Study on Fumonisin Contents of Maize Sampled From Dawanau Grains Market in Kano.
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1 Volume 4 Issue 04 April-2016 Pages ISSN(e): Website: DOI: Study on Fumonisin Contents of Maize Sampled From Dawanau Grains Market in Kano. Authors Shamsuddeen U. 1, Kabir A. 2 1,2 Department of Microbiology Bayero University Kano - ushamsudeen.bio@buk.edu.ng ABSTRACT A study was carried out on the Aflatoxin content of maize sourced from Dawanau grains market in Kano. A total of 25 maize samples were collected and subjected to moisture content determination, moulds isolation as well as aflatoxin extraction which were followed by the analysis of the toxin. Of the 25 maize samples 9 (36%), 6 (24%), 19 (76%), 7 (28%), 4 (16%), 8 (32%), 2 (8%) and 4 (8%) yielded A. flavus, A. parasiticus, Fusarium, A. niger, Penicillium, Mucor, NigrosporaandTrichodermarespectively. Analysis of the extracts using thin layer chromatography yielded blue and blue green fluorescence on the TLC plate after U.V light illumination withrf values which ranged between Analysis of the extracts using ELISA kit showed the aflatoxin content of the maize to be in the range of The study stressed the need for public enlightenment on the dangers and potential hazards associated with the consumption of mycotoxins. Key words: Fumonisin, maize, dawanau, market, Kano INTRODUCTION Maize is a very versatile cereal and is used in the production of food, feed, adhesive, oil, syrups, flakes, alcoholic and non-alcoholic drinks, starch and ethanol among others while locally it is used to make pap, nakia, tuwo, pop corn etc. In addition Nigeria is the 10 th largest producer of maize globally; producing about 98.8% of its domestic maize needs locally (El-Imam, et., al. 2012). In the field as well as in the store, many pests and parasites attack maize. Fungi rank second as the cause of deterioration and loss of maize. Fungi could cause about 50 80% of damage on farmers maize storage period if conditions are favourable for their development. The major genera commonly encountered on maize in tropical regions are Fusarium, Aspergillus and Penicillium (Fandohan et al., 2003). These genera of fungi are very important not only because they infect crops, but because of their ability to produce extracellular toxic metabolites called Mycotoxins Mycotoxins are low molecular weight compounds that do not produce symptoms in only a few hours. Mycotoxins have attracted worldwide attention due to the significant losses associated with their impact on human and animal health and consequent national economic implications. Mycotoxicosis is the consequent or effect (Disease) of ingesting toxin contaminated foods by man and animals. It may also result indirectly from consumption of animal products such as milk from livestock exposed to contaminated feed (Bankole and Adebanjo, 2003). Fumonisins are the mycotoxins produced by Fusarium spp. They are a group of non-fluorescent, water soluble and polar mycotoxins, and at least 15 related fumonisin compounds have been identified. The most Shamsuddeen U, Kabir A. IJSRE Volume 4 Issue 4 April 2016 Page 5196
2 important species producing this toxin include, Fusarium moniliforme, Fusarium proliferatum, Fusarium nygamai. Other species includes F. anthophilum, F. diamini, F. napiforme, F. thapsinum, F. globosum (Fandohan et al., 2003). Amongst these F. verticillioides and F. proliferatum are by far the most prolific fumonisin producers. They produce the highest amounts of toxins: up to 17,900µg/g. Fumonisins can contaminate maize foods and feeds as a result of fusarium invasion before and after harvest. According to Fandohan et al., 2003, Maize is the product in which fumonisins are most abundant. Fumonisins have emerged as a highly visible animal and human health safety concern since they have been associated with many animal diseases such as leukoencephalomalacia (LEM) in horses, pulmonary oedema syndrome (PES) in pigs and hepatocarcinogenesis in rats. With respect to humans, studies on the prevalence of oesophageal cancer in regions of South Africa, China, Italy and Iran revealed an association between this disease and the consumption of maize contaminated by Fusarium spp (Fandohan et al., 2003). Based on recent research results obtained so far, FB 1 has been evaluated as possibly carcinogenic to humans (Class 2B) (IARC, 2002). The aim of this work is therefore to assess the level of contamination of maize grains with the fungal metabolite, the Fumonisin. METHODOLOGY Sample Collection A total of twenty five samples of white maize grains were collected from different bags at Dawanau grains market Kano. The collected grains were stored in paper bags to avoid moisture build up which could result in increased fungal growth and mycotoxin production (El-Imam et al., 2012). Moisture Content Determination Hot air oven was switched on until the temperature reached 130 o C and allowed to heat for 30mins before the samples were placed in it to ensure uniform heat distribution. The sample was placed in a dry mill blender and grounded to powder. Ten grams of each ground sample was weighed, this was done in duplicate for each sample. The weighed samples were placed in a pre weighed crucible and placed in the pre-heated oven. The samples were heated until a constant weight was obtained. The moisture content of the samples was obtained by the following relation (ISTA, 1996). Moisture content = ( ) ( ) ( ) X 100 (ISTA, 1996). Fungal Isolation The maize seeds were thoroughly mixed to obtain homogeneity. Twenty (20) maize seeds, Per sample were randomly selected and surface sterilized using 5.25% sodium hypochlorite solution (Reckitt and Colman Nig. Ltd). The seeds were aseptically rinsed five times with sterile distilled water (Makun et al., 2009). Five (5) seeds per plate were plated on Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA). Plating was done in duplicate for each agar. Chloramphenicol (0.5mg/ml) was added to the culture media before autoclaving to inhibit bacterial growth (Aljouraifani, 2011). The plates were incubated at room temperature for 5-7 days. Following incubation, pure culture of the fungi was obtained by repeated subcultures, which were preserved on PDA slants till identified (Al-Rahmah et al., 2011). Fungal counts of maize samples Ten grams (10g) of ground sample was placed in a sterile 500ml Erlenmeyer flask containing 90ml of sterile distilled water and was homogenized by shaking thoroughly for 10mins. The homogenate was left to stand Shamsuddeen U, Kabir A. IJSRE Volume 4 Issue 4 April 2016 Page 5197
3 further for 30mins. Ten (10) fold serial dilution was then prepared. All dilution tubes were labeled accordingly. From each dilution, 1.0ml was pipette aseptically into duplicate and appropriately labeled petridishes. This was followed by pouring aseptically 15-20ml of molten Malt Extract Agar (MEA) fortified with 0.5µg/ml of chloramphenicol into each of the petridishes. The plates were allowed to solidify, then were incubated at room temperature for 24 48hours. Following incubation plates with colonies were selected, and the colonies counted. The average number of the colonies was multiplied by the inverse of the dilution factor and the result expressed as cfu/g of the sample (Shamsuddeen et al., 2012). Quantitative Evaluation/Estimation of Fumonisin using ELISA based Kits Elisa kits were purchased from Shenzhen Lvshiyuan Biotechnology Ltd China. The samples were prepared and analyses were carried out according to the manufacturers instruction. Sample extract solution was prepared by mixing 60 parts of methanol with 40 parts of deionized water. One gram of pulverized sample was dispensed into 7ml plain vacutener bottle; 5ml of sample extract solution was added. The mixture was shaken thoroughly for 5mins this was then centrifuged at 4000r/min for 5min, from the filtrate 0.5ml was pipette and 4.5ml of deionized water was added to it.from this solution 50µl was obtained for analysis. The micro wells were labeled and all samples were analyzed in duplicates alongside standards. Using a micropipette 50µl of the prepared sample above was dispensed into the micro well, then 50µl of FB 1 enzyme conjugate was added, this was followed by the addition of 50µl of FB 1 antibody working solution. The mixture was shaken gently and incubated at room temperature in the dark for 30mins. Following incubation the solution in the microwell was discarded, the microwells were washed five times each time with 300µl of washing solution. After washing 50µl/well of substrate A was dispensed, then 50µl/well of substrate B. the plate was shaken and incubated at room temperature in the dark for 15mins. Following incubation 50µl/well of stop solution was added. The microplate was then placed in a microplate reader and the optical density (OD) value obtained/read at a wavelength of 4500nm. RESULT The result of the moisture content of the maize samples is presented in table The moisture content range from 1% to 6% with sample 25 having the highest value of 6 and 3%. Table 1: Moisture contents of maize samples Sample Moisture (%) Sample Moisture (%) Shamsuddeen U, Kabir A. IJSRE Volume 4 Issue 4 April 2016 Page 5198
4 Table 2: Percentage of Occurrence of Fungi S/No Fungal Specie Frequency of Occurrence out of 25 samples % Occurrence out of 25 samples 1. A. flavus A. parasiticus Fusarium spp A. niger Penicillium spp Mucor Nigrospora Trichoderma 2 8 The fungal count (cfu/g) is shown in table 4.4 below, which showed various counts. The least being sample 20 with 1.20x10 3 cfu/g and the highest being sample 6 and 18 both of which have count in the order Table 3: Fungal Load of Maize Samples Sample Fungal Count cfu/g x x x x x x x x x x x x x x x x x x x x x x x x x10 4 The result of the aflatoxin and fumonisin content (ppb) of the samples is presented in table 4.6. The mean aflatoxin and fumonisin content are and ng/ml respectively. Shamsuddeen U, Kabir A. IJSRE Volume 4 Issue 4 April 2016 Page 5199
5 Table 4: Estimated Fumonisin Content Samples Fumonisin (ppb) DISCUSSION A total of 25 maize grain samples were collected from Dawanau grains market Kano. From Table 1 the moisture content of all samples ranged from1-6%. Samples 14 and 6 had the lowest moisture content value of 1%. While the highest moisture content value of 6% was obtained in samples 3 and 25. The low moisture content value of the grains which ranged from 1-6% shows that the maize grains were well dried to the appropriate moisture content level of below 12% In table 2 the fungal flora isolated include; Fusarium spp, Aspergillus flavus, A. parasiticus, A. niger, Penicillium spp, Mucor spp., Trichoderma and Nigrospora spp. Sources of fungal contamination of cereals are many but all are traceable to the environment in which these grains are grown, handled and processed, other factors that influence fungal contamination of grains include; rainfall, drought, temperature and humidity, soil conditions and wind, others are harvesting equipment, storage and handling as well as moisture control (Sauer et al 1992). Presence of these fungi in these grains could also be attributed. To the climate of West African countries, Nigeria inclusive, the climate is tropical, with an all year round high ambient temperature and relative humidity that provides optimal growth conditions for these molds. Climatic factors such as fluctuations in rainfall, relative humidity and temperature, results in plant stress, thus making the crops susceptible to fungal contamination. Shamsuddeen U, Kabir A. IJSRE Volume 4 Issue 4 April 2016 Page 5200
6 Poor and improper agricultural practices such as overcrowding and improper spacing of plants, late planting of maize, harvesting of maize grains in wet conditions favour the proliferation of these molds. In addition repeated planting of maize and other cereal crops in the same field or in nearby field favours fungal flora by increasing the fungal inocula in fields. Vectors such as insects that attack these maize grains also aid in the infection by creating wounds and openings on the grains, these insects as well serve as agents that transmit these fungi or their spores from one cob/plant to the other (Bilgrami and Choudhary 1998). Presence of these molds in the grains is of significance because their occurrence could result in spoilage, decreased germinability of the grains, moreover development of visible mold growth results in discoloration of the grains, and also a decrease in the quality, of the grains as well as a loss in the nutritional value of grains as reported by Fandohan et al (2003). It has been reported that, the major genera of fungi commonly encountered on maize in tropical regions are Fusarium, Aspergillus and Penicillium (Fandohan et al., 2003). Makun et al reported that out of 93 maize samples collected from Niger and Kogi States, he isolated Aspergillus flavus, A. fumigates, A. nidulans, A. niger, A. parasiticus, A. terreus, Fusarium spp, Mucor spp, Rhizopus spp and Penicillium spp. The high incidence ofa. Flavus and Fusarium spp is in line with the findings of Mohana et al.(2014) which found 25 maize samples showing high incidence of both A. flavus and F. verticilliodes out of 45 maize samples collected from different regions of southern Karnataka India. Infection of maize with Fusarium species and its contamination by Fumonisins are generally influenced by many factors including, environmental conditions (such as climate, temperature, humidity, and rainfall), insect infestation and pre and post-harvest handling. Presence of these mycotoxins in the grains despite the low moisture content of 1% - 6% suggest that the toxins were probably formed in the field when moisture content was higher, or due to isolated rain showers late in the season coupled with hot humid days and cold dry nights. It could also be during harvest due to poor agricultural practices such as threshing which may cause grain damage thus favouring the proliferation of fungi mycoflora and their toxins. In addition maize left in the fields to dry before harvesting does not dry effectively because it is not fully exposed to sunlight and residual moisture is trapped in the cob. Even though 24 samples yielded fumonisins, None of the samples had the toxin content greater than 4ppm (4,000ppb) as recommended by United States Food and Drug Administration (FDA); which recommends that Fumonisin levels in human foods should not be greater than 4ppm (4,000ppb) while in degermed dry milled corn products fumonisins levels should not exceed 2ppm (2,000ppb) (FDA, 2000). This is in line with the findings of Fandohan et al. (2005) which found all maize samples tested from four local storage systems in Benin Republic to contain fumonisins, with levels ranging from 0.6 to 2.4mg/kg. Fandohan et al. (2005) also found fumonisin levels decreased over the storage period, but not significantly. Rensburg (2012) also found the natural occurrence of Fumonisin and Fusarium spp in maize samples from 29 localities over a 3 year period in South Africa. CONCLUSION In conclusion the maize grains were found to be contaminated with either one or more molds. All samples were contaminated with Fumonisin. However none of the samples had fumonisin levels greater than benchmark figures. RECOMMENDATION Mass awareness and enlightment campaign to educate the farmers, about good agricultural practices, storage, harvest and handling of the grains to reduce the level of mycotoxin contamination. Shamsuddeen U, Kabir A. IJSRE Volume 4 Issue 4 April 2016 Page 5201
7 It is recommended that grains should be dried immediately after harvest within 24 48hrs to below 14% moisture content. Then grains should be further dried to a moisture content of below 12% before storage Educating the populace on the dangers of consuming mycotoxin contaminated food. Development of affordable and easy to use kits for testing and quantification of mycotoxins in African countries. There is urgent need for inspection and enforcement services to be put in place bynational Agency for Food Drug Administration and Control(NAFDAC) to ensure compliance to the set Aflatoxin level. While NAFDAC needs to set benchmark levels for Fumonisins and other health implicated mycotoxins. Farmers should avoid harvesting in wet conditions REFERENCES 1. Al-juraifani A.A. (2011). Natural Occurrence of Fungi and Aflatoxins of Cinnamon in Saudi Arabia. African Journal of Food Science Vol. 5 (8) Pp Bankole S.A., Adebanjo A. (2003). Mycotoxins in Food in West Africa: Current situation and possibilities of controlling it. African Journal of Microbiology Research.Vol 2 (9) Pp Bilgrami K.S., Choudhary A.K., (1998). Mycotoxins in preharvest contamination of agricultural crops,in:sinha KK, Bhatnagar D. eds. Mycotoxins in agriculture and food safety. Marcel Dekker,Newyork. Pp El-imam A.A., Joseph B.J., Abdullahi I.O. (2012).Occurrence of fumonisins and deoxynivalenol of fumonisins and deoxynivalenol in stored maize used in industrial productions in Zaria Nigeria.African Journal of Food Science Vol 6 (9) Pp ( ). 5. Fandohan P., Gnonlonfin B., Hell K., Marasas W.F.O and Wingfield M.J. (2005).Impact of indigenous storage systems and insect infestation on the contamination of maize with fumonisins.african Journal of Biotech.Vol. 5 (7) Pp Fandohan P., Hell K., Marasas W.F., Wing-Field M.J. (2003).Infection of Maize by fusarium species and contermination with Fumonisin in Africa.African Journal of Biotechnology Vol. 2 (12). Pp FDA (2000).Background paper in support of fumonisin levels in Animal Feeds (Draft). Guidance for Industry: Fumonisin level in Human Foods and Animal Feeds: dms/fumonbg2.html. 8. FDA (2000). URL: 9. IARC (2002). IARC monographs on the evaluation of carcinogenic risks of humans, some traditional herbal medicines, mycotoxins, mycotoxins, naphthalene and styrene. Vol (82) International Agency for Research on Cancer Lyon, France Pp Ismail Y. R.,(1997) Food chemistry,vol.59 No.1, Pp ISTA(International seed testing Association) International rules for seed testing. Seed science and Technology 21 (suppl.) Makun H.A., Anjorin S.T., Morofnoye. B., Adejo F.O., Afolabi A.O., Fagbayibo H., Balogun B.O., Surjucleen A.A. (2010). Fungal and Aflatoxin contermination of some human food commodities in Nigeria.African Journal of Food Science Vol. 4 (4) pp Mohana C.D., Thrippeswamy, S., Abhishek U.R., Manjunath K. (2014).Natural occurrence of A. flavus and F. verticilliodes and AFB 1 and FB 1 contamination in maize grown in southern Katnatka (India).Canadian Journal of Plant Protection (CJPP) Vol. 2 No Rensburg B.J. (2012). Modeling the incidence of Fusarium and Aspergillus toxin producing species in maize and sorghum in South Africa. Pp Shamsuddeen U, Kabir A. IJSRE Volume 4 Issue 4 April 2016 Page 5202
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