Aspergillus (A.) including A. flavus and A. parasiticus have attracted general public attention because it is the most toxic and potent carcinogen even in small quantities (FAO, 1979 and De Campos, 1987). According to The International Agency for Research on Cancer (IARC), AFB1 is classified as group I carcinogens (Humans et al., 2002). Epidemiologically, AFB1 can lead to be genotoxicity and carcinogenic toxicity for both human and animal, especially the development of liver cancer (hepatocellular carcinoma) and higher risk to hepatic B virus infected individuals (WHO, 1998; Hansen et al., 2011 and Shephard, 2008). In fact, AFB1 is very susceptible in nuts and cereal, especially maize and groundnut during the harvest, or gradually susceptible while storing and processing (Reddy et al., 2009). Therefore, it is extremely important to detect aflatoxins in food and animal feed for assurance of human and animal health as well as food hygiene and safety. In Vietnam, maize is the second only to rice as staple food for the consumption of humans and animals, especially in the rural and mountainous areas. It is regarded as the major energy feed source for Vietnam's livestock industry (Ha et al., 2004). Mountainous North-west provinces, such as Son La, are still listed as belong to the poorest in Vietnam with limited development opportunities over the past years. Therefore, maize is also considered as one of the most important crops. It grows in different ecological regions to alleviate poverty in the provinces with difficult economic conditions. Nevertheless, maize was reportedly extremely good substrate for fungal development producing mycotoxins, especially aflatoxins (Wang and Liu, 2006 and Zinedine et al., 2007). Meanwhile, Son La province is the largest maize producing area in Vietnam but few comprehensive AFB1 investigations had been carried out to 1 establish the prevalence of AFB1 contamination in maize in Son La. Previously, some studies on the occurrence of AFB1 in maize in Vietnam have provided some insight; however, these were based on small sample sizes or limited-scale geographical areas or relied on the data from one point in the maize value chain. For instance, AFB1 was surveyed in 32 maize samples in a Wang et al.’s study (1995), Trung et al. (2008) only conducted in 25 maize samples whereas a bigger sample size (97 samples) were researched by Thieu et al. (2008) and Phuong et al. (2015) (Wang et al., 1995; Trung et al., 2008; Thieu et al., 2008 and Phuong et al., 2015). This would be difficult to generalize the population. Moreover, few comprehensive AFB1 contamination research to establish the prevalence of AFB1 contamination in maize and unique study about perception and knowledge of aflatoxins had been conducted in Son La (Lee et al., 2017). Therefore, an investigation extended greater sampling was conducted to assess AFB1 contamination in maize and awareness of aflatoxins in Son La province. 1.2. RESEARCH OBJECTIVES General objectives This research conducted is to evaluate the prevalence of aflatoxin B1 contamination in maize and residents’ awareness of aflatoxins in Son La province. Specific objectives - To evaluate the prevalence of aflatoxin B1 in maize in Son La province in an attempt to give recommendations to mitigate moldy contamination in maize. - To evaluate the awareness of aflatoxins among people so as to improve their understanding of health risks for both human beings and animals relating to aflatoxins contamination by consuming maize in Son La. 2 PART 2. 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 As in most of the Asian countries like China, Thailand, Indonesia, Vietnam, maize production have been dramatically increasing during the last decade along with the rapidly increasing demand for animal feed. The Vietnamese maize production boosted from 1,177.2 thousand tons in 1995 to 2,005.9 thousand tons in 2000 and drastically increased 5,281.0 thousand tons in 2015 (GSO, 2017). This rapid increase leads to both expansion in area and yield. Maize production system was extended in all the eight Vietnamese agroecological zones included North West, North East, Red River Delta, North Central Coast, South Central Coast, Central Highlands, South East and Mekong River Delta (Figure 2.1). In 2000, maize was grown on 730.2 thousand hectares and obtained 1,179.30 thousand hectares in 2015 (GSO, 2017). Maize developed in all agro-ecological zones, especially in Northern mountainous provinces. The recent development of production particularly comes from intensive areas of maize production such as Son La, Dong Nai, Nghe An, Thanh Hoa and so on, where maize is grown on sloping and wet land as well as invested in new seeds dissemination (especially hybrid seeds like LVN10, LV885, DK88 and so on). Figure 2.1. Agro-ecological regions in Vietnam Source: Quyet (2013) 3 2.1.2. Study location - Son La province Son La province is one of the largest maize producing areas in Vietnam (GSO, 2017. It belongs to Northwestern agro-ecological zone. It is the fifth largest province in Vietnam with the area of 14,174.4 km2 (GSO, 2017). It is located in the northwestern region; it is bordered by Laos to the south, with Yen Bai, Lao Cai, Lai Chau provinces to the north, Dien Bien province to the west and Phu Tho and Hoa Binh provinces to the east (Figure 2.2). The provincial capital is Son La city, which is located approximately 300 km from Hanoi. There are 11 rural districts: Quynh Nhai, Muong La, Thuan Chau, Phu Yen, Bac Yen, Mai Son, Song Ma, Yen Chau, Moc Chau, Sop Cop and Van Ho. Nearly 80% is mountainous and its average elevation is 600 -700m above sea level. Figure 2.2. The administrative map in Son La province Source: GSO (2017) 4 The climate in Son La province is influenced by the tropical monsoon climate, it is therefore charactered by both hot summer and cold winter with a small amount of rain. Dry winter lasts from October to next March while rainy summer lasts from April to September and shows its humidity and heat. Most of the precipitation in years concentrates in June, July, August and September (Appendix 2). The average annual temperature is 23°C (highest 35°C, lowest 11.5°C). The average rainfall in recent years ranges from 120 to 150 mm, and the average humidity is 79% (GSO, 2017). Son La province had a population of nearly 1,182.4 thousand people in 2005 with the density of 83 people/km2 (GSO, 2017). Similar to other Northwest provinces, Son La’s population comprises many ethnic minorities: 54% are Thai, 13% are H'Mong, whereas Kinh or Viet (the major ethnic group nationwide) accounts for only about 18% (Zohova, 2011). Other ethnics are Ma, Dao, Muong, Khmer, Tay and Tai. The province has a total land area of about 1.4 million hectares whereas 64% is agricultural land. Son La has a unique topography as it is partitioned deeply by a high mountain range. Mountains and plateaus cover 75% of the province (Karimov et al., 2016). Maize area and production in Son La have increased significantly and now occupy a large part of the total maize production area of the entire Vietnam, owing to its advantageous geography and climate. Maize production in Son La increased mostly because of maize field expansion and with a slight boost in maize yield. The province production satatistics based on the area cropped and the corresponding quantity produce from 2004 to 2014 was shown in Figure 2.3 and Appendix 2.1. 5 Figure 2.3. Area under maize, maize production and yield in Son La province (2004 - 2014) Source: GSO (2017) Maize is planted in Son La in two main seasons. Maize is grown in swidden fields in the summer season (it is planted in April or May and harvested in July and August or maybe extended until September) while in the winter season maize is cultivated in alluvial lands near rivers or stream banks. Furthermore, it has also been grown in fields during the spring (Tran, 2005). Basically, there are two kinds of maize that are cultivated. Local maize has been grown for years and hybrid maize has been newly introduced as a part of the province’s overall agricultural development strategy. Hybrid maize yields have higher productivity than local ones but it is more vulnerable to the climate. Nevertheless, it is more suitable to produce livestock feed. High domestic demand for maize leads to a tremendous increase in maize imports. Therefore, it should be noted that there is a high demand for maize in Son La because, according to the stakeholders at the value chain assessment workshop, it is considered to be of better quality in contrast to imported maize from China. It is one of the most important cash crops for ethnic minorities and a big number of poor households in the uplands. 6 The maize grown area is not evenly distributed across Son La province, partly due to inadequate agricultural land. The output is highest in Moc Chau, Mai Son and Song Ma district, which also have the highest share of planted area (Table 2.1). A majority of maize producers are small-scale farmers who depend on maize sales for their livelihoods. In 2013, maize yields in the districts of Son La ranged between 3 and 5 tonnes/ ha on average (Table 2.1). Farmers use fertilizers to keep yields above average. Table 2.1. Area under maize, maize production and yield of districts in Son La in 2013 Area under maize (thousand ha) Maize production (thousand tons) Maize yields (tons/ha) Son La city 3.43 18.23 5.31 Quynh Nhai 4.35 13.35 3.07 Thuan Chau 8.73 33.1 3.79 Muong La 13.5 47.26 3.5 Bac Yen 11.89 42.83 3.6 Phu Yen 21.03 64.65 3.07 Moc Chau 22.75 97.21 4.27 Yen Son 17.87 77.81 4.35 Mai Son 21.16 99.15 4.69 Song Ma 23.88 101.12 4.24 Sop Cop 2.11 6.96 3.3 Van Ho 12.08 53 4.39 Districts Source: Statistic Yearbook of Son La (2013) Note: Van Ho district was recently separated from Moc Chau district. Maize products particularly hybrid maize produced in Son La are mainly supplied to enterprises in the livestock sector (for example CP Thailand joint venture company). Moreover, Tran (2005) revealed that Son La maize was sold in many markets such as Ha Tay, Thanh Hoa, Nam Dinh, Nghe An and so on. That also means that maize in Son La became a highly market-oriented product. 7 2.2. AFLATOXINS AND THEIR IMPACTS ON HUMAN AND ANIMAL HEALTH 2.2.1. Aflatoxins Aflatoxins are toxic secondary metabolites produced by filamentous, which can occur in host crops contaminated by some species of Aspergillus (Calvo et al., 2002). Among Aspegillus spp., two strains such as A. flavus and A. parasiticus can produce aflatoxins B1, B2, G1, G2, and M1 (Busby and Wogan, 1984). Worldwide, around 25% of agricultural products were contaminated with aflatoxins (Wild and Gong, 2010 and Yard et al., 2013), while a number of agricultural products are contaminated such as groundnut, maize, and cereal grains with cereals, and groundnuts being the most susceptible (Wilson and Payne, 1994).  Properties of aflatoxins Hell (1997) stated that four major groups of aflatoxins are identified: B1, B2, G1 and G2. These abbreviations are indicative of the colours these fluorescence under the ultraviolet light (385 nm); thus B is for blue and G is for yellow-green. The M is a hydroxylated metabolic product of B (Bankole and Adebanjo, 2003). Aflatoxins are crystalline substances. They not only dissolve freely in some moderately polar solvents such as methanol, chloroform, dimethyl sulfoxide but they also dissolve in water to the extent of 10-20 mg L-1. Several important physical and chemical properties of aflatoxins are shown in Table 2.2 (Cole and Cox, 1981). Table 2.2. The property of aflatoxins Property Chemical Formular Molecular weight Melting point ( oC ) Sorbent, + Pentane Flourescence Aflatoxins B1 B2 G1 G2 M1 C17H12O8 312 268-269 (D) C17H14O8 314 287-289 (D) C17H12O7 328 244-249 (D) C17H14O7 330 C17H12O7 328 230 Ethyl acetate 425 nm 299 (D) Chloroform Chloroform Chloroform 425 nm 425 nm 450 nm Methanol 425 nm Source: Cole and Cox (1981) D= Decomposition 8  Chemical structures of aflatoxins. The chemical structures of some aflatoxins are shown in figure 2.4 (Cole and Cox, 1981). Figure 2.4. Chemical structure of aflatoxins Aflatoxins are highly carcinogenic and can be acutely toxic or fatal if ingested in sufficient quantities for both livestock and humans (Shephard, 2008). 2.2.2. The impact of aflatoxins on human and animal health  On human health Aflatoxins are genetoxic, carcinogenic and immunosuppressive substances, and cause both acute and chronic toxicity. Associated health problems are difficult to diagnose, mainly due to cryptic, long-term and chronic exposures. Nevertheless, in 2004 and 2005, the Kenyan government showed and recognized that hundreds of human death cases were ascribed to the consumption of aflatoxin contaminated maize products (Lewis et al., 2005). Among worldwide countries, populations of developing countries are the most susceptible to aflatoxicosis sickness. This is because security blankets of developed countries in crops at pre-harvest and post-harvest level are quite strict. The same occurs with milk derivates, because developing countries have 9 not accepted and assumed amenities as quick as developed countries. Shephard (2003) and Williams et al. (2004) reported in developing countries worldwide that there were more than 5 billion people at risk of chronic exposure to aflatoxins through contaminated food. Because of being an alarming number, aflatoxins have been recently considered as an important public health issue. Adult humans usually have a high tolerance of aflatoxins, and, in the reported acute poisonings, there are usually children who die (Cullen and Newberne, 1994). The adverse impacts of aflatoxins in human beings and animals have been categorized in two common forms:  Acute aflatoxicosis It is produced when moderate to high levels of aflatoxins are consumed. Specific, acute episodes of disease include hemorrhage, acute liver damage which manifests as severe hepatotoxicity with a case fatality rate of approximately 25%, edema, absorption and/or metabolism of nutrients and alteration in digestion. The early symptoms of hepatotoxicity from aflatoxicosis can include anorexia, malaise, and low-grade fever. Acute high-level exposure can progress to potentially lethal hepatitis with vomiting, abdominal pain, jaundice, fulminant hepatic failure and death (Walderhaug, 1992; Cullen and Newberne, 1994 and Strosnider et al., 2006).  Chronic aflatoxicosis It results from ingestion of low to moderate levels of aflatoxins. The effects are usually subclinical and difficult to recognize. Some of the common symptoms are impaired food conversion and slower rates of growth with or without the production of an overt aflatoxin syndrome (Walderhaug, 1992). Aflatoxicosis is not only caused by inhalation, but also, as mentioned before, is caused by aflatoxin ingestion. In places like Brazil and Abu Dhabi, there have been found lots of cases in which infants were exposed to aflatoxin M1 from mother’s breast milk. Aflatoxins have also been found in infant formula (Aksit et al., 1997; Saad et al., 1995 and Navas et al., 2005). There are lots of earlier studies reporting the presence of aflatoxins and derivatives in human urine, blood, and human cord blood that apparently can enter the developing fetus in humans and animals (Denning et al., 1990). The 80’s and 90’s were 10