Length of Fermentation and Detection Methods of Aflatoxin B i During Fermentation of Maize into Ogi.

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1 Nigerian Journal of Microbiology, Vol. 22(1): Length of Fermentation and Detection Methods of Aflatoxin B i During Fermentation of Maize into Ogi. *Oluwafemi, F. and Ikeowa, M.C. Department of Microbiology, University of Agriculture, Abeokuta, Nigeria. Abstract The length of fermentation and detection methods of aflatoxins in fermented maize (Ogi) was investigated. The effect of spontaneous fermentation and storage under refrigeration temperature (4 o C) and ambient tropical condition (28 o C) was studied in the first experiment. Levels of aflatoxin as high as 50µg/kg was recorded for the control sample. Aflatoxins present in the maize grains persisted after 24 to 48 hours period of fermentation. However, there was a 50% reduction after 72 hours of fermentation of maize ( 50 µg kg -1 to 25 µg kg 1 ) which was statistically significant at P = 0.05 using thin layer chromatography. Analysis of the bran showed that there was no adsorption of aflatoxin at 24 and 48 hours of fermentation. There was 50% adsorption of aflatoxin to the bran from the fermented maize grains after 72h fermentation period. Organisms isolated from fermented maize grains were Corynebacterium spp., Fusarium spp., Aspergillus spp., Penicillium spp., Lactobacillus spp., Leucosnostoc spp., Lactococcus spp. The ph values of 0-72h fermented maize grains range between 2.95 and There was no effect of storage on aflatoxin content of ogi at ambient and refrigeration temperatures. Maximum degradation of aflatoxin was after 72 hours and over 95% degradation was achieved. Lactic acid bacteria count range from zero to 9 x10 4 cfu/g while fungal count decreased from 12 x 10 4 to 2 x 10 4 cfu/g. Titrable acidity increased from 0.124M to 0.169M while ph values decreased from 6.8 to 4.0. Key words, Fermentation, detection, food, aflatoxin, contamination *Corresponding author; foluwafemi2000@yahoo.co.uk Introduction Fermented maize ( Ogi ) is a staple cereal of the Yorubas in Nigeria and is the first food given to babies at weaning (Akinrele, 1968). It is produced by soaking corn in water for 2 to 3 days, followed by milling and sieving through a screen mesh. The sieved material is allowed to sediment and ferment and is marketed as wet cakes or Ogi (Akinrele1968). Fermentation is the slow decomposition process of organic substances induced by microorganisms or by complex nitrogenous substances (enzymes) of plants and animal origin (Walker, 1998). It occurs naturally by the action of microorganisms. It can be controlled to give beneficial results. It is a relatively efficient, low energy preservation process which increases the shelf life and decreases the need for preservation technology (Adams and Moss, 1999). Aflatoxin is a common contaminant of foods, particularly in Nigeria and other West African countries (Williams et al, 2004).The toxin is produced by Aspergillus flavus and A. parasiticus during production, harvest, storage and food processing and it is considered by the U.S. Food and Drug Administration (FDA) to be an unavoidable contaminant of foods. Aflatoxicosis is the poisoning that results from ingesting aflatoxins. Two forms of aflatoxicosis have been identified, the first is acute severe intoxification, which results in direct liver damage of humans and farm animals consuming aflatoxin- contaminated foods and subsequent illness or death and the second is chronic subsymptomatic exposure. Large doses of aflatoxin lead to acute illness and death usually through liver cirrhosis (CDC, 2004), chronic sublethal doses of aflatoxin have nutritional and immunologic consequences (Oluwafemi, 2000). All doses of aflatoxins have cumulative effect on the risk of cancer. Aflatoxins have been reported to be produced in cereals, peanuts, figs, seeds, and fermented products including cheese (Bankole and Adebanjo, 2003) and fermented meats such as Salami sausage and country hams (Alvurez-Barra et al, 2000). Aflatoxin contaminated diet has been linked with the high incidence of liver cancer in Africa (Bankole and Adebanjo, 2003). Oluwafemi (2000) also linked aflatoxins with male infertility. In her work, she found out that 37% of infertile men had aflatoxins in their blood and semen suggesting that aflatoxins may be a contributory factor in male infertility. In a recent review, Bankole and Adebanjo (2003) found that the level of aflatoxin B 1, B 2 and G 1 was significantly higher in corn from the high incidence area for human hepatocellular carcinoma and the average daily intake of aflatoxin B 1 from the high risk area 1698

2 was µg/kg aflatoxin. Udoh et al (2000) reported 33% of maize sample from ecological zones of Nigeria contaminated with aflatoxins. During the steeping of corn, Corynebacterium, becomes prominent and appears to be responsible for the diastatic action necessary for the growth of yeasts, and lactic acid bacteria (Banigo and Muller, 2000). Other microorganisms associated with ogi fermentation are Cephalosporium, Fusarium, Aspergillus and Penicillium (Banigo and Muller, 2000).. Most of the acid produced as lactic acid depress ph of desirable products to around 3.8. The Corynebacteria developed early and their activities cease after the first day while those of the Lactobacilli and yeasts continue beyond the first day of fermentation (Banigo and Muller, 2000). Fermented foods generally have beneficial roles in the lives of people (Dirar. 1993). The benefits to people in developing countries consuming fermented foods include improving food security, food preservation, increasing income and employment generation, improving nutrition, vitamins, digestibility but more importantly removing anti-nutritional factors. Many foods contain naturally occurring toxins and anti-nutritional compounds. These can be removed or detoxified by the action of microorganisms during fermentation. Numerous detoxification methods (Coomes et al., 1966; Bullerman, 1974; Doyle et al., 1982; Paster et al., 1988; Adegoke et al., 1991; Adegoke et al., 1996; Atanda et al., 2007) have been proposed ranging from chemical detoxification to the recent use of plant extracts. The practical applicability in developing countries of these novel procedures still remains a dream probably due to the cost of detoxification, and the high technology involved. Since heavily aflatoxin-contaminated grain is a common feature in Nigerian markets, the aim of this study is to determine the effect of length of fermentation on aflatoxin levels, to determine the effect of storage at refrigeration temperature and normal tropical ambient condition and thirdly to isolate and characterize the organisms involved in maize fermentation. Materials and methods Sample collection Maize grains were bought from the open markets in Abeokuta, Nigeria. Two hundred grammes of the samples were weighed into three bowls each and 1000ml of water added. Each bowl of maize grains was fermented 24 h, 48 h and 72 h respectively. The zero hour sample was milled and analysed immediately and was used as control. At the end of each fermentation period, maize grains were removed from the water and milled in the laboratory using a warring blender. At the end of the milling, the sample was filtered to separate the Ogi from the bran using Muslim filter. The sediment was put in white cloth and tied so as to remove excess water to get a solid cake of the maize i.e. Ogi. ph measurement: The ph values of the 24, 48,and 72 hour fermented steep water weredetermined using ph meter (Mettler Delta 340) The electrode was standardized using buffer 4 and 6.8. Three readings were taken for each sample and the average was recorded. Microbial Analysis: The initial and final microbial profiles were studied. One gram of sample that had been milled was serially diluted with saline. Serially diluted samples (0.1 ml) were plated on man Rogosa sharpe agar and potato dextrose agar. The plates were incubated, observations recorded and isolates identified using standard procedures (Klich, 2002). The results were expressed as colony forming units (cfu) per gram. Aflatoxin extraction and quantification Ten grammes of ogi and bran were weighed into different Erlenmeyer flasks and the following added: 25ml of H 2 O, 25g of diatomaceous earth and 250ml chloroform (CHCl 3 ). The flasks were covered and shaken for 30 minutes in a rotary shaker. The supernatant was filtered and evaporated to dryness in a water bath. Precoated silica gel (20cm by 20cm) plates were used for spotting the redissolved dried extracts of the samples (Oluwafemi and Da-Silva, 2005). Redissolved dry extract (2.5 µl, 10 µl) were spotted on a 4cm line from the bottom of the thin layer chromatography (TLC) plate. On the same plate, 2.5 and 10 µl aflatoxin B 1 standard were spotted for quantification analysis. 1699

3 The plates were developed in a tank containing: diethylether, chloroform and water (95:4:1). The spots were visualized at 366nm using U.V light. Quantification of aflatoxin B 1 was done by comparison of the peak heights with those of known standard and the exact aflatoxin content was calculated as follows: Ug/kg AFB 1 =SxYxV divided by X x W S=ul aflatoxin B 1 standard equal to test solution spotted Y= concentrate on of aflatoxin B 1 standard,ug/kg V=ul test solution spotted giving fluorescent intensity equal to S W=test portion applied to column Enzyme linked Immunosorbent Assay. This was carried out in the second fermentation using pre-coated microtitre plates coated with antibody to aflatoxin. Twenty grammes of maize samples including unfermented and fermented grains were milled. Extraction was done using 100ml of chloroform. Optical densities of standard aflatoxin was compared with those of samples and aflatoxin levels extrapolated. Results The initial aflatoxin level of unfermented maize grains in the first experiment was 50ug/kg. After 24 and 48 hours, there was no difference in the aflatoxin B 1 (AFB 1 ) level as there was no aflatoxin in the bran of both 24 and 48h fermented maize (Table 1). However there was a significant reduction of 50% AFB 1, after 72 hours of fermentation. The 72h-fermented ogi sample had 25 µg/kg AFB 1 and the remaining aflatoxin had adsorbed unto the bran. This was confirmed when the bran was extracted with chloroform and the aflatoxin level quantified. The effect of storing the fermented ogi further under ambient tropical conditions and at 4 o C (refrigeration temperature) did not give any significant difference at P = Lactic acid bacteria count (Table 1) increased with increase in length of fermentation. This increase brought about increase in lactic acid production with its consequent effect on the ph. In the second experiment carried out (Table 2), lactic acid production continued to increase as the fermentation progressed (zero to 9 x 10 4 cfu/g). Consequently, the ph dropped from 6.8 to 4.0 and the titrable acidity increased from 0.124M to 0.169M. Fungal counts decreased from 12 x 10 4 to 2 x Fig. 1. Aflatoxin level of maize grains before fermentation using ELISA Table1. Effect of fermentation period on levels of aflatoxin B 1 in maize. Time AFB 1 in Ogi (ug/ml) AFB 1 in Bran(ug/kg) LAB count 0h 50 < a 24h 50 < x 10 4b 48h 50 < x 10 4c 72h x 10 4d Mean values carrying the same superscript in the same column are not significantly different using Duncan multiple range test. 1700

4 Fig 2. Aflatoxin level of maize after 72h of fermentation of maize grains using ELISA Fig 3. Aflatoxin level of maize grains after 96h fermentation using ELISA Table 2. Effect of 96 h fermentation on levels of aflatoxin, ph, fungal count, titrable acidity and LAB count Time 0h 24h 48h 72h 96h ph Titrable Acidity (M) LAB count 1 x x x x x 10 4 (cfu/ml) Fungal count 12 x x x x x 10 4 (cfu/ml) Total aflatoxin (ug/kg) Discussion There are many traditional beliefs about the medicinal properties of fermented food products. The fur ethnic group in Sudan strongly believes that the consumption of fermented foods protect them from disease (Dirar, 1993). Kombacha (a fermented milk product in Russia) has been used to treat tuberculosis (Greenwalt et al., 2000). The lowered ph inhibits the growth of food spoiling or poisoning microorganisms as seen in this study. This was in agreement with findings of Gourama and Bullerman (1995). Fungal count in both experiments was on the decrease, although Aspergillus sp. persisted throughout the study. Wiseman and Marth (1981) reported the effect of lactic acid bacteria on A. parasiticus growth. They reported that there was decrease in aflatoxin production due to the presence of lactic acid bacteria. Gourama and Bullerman, (1995) reported that Lactobacillus spp. were found to inhibit aflatoxin biosynthesis whereas L. lactis was found to stimulate aflatoxin production. Aflatoxin levels decreased from50 ug/kg to 25 ug/kg in the first experiment while in the second experiment reduction of aflatoxin was from 160 ug/kg to ug/kg. Although values obtained from the two experiments vary, however there was a strong indication that there was aflatoxin reduction irrespective of detection method. Oluwafemi (2000) confirmed variation in detection methods especially with reference to thin layer chromatography and enzyme linked immunosorbent assay. Other workers (Branem et al., 1975; Wiseman and Marth, 1981; Sutic et al., 1997) agreed that the inhibitory compound produced by lactic acid bacteria was a polypeptide compound that can be inactivated by pronase E and trypsin. Gourama (1995) found that Lactobacillus sp totally inhibited the germination of mould spores. Megalla and Hafez (1984) found that aflatoxin B 1 was transformed to aflatoxin B 3 in acidic environment. The relevance of the findings of Hafez and Megalla (1984) is a confirmatotion 1701

5 that lactic acid bacteria are known to detoxify aflatoxin in fermented foods such as maize. The antiaflatoxigenic effect of lactic acid bacteria might also be as a result of the secretion of bacteriocins (Yurdugul and Bozoglu, 2002) and some other metabolites such as hydrogen peroxide. The benefits of fermented foods apart from degradation of aflatoxins include improved food security, cheap and energy efficient means of preservation and increased levels of vitamins (Arthur, 1986). Conclusion Fermentation of maize for a minimum of three days is a relatively effective form of detoxification method that can reduce aflatoxin by 50%. From the results obtained in this study, it is apparent that lactic acid bacteria have the potential to be used as biological control agents in foods to prevent mould growth and aflatoxin production. This efficacy depends to a great extent on the initial level of contamination and degree of association of aflatoxins with the food constituents, moisture content and type of aflatoxins. This low cost technology although practised unknowingly by the West-African people is now been truncated due to poverty. This current study has confirmed that a minimum of 72hours fermentation can be the first aid solution to help populations in the continent to face mycotoxin problems by reducing the aflatoxin contents of foods to significantly lower levels. References Adegoke, G. O.; Babalola,A. K. and Akanni,A. O Effect of sodium metabisulphite,hydrogen peroxide and heat treatment on aflatoxin B 1 in lafun and gari-two cassava products. Die Nahrung 35: Adegoke, G. O., Olojede, F., Engelhardt, G. and Wallnofer, P. R Inhibition of growth and aflatoxin production in Aspergillus parasiticus NRRL 2999 by Garcinia kola (Family Guttiferrae). Adv. Food Sci. (CMTL) 13(3/4): Akinrele, I. A Fermentation studies on maize during the preparation of a traditional African starch cake food. J. Sci. Food Agric 21: Alvurez-Barra, V. A. M, Price, J. F, Gray, J. I. and August, S. D Some factors influencing aflatoxin production in a fermented sausage. J. Food Sci. 47: Atanda, O. O., Akpan, I. and Oluwafemi, F The potential of some spice essential oils in the control of A. parasiticus CFR 223 and aflatoxin production. Food Cont. 18: Banigo, E. O. I and Muller, H. G Carboxylic acid patterns in Ogi fermentation. J. Sci Food Agric. 23: Bankole, S. A. and Adebanjo, A Aflatoxin contamination of dried yam chips marketed in Nig. Trop. Sci. 43: 3-4. Batish, V. K., Grover, S. and Ram, L Screening lactic acid starter culture for antifungal activities. Cult. Dairy Prod. J. 24(2): Branem, A. L., Go, H. C. and Genske, R. P Purification and properties of anti microbial substances produced by Streptococcus diacetylactis and Leuconostoc citrovorum J. Food Sci 40: Bullerman, L. B Inhibition of aflatoxin production by cinnamon. J. Food Sci. 29: Center for Disease Control and Prevention (CDC) Outbreak of aflatoxin poisoning- Eastern and Central Provinces, Kenya. Morb. Mortal. Wkly. Rep. 53: Coomes, T. J., Crowther, P. C., Feuell, A. J. and Francis, B. J Experiment al detoxification of groundnut meals containing aflatoxin. Nature, 209: Dirar, H. T The Indigenous Fermented foods of the Sudan: A Study in the African food and Nutrition; CAB International UK. Doyle, M. P., Applebaum, R. S., Brackett, K. E. and Marth, E. N Physical, chemical and biological degradation of mycotoxins in foods and agricultural commodities. J. Food Prot. 45:

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