A Proposal on: Total Diet Study (TDS) on Total Aflatoxins (B1, B2, G1 and G2) Consumed through Selected
Foods from Selected Clusters of Nepal
1. Introduction Aflatoxins are naturally occurring cytotoxin, which are toxic compounds produced as secondary metabolites by the fungi Aspergillus flavus and Aspergillus parasiticus growing on a variety of food and feed products under favorable temperature and humidity and are known to be carcinogenic, mutagenic, teratogenic and immunosuprresive. Contamination of aflatoxins can take at any point along the food chain from the field, harvest, handling, shipment and storage. They have been found to contaminate a wide variety of important agricultural and dairy products world-wide, e.g. corn, wheat, rice, spices, dried fruits, and nuts, oilseeds, spices, herbs and coffee etc (Khayoon, et al., 2010). Aflatoxins are mainly colonized and contaminate crops before harvesting or during improper storage. The ambient temperature in the range of 28-350C is favorable for Aflatoxin growth. Aflatoxins are normally refers to the group of difuranocoumarins and classified into two broad groups according to their chemical structures; the difurocoumarocyclopentenone series (AFB1, AFB2, AFB2A, AFM1, AFM2, AFM2A and aflatoxicol) and the difurocoumarolactone series (AFG1, AFG2, AFG2A, AFGM1, AFGM2, AFGM2A and AFB3). The aflatoxins fluoresce strongly in ultraviolate light (i.e. 365nm); B1 and B2 produce a blue fluorescence where as G1 and G2 produce green fluorescence (Anonymous, 2011). However, there are four major naturally occurring Aflatoxins i.e. AFB1, AFB2, AFG1 and AFG2, where AFB1 and AFB2 are major aflatoxins found in cereals and nuts products including maize, peanuts etc. It is most toxic and carcinogenic to human beings and animals. It may cause liver cancer.
Figure1: Terminal portion of a conidiophore of A. flavus showing the basal portion of the vesicle and distribution of radiation phialides (arrows). X 1000.
Figure 2: Important chemical structures of different Aflatoxins in foods
Figure 3: Multiple effects of Aflatoxins in animals 1.1 Some preliminary work done in DFTQC, CFL, Kathmandu Some preliminary work on Aflatoxins has been done in DFTQC, CFL, Kathmandu, Nepal. Based on the work the summary of results has been presented in Table 1. Table 1: Results of Aflatoxin B1 in different foods of Nepal S.N. Commodity Total no. Concentration % of ppb contamination 1 Lito 11 3.88-19.08 100% 2 Maize/Corn 8 4-39.95 100% 3 Soyabean 3 3.78- 9.91 100% 4 Wheat 4 3.75-19.67 100% 5 Anta/maida 6 3.8-19.23 100% 6 Suji 4 3.94-19.93 100% 7 Ground nuts 11 3.78-39.69 100% 8 Kaju 6 0-9.89 66.67% 9 Miscellaneous 8 3.73-39.67 10 Total 61 96.72% Source: Rai and Chaudhari, (2011) Remarks AFB1 contamination determined by ELISA Kits method
The above table showed that the contamination Aflatoxin in Nepalese foods is almost cent percent. Therefore, there is a immediate requirement of such extensive research in major food commodities to find the Total Dietary Intake of such contaminants. 2. Objectives of Study The major objective of this study is to explore the dietary consumption of total Mycotoxins (AFB1, AFB2, AFG1 and AFG2) among Nepalese people through cereals and nuts food sources. 3
2.1 Specific Objectives i. To collect the food samples as per sampling plan ii. Conduct a survey in fields regarding to the dietary consumption of Aflatoxins iii. Screen test by ELISA Kits method iv. Confirmatory Test by Reference Lab of India or Others v. Analysis of Aflatoxin B1, B2, G1 and G2 in CFL vi. Report preparation and submission vii. Report dissemination through a Seminar 3. Methodology 3.1 Sample Collection Maize, Peanuts and their products; Suzi grits, cheese-balls, pop-corn, corn-flakes, Lito etc. will be collected from different parts/clusters of Nepal as mentioned in Table 2. Table 2: Sampling plan S.N. Region District 1 Eastern Mountain: Sankhuwasabha Hill: Terathum, Dhankuta, Udayapur, Ilam Terai: Jhapa, Sunsari, Morang 2 Central Mountain: Rasuwa, Hill: Sindhupalanchowk, Kathmandu, Bhaktapur, Sindhuli, Kavre, Terai: Chitwan, Makwanpur 3 Western Mountain: Mustang, Manag Hill: Kaski, Tanahun, Gorkha, Palpa Terai: Nawalparasi, Rupandehi 4 Midwestern Mountain: Jumla, Kalikot Hill: Pyuthan Terai: Dang, Surkhet 5 Far-western Mountain: Bajura Hill: Dadeldhura, Baitadi, Dharchula Terai: Kailali, Kanchanpur, Total 5 5- Regions 33 320 samples 6 6 districts 9 samples = 54 5 5 districts X 10 samples = 50 8 8 districts X 9 samples = 72 8 8 districts X 9 samples = 72 No. of No of samples Districts 8 8 districts X 9 samples =72
3.2 Survey Questionnaire A descriptive study by questionnaire survey will be conducted in all sampling clusters regarding to the consumption of cereals and nut crops and their products during the field sampling.
4. Budget Estimation of Study The tentative budget for carrying out the proposed study is given in Table 3. Table 3: Budget Estimation Activities Particulars Sample DSA: collection: No of Persons 10x7x1800 Travel Allowance: Eastern region two way plan and local transportation (2x 15,000) Central Region two way plan and local transportation (2x 10,000) Western Region for two way plan fare and local transportation (2x 12,000) Mid western Region for two way plan fare and local transportation (2x 15,000) Far western Region for two way plan fare and local transportation (2x 18,000) Sample cost: Maize: Sample size: 1.5 Kg No of Samples: 200 Price per Kg: 50 Peanut: Sample Size: 1.5 Kg No of Sample: 80 Price per kg: 200 Maize and peanut products etc. Sample Size: 1.5kg No of Sample: 40 Price per kg: 200 No of Sampling Bag- 50 Per piece @ 50x 200= Rs. 10,000.00 PE Bags 4 kg x 300 = 1,200.00 Local transportation currier cost etc. Per district @ 500x 33= 16,500.00 Aflatoxins B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2 2 kits x 60,000 3 samples Testing fee (3x 4000) Currier cost for delivering sample to India HPLC grade methanol= 5 bottles (5x2250) 5 Cost 1,26,000.00 30,000.00 20,000.00 24,000.00 30,000.00 36,000.00 Total 2,66,000.00
15,000.00 51,000.00 24,000.00
Sampling Accessories
12,000.00 27,700.00
27,700.00
Certified Raw Material ( CRM) Purchase ELISA Screening Kit Verification/V alidation Chemicals and
30,103.00 30,103.00 24,352.00 37,431.00 120,000.00 12,000.00 10,000.00 11,250.00
121,989.00
120,000.00 22,000.00 115,375.00
Glasswares etc. HPLC Grade water 15 L(25x 1075) Chloroform 10 bottles (10x2,125) Hexane: 5 bottles (5x2125) Micropipette (2 pcs x 9700.00) Acetonitrile bottles (4 x 5125) Separating funnel (3x1825) Printing, Printing 60 pages x Rs. 50 binding & Designing per page Rs. 250 x Rs 60 per page Photocopying Binding 20 pieces x Rs. 50 expenses Stationary Stationary: 20 Persons x Rs 250
26,875.00 21,250.00 10,625.00 19,400.00 20,500.00 5,475.00 3,000.00 15,000.00 1,00.00 5,000.00
19,000.00
5,000.00
Total 748,064.00 In Word: Seven Lakhs Fourty Eight Thousands and Sixty Four Nepali Currency, which is equivalent to Approx. USD 9,218.00 * 1USD= NRs 81.15 5. Facility Available in DFTQC, Central Food Laboratory a. Thin Layer Chromatography b. ELISA Reader c. HPLC Analysis d. Competent Analysts e. Infrastructure for study 6. Team Involvement in Study 1. Binaya P. Shrestha Lab Chief, CFL 2. Sangita Swar SFRO, Co-ordinator 3. Krishna P. Rai FRO, Technical Co-ordinator 4. Jyoti Joshi FRO 5. Hareram Pradhan FRO 6. Shiv Sagar Chaudhary FRO 7. Sovita Shrestha FRO 8. Bal Kumari Sharma TA 9. Laxmi Paudyal TA 10. Ghananath Adhikari TA 7. Outputs i. Total Dietary Intake of total Aflatoxins B1, B2, G1 and G2 in Nepalese people will be established in selected commodity ii. General people will be aware of such natural toxin as they are consuming since long time. iii. A original research article will be published in International Journal iv. Support for method establishment, validation and scope extension for accreditation. 7. Expected Outcomes i. TDS study on total aflatoxins in cereals and nuts products in clusters zones of Nepal will be carried out. ii. Support to make a national policy on formulation of standard on total aflatoxins in foods.
8. Action plan (January - December, 2012) for study S.N. Months 1 Proposal prepare and Submission 2 Sample collection and survey 3 Procurement of Chemicals and Laboratory analytical works 4 Verification from Out side lab 5 Data analysis/ Result interpretation 6 Report Writing and submission Jan Feb March April May Jun July Aug Sept Oct Nov Dec
9. Limitations of this study i. All types of Mycotoxins probably found in different foods will not be covered ii. All the food commodities will not be covered by this study iii. Random sampling and cluster sampling will be carried out 10. References 1. 2. 3. Anonymous (2011). Aflatoxin, available at: http://en.wikipedia.org/wiki/Aflatoxin
Khayoon W.S., Bahruddin S., C.B. Yan, N.H. Hashim, A.S.M. Ali, M.I. Salleh and Baharuddin S. (2010). Determination of aflatoxins in imported rice to Iran, Food and Chemical Toxicology, 47:2064-2066. Rai and Chaudhari, (2011). A research report on Mycotoxins (Aflatoxin B1) contamination in food products available in Nepalese markets. DFTQC, Central Food Lab, Kathmandu, Nepal.
