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Occurrence of aflatoxin B1 in food products derivable from ‘egusi’ melon seeds consumed in southwestern Nigeria
Food Control 21 (2010) 974–976
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Occurrence of aflatoxin B1 in food products derivable from ‘egusi’ melon seeds consumed in southwestern Nigeria Samuel A. Bankole a,*, Adedotun A. Adenusi b, O.S. Lawal c, O.O. Adesanya a a
Department of Microbiology, Olabisi Onabanjo University, PMB 2002, Ago-Iwoye, Ogun State, Nigeria Department of Plant Science and Applied Zoology, Olabisi Onabanjo University, PMB 2002, Ago-Iwoye, Ogun State, Nigeria c Department of Chemical Sciences, Olabisi Onabanjo University, PMB 2002, Ago-Iwoye, Ogun State, Nigeria b
a r t i c l e
i n f o
Article history: Received 22 July 2009 Received in revised form 20 November 2009 Accepted 28 November 2009
Keywords: Aflatoxin B1 Egusi melon Egusi soup Ogiri Robo
a b s t r a c t Aflatoxin, the toxic secondary metabolite of Aspergillus flavus and Aspergillus parasiticus has been considered as one of the most serious food safety problems in sub Saharan Africa. Egusi melon seeds is susceptible to these fungal infection during postharvest. Therefore, the content of aflatoxin B1 in three food products derivable from ‘egusi’ melon seeds ogiri’ (fermented melon seed condiment), ‘robo’ (melon ball snacks) and egusi soup destined for human consumption in Nigeria in 2005 and 2006 were determined. Aflatoxin B1 was analysed by thin layer chromatography (TLC) with fluorescent detection. The percentage of samples positive for aflatoxin B1 were for 25% robo, 31.8% for ogiri, and 19.5% for ‘egusi’ soup. Aflatoxin B1 ranged from 2.3 to 15.4 ppb in all samples. The overall mean levels of aflatoxin B1 were for 8.9 ppb for ogiri, 9.7 ppb for ‘robo’ and 7.2 ppb for ‘egusi soup’. All positive melon seeds derived food products analysed in this study contained aflatoxin B1 at concentrations much lower than the 20 ppb permissible limit recommended in Nigerian food and suggests that melon seed derived foods present less risk by human exposure to aflatoxin through their consumption. However, it is important to consider these levels in terms of their contribution to overall daily exposure to aflatoxin levels in food. This is the first report of aflatoxin determination in melon seed food products. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction ‘Egusi’ melon (Colocynthis citrullus L.) is a widely cultivated and consumed oil seed crop in West Africa. The seeds popularly called ‘egusi’ contain about 53% oil, 28% protein and some other important mineral nutrients (Abaelu, Makinde, & Akinrimisi, 1979; Oyolu, 1977). They are consumed in ‘egusi soup’, melon ball snacks and ogiri, (a fermented condiment) (Odunfa, 1981; Oyenuga, 1968). In certain parts of southeastern Nigeria, ground mixtures of melon seeds and the fungus Pleurotus tuber-regium are moulded into stabilized balls to substitute. meat in the diet of the inhabitants (Nwokolo & Sim, 1987). In west Africa, the development of fungi particularly Aspergillus spp. in storage is a serious problem exasperated by the humid tropical climate that promote fungal growth (Bankole, Schollenberger, & Drochner, 2006). These fungi can grow in certain food and feed and under conducive environmental conditions produce toxic secondary metabolites called mycotoxins of which aflatoxins are the most serious and well known examples. The four principal aflatoxins are aflatoxin B1, B2, G1 and G2, of which aflatoxin B1 is the
* Corresponding author. E-mail address: [email protected] (S.A. Bankole). 0956-7135/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2009.11.014
most common and most toxic (IARC, 1993). Heavy dietary exposure to food-borne mycotoxins has been linked to the high incidence of liver cancer, growth retardation, decreased immunity and vaccination failure in Africa (reviewed in Bankole, Schollenberger, et al., 2006). There are also several reports of human intoxication due to consumption of aflatoxin contaminated agricultural products (CDC, 2004; Krichnamachari, Nagarajan, Bhat, & Tilak, 1975; Ngindu et al., 1982). Aflatoxin is considered to be one of the most potent hepatocarcinogens known to several mammalian species including man (CAST, 2003). The high levels of aflatoxin markers in the blood serum of Africans has often been adduced to the frequent consumption of maize and groundnut but other commonly consumed foods such as melon seeds have now been shown to be naturally contaminated with aflatoxins (Bankole, Ogunsanwo, & Mabekoje, 2004; Bankole, Ogunsanwo, Osho, & Adewuyi, 2006; DiProssimo & Malek, 1996). Aspergillus flavus has been found to have the highest species count among the moulds associated with stored melon seeds (Aboaba & Amasike, 1991; Bankole, 1993; Bankole, Ikotun, & Ekpo, 1999; Bankole, Ogunsanwo, et al., 2006). Nigeria is listed as regulating aflatoxin B1 at 20 ppb in all classes of food for human consumption (FAO, 2004). Thus, there is the urgent need for Africans to find lasting solution to mycotoxin problem for them to continue to enjoy patronage by the competitive EU markets.
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Food processing operations such as cooking, soaking and fermentation impact on aflatoxin contamination levels to varying degrees in foods (Westby, Riley, & Bainbridge, 1997). Case studies of aflatoxin levels in some processed foods such as ‘kenkey’ (fermented maize product) have been reported (Kpodo, Sorensen, & Jakobsen, 1996), while Fandohan et al. (2005) presented the impact of processing of maize into food products on aflatoxin levels in Benin. Processing of melon seeds into foods involve steps such as milling, fermentation, roasting and cooking, but the effect of these operations on African foods are not yet clear (Westby et al., 1997). There is no information on aflatoxin contamination in processed melon seeds consumed in west Africa. The objective of this study was to survey aflatoxin B1 levels in three major food foods obtained from melon seed products in Nigerian markets that are for sale to Nigerians.
2. Materials and methods 2.1. Sample collection Forty samples each of melon seed products: ‘ogiri’ (fermented ‘egusi’ seed condiment) and ‘robo’ (melon ball snacks) were purchased from street hawkers and local markets in southwestern Nigeria in 2005. Forty samples of ‘egusi’ soup which is the most popular form in which ‘egusi’ melon is consumed was also collected in 2005 from households and food restaurants within 20 km radius to Olabisi Onabanjo University, Ago-Iwoye because the soup will develop sour taste and off flavour if transported for long distances. In 2006, additional samples of 36, 45 and 32 of ‘ogiri’, ‘robo’ and egusi soup, respectively were also collected from appropriate destinations. All the samples were stored in the refrigerator at 4 °C pending analysis. The samples were dried in a vacuum oven at 40 °C for 24 h and then milled.
2.2. Assay for aflatoxin B1 The standard of aflatoxin B1 were from Sigma (St. Louis, USA). The aflatoxin analysis was done using a modified version of that described by Schuller and Van Egmond (1982). Fifty grams of ground dried melon seed product was mixed with 200 ml of methanol/water (80:20 v/v) plus 5 g of NaCl in a high speed blender for 2 min. The mixture was filtered through a Whatman No. 1 filter paper. To 50 ml of the filtrate in a separatory funnel, 50 ml of chloroform was added, shaken gently for 1 min, and left to stand for 15 min. The chloroform layer was drained into a beaker containing 5 g of cupric carbonate, and passed through filter paper containing anhydrous sodium sulphate. Thereafter the chloroform extract was submitted to solvent evaporation on a water bath until nearly dry, and transferred into plastic vials and dried by gentle stream of nitrogen gas. For aflatoxin assay, the dried aflatoxin extracts were resuspended in 200 ll of benzene and acetonitrile solution (98:2). Five, 10 and 15 ll of the sample were spotted on silica-gel (60G) plates (0.25 mm). A series of standard aflatoxin B1 solution were also spotted on the same plate for comparison. The plates were developed with chloroform–acetone (88 + 12) in a thin layer chromatography (TLC) tank. Aflatoxin B1 concentration was quantified by visually comparing the intenties of fluorescent of extract spots with that of standard solution spot under long wave UV light (365 nm). The confirmatory test for aflatoxin B1 was carried out (using the sample extracts and standard aflatoxin B1 solution) by adding trifluoroacetic acid to fluorescent spots on dried TLC plates followed by tetra oxo sulphate (VI) acid spray as earlier described (Bankole, Ogunsanwo, et al., 2006).
Recovery experiments were carried out by spiking the various melon seed products prepared from high quality ‘egusi’ having non detectable AFB1 levels with aflatoxin B1 at (5 ppb); then, extracted and analysed by the TLC procedure described above to validate the method for the melon seed products. The recovery experiments were also carried out at different times on the same spiked samples to determine the reliability of the analytical method.
3. Results and discussion The mean recoveries of AFB1 from artificially spiked egusi melon seed food products were 86%, 78% and 82% in ogiri, robo and egusi soup, respectively. The relative standard deviation for within day repeatability was 4.6%, 7.3% and 3.8% for ogiri, robo and egusi soup, respectively. Tables 1 and 2 present the summary of number of samples analysed, the number found to contain detectable levels of AFB1, incidence rate, and the concentrations of AFB1 for 2005 and 2006. As can be seen, the incidence of AFB1 was 25%, 32.5% and 17.5% in ogiri, robo and egusi soup respectively in 2005. From the 2006 analysis, it can also be seen that the incidence of contamination was 25% for ogiri, 31.1% for ‘robo’ and 21.9% for ‘egusi soup’, The levels of aflatoxin B1 varied from 2 to 15 ppb in 2005 and 3 to 14 ppb in 2006 in all melon seed products sampled. The overall mean levels of aflatoxin B1 were for 8.9 ppb for ogiri, 9.7 ppb for ‘robo’ and 7.2 ppb for ‘egusi soup. These amounts are under permissible level of 20 ppb accepted in Nigerian foods on paper. In an earlier study, aflatoxin B1 was detected in 37% of 137 Nigerian melon seed samples with mean levels of aflatoxin B1 contamination of 14.8 ppb in the forest and 11.3 ppb in the savanna (Bankole et al., 2004). The levels found in the processed melon seeds are much below those of the raw foods. Also, while 3.5% of analysed melon seed samples had aflatoxin level above the Nigerian 20 ppb limit (Bankole et al., 2004), none of the samples in the present investigation had toxin level above 20 ppb. In this regard, the effect of food processing techniques on aflatoxin content must be taken into account, which could have accounted for the low level of occurrence of aflatoxin B1 in this study compared to
Table 1 Distribution and levels of aflatoxins in melon seed product samples in southwestern Nigeria in 2005. ‘Egusi’ product
No. of samples
Positive samples
% of positive samples
Aflatoxin B1 Mean SD
Range
‘Ogiri’ ‘Robo’ Soup
40 40 40
10 13 7
25 32.5 17.5
8.9 ± 4.5 8.3 ± 3.3 7.2 ± 2.7
2–13 3–15 3–10
SD, standard deviation.
Table 2 Distribution and levels of aflatoxins in melon seed product samples in southwestern Nigeria in 2006. ‘Egusi’ product
No. of samples
‘Ogiri’
36
9
‘Robo’
45
Soup
32
SD, standard deviation.
Positive samples
% of positive samples
Aflatoxin B1 Mean SD
Range
25
9.0 ± 3.25
14
31.1
7.6 ± 2.92
7
21.9
7.3 ± 2.72
4.1– 14.3 2.5– 11.7 4.3– 10.7
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levels previously established in melon seeds. The percentage loss of aflatoxins in food has also found to be variable depending on the techniques (Fandohan, Hell, & Marasas, 2008) which explains the variations found in aflatoxin B1 levels in various melon seed derived foods. The AFB1 incidence and levels of contamination found in the present study are also lower to those reported in other Nigerian processed foods that have so far been analysed. For instance, Kpodo et al. (1996) reported aflatoxin levels of 0.7 and 313 ppb in kenkey, fermented maize dough from two kenkey production sites in Accra, Ghana while Bankole, Ogunsanwo, and Eseigbe (2005) found AFB1 in 64.2% of 106 samples of dry roasted groundnuts with a mean of 25.5 ppb. Aflatoxin contamination in foods resulting in public health threats and economic loses is a frequently occurring problem in sub Saharan African countries (Bankole, Schollenberger, et al., 2006; Kpodo & Bankole, 2008). Some results have been obtained on aflatoxin levels in various food products such as roasted groundnuts (Bankole et al., 2005), maize based foods (Fandohan et al., 2005). It is pertinent to point out that contamination levels of aflatoxin B1 in this study were much lower that those obtained in previous studies of African processed foods cited above. In conclusion, the results of this study demonstrate that the concentrations of aflatoxin B1 in all analysed melon seed products were under permitted levels. However, this amount should be considered in regard to overall daily exposure to mycotoxins, and the overall goal should be to have no aflatoxins in food. It is therefore important to establish routine monitoring program for aflatoxin contamination in food in Nigeria with a view to establishing permanent monitoring and control program from production until consumption. However, it is important to consider these levels in terms of their contribution to overall daily exposure to aflatoxin levels in food. Acknowledgements The financial support for this study from the International Foundation for Science, Stockholm, Sweden through a grant to Dr. S.A. Bankole is thankfully acknowledged. References Abaelu, A. M., Makinde, M. A., & Akinrimisi, E. O. (1979). Melon (egusi) seed protein 1: Study of amino acid composition of defatted meal. Nutritional Reports International, 20, 605–613. Aboaba, O. O., & Amasike, J. (1991). Storage of melon seeds. Nigerian Journal of Botany, 4, 213–219. Bankole, S. A. (1993). Moisture content, mould invasion and seed germinability of stored melon. Mycopathologia, 122, 123–126.
Bankole, S. A., Ikotun, B., & Ekpo, E. J. A. (1999). Fungal deterioration of melon seeds stored in jute sacks and polyethylene bags in Ago-Iwoye, South Western Nigeria. Mycopathologia, 146, 135–146. Bankole, S. A., Ogunsanwo, B. M., & Eseigbe, D. A. (2005). Aflatoxins in Nigerian dryroasted groundnuts. Food Chemistry, 89, 503–506. Bankole, S. A., Ogunsanwo, B. M., & Mabekoje, O. O. (2004). Natural occurrence of moulds and aflatoxin B1 in melon seeds from markets in Nigeria. Food and Chemical Toxicology, 42, 1309–1314. Bankole, S. A., Ogunsanwo, B. M., Osho, A., & Adewuyi, G. O. (2006). Fungal contamination and aflatoxin B1 of ‘egusi’ melon seeds in Nigeria. Food Control, 17, 814–818. Bankole, S. A., Schollenberger, M., & Drochner, W. (2006). Mycotoxins in food systems in sub Saharan Africa: A review. Mycotoxin Research, 22, 163–169. CAST (2003). Mycotoxins: Risk in plant, animal and human systems. Task Force Report 139. Council for Agricultural Science and Technology (pp. 199), Ames, Iowa. CDC (2004). Centre for disease control and prevention. In Outbreak of aflatoxin poisoning – Eastern and central provinces, Kenya, January–July, 2004. September 3, (Vol. 53(34), pp. 790–793). DiProssimo, V. P., & Malek, G. (1996). Comparison of three methods for determining aflatoxins in melon seeds. Journal of AOAC International, 79, 1330–1335. Fandohan, P., Hell, K., & Marasas, W. F. O. (2008). Food processing to reduce mycotoxins in Africa. In J. E. Leslie, R. Bandyopadhyay, & A. Visconti (Eds.). Mycotoxins detection methods, management, public health and agricultural trade (pp. 309–316). CABI. Fandohan, P., Zoumenou, D., Hounhouigan, D. J., Marasas, W. F. O., Wingfield, M. J., & Hell, K. (2005). Fate of aflatoxins and fumonisins during the processing of maize into food products in Benin. International Journal of Food Microbiology, 98, 249–259. Food and Agriculture Organisation, FAO (2004). Worldwide regulations for mucotoxins in food and feed in 2003. Food and Nutrition paper 81. Rome, Italy: Food and Agriculture Organisation of the United Nations. IARC (1993). Some naturally occurring substances: Food items and constituents, heterocyclic amines and mycotoxins. IARC monographs on evaluation of carcinogenic risk to humans (Vol. 56). Lyon, France: International Agency for Research on Cancer. Kpodo, K., & Bankole, S. A. (2008). Mycotoxin contamination in foods in West and Central Africa. In J. E. Leslie, R. Bandyopadhyay, & A. Visconti (Eds.). Mycotoxins detection methods, management, public health and agricultural trade (pp. 103–116). CABI. Kpodo, K., Sorensen, A. K., & Jakobsen, M. (1996). The occurrence of mycotoxins in fermented maize products. Food Chemistry, 56, 147–153. Krichnamachari, K. A., Nagarajan, V., Bhat, R. V., & Tilak, T. B. (1975). Hepatitis due to aflatoxicosis – An outbreak in western India. Lancet, 305, 1061–1063. Ngindu, A., Kenya, P. R., Ocheng, D. M., Omondi, T. N., Ngare, W., Gatei, D., et al. (1982). Outbreak of acute hepatitis caused by aflatoixn poisoning in Kenya. Lancet, 319, 1346–1348. Nwokolo, E., & Sim, J. S. (1987). Nutritional assessment of defatted seed meal of melon (Colocynthis citrullus L.) and fluted pumpkin (Telfaria occidentales Hook) by chick assay. Journal of the Science of Food and Agriculture, 38, 237–246. Odunfa, S. A. (1981). Microbiology and amino acid composition of ‘ogiri’ – A food condiment from melon seeds. Nahrung, 25, 811–816. Oyenuga, V. A. (1968). Nigeria’s food and feedstuffs: Their chemistry and nutritive value (3rd ed.). Ibadan: Ibadan University Press. Oyolu, O. (1977). A qualitative and quantitative study of seed types in ‘egusi’ (Colocynthis citrullus L.). Tropical Science, 19, 51–61. Schuller, P. L., & Van Egmond, H. P. (1982). Determination of afltoxin B1. In Based upon the methods of European communities (O. J. L.102, 9–17 Dated 5.4.1976 (1982). Amended by Scchuller, P.L., & Van Egmond, H.P. Westby, A., Riley, A., & Bainbridge, Z. (1997). Review of the effect of fermentation on naturally occurring toxins. Food Control, 8, 329–339.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Occurrence of aflatoxin B1 in food products derivable from ‘egusi’ melon seeds consumed in southwestern Nigeria Samuel A. Bankole a,*, Adedotun A. Adenusi b, O.S. Lawal c, O.O. Adesanya a a
Department of Microbiology, Olabisi Onabanjo University, PMB 2002, Ago-Iwoye, Ogun State, Nigeria Department of Plant Science and Applied Zoology, Olabisi Onabanjo University, PMB 2002, Ago-Iwoye, Ogun State, Nigeria c Department of Chemical Sciences, Olabisi Onabanjo University, PMB 2002, Ago-Iwoye, Ogun State, Nigeria b
a r t i c l e
i n f o
Article history: Received 22 July 2009 Received in revised form 20 November 2009 Accepted 28 November 2009
Keywords: Aflatoxin B1 Egusi melon Egusi soup Ogiri Robo
a b s t r a c t Aflatoxin, the toxic secondary metabolite of Aspergillus flavus and Aspergillus parasiticus has been considered as one of the most serious food safety problems in sub Saharan Africa. Egusi melon seeds is susceptible to these fungal infection during postharvest. Therefore, the content of aflatoxin B1 in three food products derivable from ‘egusi’ melon seeds ogiri’ (fermented melon seed condiment), ‘robo’ (melon ball snacks) and egusi soup destined for human consumption in Nigeria in 2005 and 2006 were determined. Aflatoxin B1 was analysed by thin layer chromatography (TLC) with fluorescent detection. The percentage of samples positive for aflatoxin B1 were for 25% robo, 31.8% for ogiri, and 19.5% for ‘egusi’ soup. Aflatoxin B1 ranged from 2.3 to 15.4 ppb in all samples. The overall mean levels of aflatoxin B1 were for 8.9 ppb for ogiri, 9.7 ppb for ‘robo’ and 7.2 ppb for ‘egusi soup’. All positive melon seeds derived food products analysed in this study contained aflatoxin B1 at concentrations much lower than the 20 ppb permissible limit recommended in Nigerian food and suggests that melon seed derived foods present less risk by human exposure to aflatoxin through their consumption. However, it is important to consider these levels in terms of their contribution to overall daily exposure to aflatoxin levels in food. This is the first report of aflatoxin determination in melon seed food products. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction ‘Egusi’ melon (Colocynthis citrullus L.) is a widely cultivated and consumed oil seed crop in West Africa. The seeds popularly called ‘egusi’ contain about 53% oil, 28% protein and some other important mineral nutrients (Abaelu, Makinde, & Akinrimisi, 1979; Oyolu, 1977). They are consumed in ‘egusi soup’, melon ball snacks and ogiri, (a fermented condiment) (Odunfa, 1981; Oyenuga, 1968). In certain parts of southeastern Nigeria, ground mixtures of melon seeds and the fungus Pleurotus tuber-regium are moulded into stabilized balls to substitute. meat in the diet of the inhabitants (Nwokolo & Sim, 1987). In west Africa, the development of fungi particularly Aspergillus spp. in storage is a serious problem exasperated by the humid tropical climate that promote fungal growth (Bankole, Schollenberger, & Drochner, 2006). These fungi can grow in certain food and feed and under conducive environmental conditions produce toxic secondary metabolites called mycotoxins of which aflatoxins are the most serious and well known examples. The four principal aflatoxins are aflatoxin B1, B2, G1 and G2, of which aflatoxin B1 is the
* Corresponding author. E-mail address: [email protected] (S.A. Bankole). 0956-7135/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2009.11.014
most common and most toxic (IARC, 1993). Heavy dietary exposure to food-borne mycotoxins has been linked to the high incidence of liver cancer, growth retardation, decreased immunity and vaccination failure in Africa (reviewed in Bankole, Schollenberger, et al., 2006). There are also several reports of human intoxication due to consumption of aflatoxin contaminated agricultural products (CDC, 2004; Krichnamachari, Nagarajan, Bhat, & Tilak, 1975; Ngindu et al., 1982). Aflatoxin is considered to be one of the most potent hepatocarcinogens known to several mammalian species including man (CAST, 2003). The high levels of aflatoxin markers in the blood serum of Africans has often been adduced to the frequent consumption of maize and groundnut but other commonly consumed foods such as melon seeds have now been shown to be naturally contaminated with aflatoxins (Bankole, Ogunsanwo, & Mabekoje, 2004; Bankole, Ogunsanwo, Osho, & Adewuyi, 2006; DiProssimo & Malek, 1996). Aspergillus flavus has been found to have the highest species count among the moulds associated with stored melon seeds (Aboaba & Amasike, 1991; Bankole, 1993; Bankole, Ikotun, & Ekpo, 1999; Bankole, Ogunsanwo, et al., 2006). Nigeria is listed as regulating aflatoxin B1 at 20 ppb in all classes of food for human consumption (FAO, 2004). Thus, there is the urgent need for Africans to find lasting solution to mycotoxin problem for them to continue to enjoy patronage by the competitive EU markets.
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Food processing operations such as cooking, soaking and fermentation impact on aflatoxin contamination levels to varying degrees in foods (Westby, Riley, & Bainbridge, 1997). Case studies of aflatoxin levels in some processed foods such as ‘kenkey’ (fermented maize product) have been reported (Kpodo, Sorensen, & Jakobsen, 1996), while Fandohan et al. (2005) presented the impact of processing of maize into food products on aflatoxin levels in Benin. Processing of melon seeds into foods involve steps such as milling, fermentation, roasting and cooking, but the effect of these operations on African foods are not yet clear (Westby et al., 1997). There is no information on aflatoxin contamination in processed melon seeds consumed in west Africa. The objective of this study was to survey aflatoxin B1 levels in three major food foods obtained from melon seed products in Nigerian markets that are for sale to Nigerians.
2. Materials and methods 2.1. Sample collection Forty samples each of melon seed products: ‘ogiri’ (fermented ‘egusi’ seed condiment) and ‘robo’ (melon ball snacks) were purchased from street hawkers and local markets in southwestern Nigeria in 2005. Forty samples of ‘egusi’ soup which is the most popular form in which ‘egusi’ melon is consumed was also collected in 2005 from households and food restaurants within 20 km radius to Olabisi Onabanjo University, Ago-Iwoye because the soup will develop sour taste and off flavour if transported for long distances. In 2006, additional samples of 36, 45 and 32 of ‘ogiri’, ‘robo’ and egusi soup, respectively were also collected from appropriate destinations. All the samples were stored in the refrigerator at 4 °C pending analysis. The samples were dried in a vacuum oven at 40 °C for 24 h and then milled.
2.2. Assay for aflatoxin B1 The standard of aflatoxin B1 were from Sigma (St. Louis, USA). The aflatoxin analysis was done using a modified version of that described by Schuller and Van Egmond (1982). Fifty grams of ground dried melon seed product was mixed with 200 ml of methanol/water (80:20 v/v) plus 5 g of NaCl in a high speed blender for 2 min. The mixture was filtered through a Whatman No. 1 filter paper. To 50 ml of the filtrate in a separatory funnel, 50 ml of chloroform was added, shaken gently for 1 min, and left to stand for 15 min. The chloroform layer was drained into a beaker containing 5 g of cupric carbonate, and passed through filter paper containing anhydrous sodium sulphate. Thereafter the chloroform extract was submitted to solvent evaporation on a water bath until nearly dry, and transferred into plastic vials and dried by gentle stream of nitrogen gas. For aflatoxin assay, the dried aflatoxin extracts were resuspended in 200 ll of benzene and acetonitrile solution (98:2). Five, 10 and 15 ll of the sample were spotted on silica-gel (60G) plates (0.25 mm). A series of standard aflatoxin B1 solution were also spotted on the same plate for comparison. The plates were developed with chloroform–acetone (88 + 12) in a thin layer chromatography (TLC) tank. Aflatoxin B1 concentration was quantified by visually comparing the intenties of fluorescent of extract spots with that of standard solution spot under long wave UV light (365 nm). The confirmatory test for aflatoxin B1 was carried out (using the sample extracts and standard aflatoxin B1 solution) by adding trifluoroacetic acid to fluorescent spots on dried TLC plates followed by tetra oxo sulphate (VI) acid spray as earlier described (Bankole, Ogunsanwo, et al., 2006).
Recovery experiments were carried out by spiking the various melon seed products prepared from high quality ‘egusi’ having non detectable AFB1 levels with aflatoxin B1 at (5 ppb); then, extracted and analysed by the TLC procedure described above to validate the method for the melon seed products. The recovery experiments were also carried out at different times on the same spiked samples to determine the reliability of the analytical method.
3. Results and discussion The mean recoveries of AFB1 from artificially spiked egusi melon seed food products were 86%, 78% and 82% in ogiri, robo and egusi soup, respectively. The relative standard deviation for within day repeatability was 4.6%, 7.3% and 3.8% for ogiri, robo and egusi soup, respectively. Tables 1 and 2 present the summary of number of samples analysed, the number found to contain detectable levels of AFB1, incidence rate, and the concentrations of AFB1 for 2005 and 2006. As can be seen, the incidence of AFB1 was 25%, 32.5% and 17.5% in ogiri, robo and egusi soup respectively in 2005. From the 2006 analysis, it can also be seen that the incidence of contamination was 25% for ogiri, 31.1% for ‘robo’ and 21.9% for ‘egusi soup’, The levels of aflatoxin B1 varied from 2 to 15 ppb in 2005 and 3 to 14 ppb in 2006 in all melon seed products sampled. The overall mean levels of aflatoxin B1 were for 8.9 ppb for ogiri, 9.7 ppb for ‘robo’ and 7.2 ppb for ‘egusi soup. These amounts are under permissible level of 20 ppb accepted in Nigerian foods on paper. In an earlier study, aflatoxin B1 was detected in 37% of 137 Nigerian melon seed samples with mean levels of aflatoxin B1 contamination of 14.8 ppb in the forest and 11.3 ppb in the savanna (Bankole et al., 2004). The levels found in the processed melon seeds are much below those of the raw foods. Also, while 3.5% of analysed melon seed samples had aflatoxin level above the Nigerian 20 ppb limit (Bankole et al., 2004), none of the samples in the present investigation had toxin level above 20 ppb. In this regard, the effect of food processing techniques on aflatoxin content must be taken into account, which could have accounted for the low level of occurrence of aflatoxin B1 in this study compared to
Table 1 Distribution and levels of aflatoxins in melon seed product samples in southwestern Nigeria in 2005. ‘Egusi’ product
No. of samples
Positive samples
% of positive samples
Aflatoxin B1 Mean SD
Range
‘Ogiri’ ‘Robo’ Soup
40 40 40
10 13 7
25 32.5 17.5
8.9 ± 4.5 8.3 ± 3.3 7.2 ± 2.7
2–13 3–15 3–10
SD, standard deviation.
Table 2 Distribution and levels of aflatoxins in melon seed product samples in southwestern Nigeria in 2006. ‘Egusi’ product
No. of samples
‘Ogiri’
36
9
‘Robo’
45
Soup
32
SD, standard deviation.
Positive samples
% of positive samples
Aflatoxin B1 Mean SD
Range
25
9.0 ± 3.25
14
31.1
7.6 ± 2.92
7
21.9
7.3 ± 2.72
4.1– 14.3 2.5– 11.7 4.3– 10.7
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levels previously established in melon seeds. The percentage loss of aflatoxins in food has also found to be variable depending on the techniques (Fandohan, Hell, & Marasas, 2008) which explains the variations found in aflatoxin B1 levels in various melon seed derived foods. The AFB1 incidence and levels of contamination found in the present study are also lower to those reported in other Nigerian processed foods that have so far been analysed. For instance, Kpodo et al. (1996) reported aflatoxin levels of 0.7 and 313 ppb in kenkey, fermented maize dough from two kenkey production sites in Accra, Ghana while Bankole, Ogunsanwo, and Eseigbe (2005) found AFB1 in 64.2% of 106 samples of dry roasted groundnuts with a mean of 25.5 ppb. Aflatoxin contamination in foods resulting in public health threats and economic loses is a frequently occurring problem in sub Saharan African countries (Bankole, Schollenberger, et al., 2006; Kpodo & Bankole, 2008). Some results have been obtained on aflatoxin levels in various food products such as roasted groundnuts (Bankole et al., 2005), maize based foods (Fandohan et al., 2005). It is pertinent to point out that contamination levels of aflatoxin B1 in this study were much lower that those obtained in previous studies of African processed foods cited above. In conclusion, the results of this study demonstrate that the concentrations of aflatoxin B1 in all analysed melon seed products were under permitted levels. However, this amount should be considered in regard to overall daily exposure to mycotoxins, and the overall goal should be to have no aflatoxins in food. It is therefore important to establish routine monitoring program for aflatoxin contamination in food in Nigeria with a view to establishing permanent monitoring and control program from production until consumption. However, it is important to consider these levels in terms of their contribution to overall daily exposure to aflatoxin levels in food. Acknowledgements The financial support for this study from the International Foundation for Science, Stockholm, Sweden through a grant to Dr. S.A. Bankole is thankfully acknowledged. References Abaelu, A. M., Makinde, M. A., & Akinrimisi, E. O. (1979). Melon (egusi) seed protein 1: Study of amino acid composition of defatted meal. Nutritional Reports International, 20, 605–613. Aboaba, O. O., & Amasike, J. (1991). Storage of melon seeds. Nigerian Journal of Botany, 4, 213–219. Bankole, S. A. (1993). Moisture content, mould invasion and seed germinability of stored melon. Mycopathologia, 122, 123–126.
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