Root and tuber crops are of immense importance to the feeding habit of African populace [1]. Cassava (Manihot esculenta Crantz) is consumed in various forms including Attiéké. Attiéké is becoming a daily intake for people in West and Central Africa that has a bed rock in it as an energy source [2]. It is a starchy-couscous dish derived from fermented Cassava dough processing and most importantly produced by Ivorian particularly the coastal population of the country [3]. Its appreciation is going beyond boundaries as a staple food due to black African-diaspora immigration [4]. In Cote d'Ivoire, Attiéké plays a large part in household sustenance with regards to its combat against hunger and also its nutritional supplements [5]. Attiéké is acknowledged to be representing 5% food expenditure and 20.5% Calories diet daily intake for consumers and its production is approximately estimated around 18965 to 40000 tons and consumption per inhabitants lies between 28 and 30 kg annually [5-8]. Its production still follows conventional procedure and not on modern procedure [9-11].

The preparation of attieke from cassava varies and numerous across different communities, though, the most complex aim is eliminating its bitterness and toxicity [12]. To avoid this, fermentation is carried out in numerous traditional transformation technologies of Cassava roots [13]. Its production proceed in the unit operations including peeling, grating, fermenting, pressing, crumbling, sieving, semolining, drying, air winding and cooking [14,15]. These units of operations proceeds traditionally, but unhygienic handling may lead to faster fungal deterioration and a resultant mycotoxicity.

Fungi which were ignorantly believed to be an anaesthetic organism growing on food had been lime lighted when the famous Turkey X diseases claimed lives of approximately 10,000 Turkish and lesser domestic birds in Great Britain [16]. This became informative that spoilage fungi could produce toxins named Aflatoxin: “A” obtained from Aspergillus and “fla” from flavus, where the name of the organism Aspergillus flavus was derived. Conditions that predispose food to these organisms include; hot and humid climate, damage by insects that decrease host’s immunity, moisture content of 16% and above [17].

This research was embarked upon to investigate food values, spoilage moulds and aflatoxin detection in Attiéké from Nigeria and Ivory Coast.

Collection of samples

Samples of prepared ready for cooking ‘Attiéké’ (i.e., processed cassava dough) were randomly selected from two major table top food sellers in three (3) locations where ‘Attiéké’ has its best cook and very demanding consumers as shown below:

1. a) Ejigbo, Beulah Church (6°33’8’’N, 3°18’26’’E)

b) Ejigbo (Ore meji), (6°30’8’’N, 3°18’20’’E)

2. a) Iwo, BHS (7°47’00N, 4°12’00’’E)

b) Iwo, Odo, ori market (7°46’00N, 4°12’00’’E)

3. a) Adjame Bromokoute 1 (5.36°N, 4.02°W)

b) Adjame Bromokote 2 (5.36°N, 4.02°W)

These locations are tropics, according to climate data; they have an annual rainfall of 1247 mm, 1264 mm and 1781 mm, respectively which last from April to October in Ejigbo and Iwo and from January to June in Adjame.

Variations due to different days of cooking were worked out such that they were all steamed at the same time. A total of six (6) samples were collected (2) samples from each location. The cooked samples were placed in clean sterile polythene bags, securely tied, labeled and transported to the laboratory.

Research treatments and design

Appreciable gram of each sample was taken and divided into three (3); one-third was stored in the freezer at subzero degree for aflatoxin analysis, the second fraction was stored at room temperature for nutrient analysis and the other was used immediately for isolation of fungi. The experiment was laid in Completely Randomized Design with three replicates.

Isolation and characterization of fungi biota

The isolation of fungi was carried out according to procedure described by Jimoh and Kolapo [18]. All samples collected were conditioned in a sterile package. About 2 g of each of the samples taken at random were aseptically placed in three replicates of Petridishes containing Potato Dextrose Agar and Lactic acid (10.53 gL^-1). The dishes were incubated at 27 ± 2°C for 3-6 days. Fungal cultures obtained were subsequently sub-cultured for purification. Upon fungi maturation, they were characterized based on cultural and morphological features such as colony diameter, colony color on agar, front and reverse and colony texture. Slide culture was then prepared and incubation in moist chambers at 26 ± 2°C for 4 days before observation under a light microscope (labomed, model CxL). Mycological keys and manuals were used for macro and microscopic features that are commonly used identification of fungi, which were conidiophores, conidial shape, phialides and metulae, presence and shape of vesicles [19].

Aflatoxin detection

The modified method of using HPLC chromatography was followed in determining aflatoxin concentrations of Aflatoxin B1 (AFB₁), Aflatoxin B2 (AFB₂) and Aflatoxin G1 (AFG₁) [20]. The samples were injected into the HPLC column heated to 40oC using mainly reversedphased columns, with mobile phases composed of water: methanol solution (60:40, v/v). To 1 L of mobile phase were added 119 mg of Potassium bromide and 350Bl of 4 M nitric acid (required for post/ column electrochemical derivatisation with Kobra Cell, ROBiopharm Rhone). This method is used after an extraction with acetonitrile and water, reaching limits of qualification between 0.012 and 0.073 μg kg^-1 was used. The coupling of HPLC to mass spectrometry was also used for the detection technique at the excitation wavelength of 362 nm and the emission wavelength was 425 nm. The ionization sources employed was based on atmospheric pressure [21,22]. The sample was calculated by analyzing triplicates of six samples. The method showed recoveries between 95% and 114% with a LOQ of 1.5 ngmL^-1.

Crude protein determination

The crude protein in the samples was determined by the routine semi-micro Kjeldahl, procedure. 0.5 g of each finely ground dried sample was weighed carefully into the Kjeldahl digestion tubes, 1 tablet of selenium catalyst and 10 ml of conc.H₂SO₄ was added into the digestion preset at 500°C. The digestion was left for 4 h in a fume cupboard. The tube was placed in a distilling unit and 5 ml of 40% NaOH was added to it. The mixture was steam distilled for 2 min into a 50 ml flask containing 10 ml of 2% Boric acid, mixed with indicator solution which was then titrated against 0.01 N HCl until a wine color was obtained. The quantification was done using AOAC procedure [23].

Crude fat determination

1 g of each dried sample was weighed into fat free extraction thimble and plug lightly with cotton wool. A soxhlet flask was then filled to ¾ of its volume with petroleum ether and the ether was left on heater to siphon until it was short of siphoning. Ether content of the extractor was drained into the ether stock bottle. The thimble containing sample was then removed and dried on a clock glass on the bench top. The extractor, flask and condenser were replaced and the distillation continued until the flask was practically dry. The flask containing the fat was detached; its exterior cleared and dried to a constant weight in the oven, and the crude fat was determined [23].

Dry matter and moisture determination

2 g of the sample were weighed into a previously weighed crucible (Wo). The crucible plus sample (W₁) taken was then transferred into the oven set at 100°C to dry to a constant weight for 2 h. At the end, the crucible plus sample was removed from the oven and transferred to desiccators, cooled for ten minutes and weighed (W₃) [23].

Ash determination

2 g of the sample was weighed into a porcelain crucible. This was transferred into the muffle furnace set at 550°C and left for about 4 h. About this time it had turned to white ash. The crucible and its content were cooled to about 100°C in air, then room temperature in a desiccator and weighed [23].

Fibre determination

2 g of the sample was weighed accurately into the fibre flask and 100 ml of 0.255 N H₂SO₄ was added. The mixture was heated under reflux for 1 h with the heating mantle. The hot mixture was filtered through a fibre sieve cloth. The residue was returned to the fibre flask to which 100 ml of (0.313 N NaOH) was added and heated under reflux for another 1 h. The mixture was filtered through a fibre sieve cloth and 10 ml of acetone added. The residue was washed with about 50 ml hot water on the sieve cloth before it was finally transferred into the crucible. The crucible and the residue were oven-dried at 105°C overnight to drive off moisture. The oven-dried crucible containing the residue was cooled in a desiccator and weighed to obtain the weight W₁. The crucible with weight W₁ was transferred to the muffle furnace for ashing at 550°C for 4 h. The crucible containing white or grey ash was cooled in the desiccator and weight to obtain W2. The difference W₁ – W₂ gives the weight of fibre [23].

Data were subjected to Statistical Analysis of Variance (ANOVA) at 95% and 99% probability levels using SAS 9.3 statistical package and means were separated using Duncan Multiple Range Test.

The result in Table 1 shows the Mean Nutritional composition in wet Attiéké samples collected from different location under different storage periods. The samples were highly significant (P<0.01) for crude protein and moisture contents, but significant (P<0.05) for Crude fibre, Crude Fat and ash contents. It was observed that that all the samples were significantly different from each other for crude protein. The crude protein obtained from samples from Ivory Coast stored for three days was significantly higher (P<0.05) than other samples while the least mean value was obtained from sample collected from Nigeria with storage period of a day (Table 1).

Table 1: Nutritional composition of wet attiéké sample.

The result also shows that the Crude fibre of sample collected from Ivory Coast stored for one day is significantly higher but not different from Ivory Coast samples stored for two days. Also, sample collected from Nigeria stored for one day and three days were non significantly different from each other, but different from sample collected from Nigeria stored for two days and sample collected from Ivory Coast stored for three days were not significantly different from each other.

The Crude Fat of samples from Nigeria stored for three days were not significantly (P>0.05) different from Ivory Coast samples stored for three days. The highest mean value of Wet Attiéké Crude Fat was recorded from Nigeria stored for one day. The ash content of samples collected from Ivory Coast stored for two days and three days were not significantly different from each other while the least ash content of Wet Attiéké was obtained from Nigeria sample stored for two days.

Highest moisture content was observed from samples collected in Ivory Coast stored for three days while the least moisture content of Attiéké was obtained from Nigeria sample stored for two days.

Nigeria stored for one day; A18, Nigeria stored for two days; A19, Nigeria stored for three days; A20, Ivory Coast stored for one day; B18, Ivory Coast stored for two day; B19, Ivory Coast stored for three day;B20.

Results obtained in this study showed that the spoilage fungi found associated with Attiéké include; Penicillium chrysogenum, Aspergillus niger, Candida albicans, Aspergillus flavus and Mucor hiemalis as shown in Figures 1a-1f.

Figure 1: Bio-deteriorating fungal contents in ‘Attieke’ samples. a: Photomicrograph of Penicillium chrysogenum. b: Photomicrograph of Aspergillus niger. c: Pure culture of Candida albicans in PDA media plate. d: Photomicrograph of Aspergillus flavus. e: Photomicrograph of Mucor hiemalis. f: Pure culture of Mucor hiemalis in PDA media plate.

It was also observed in Table 2 that, Attiéké samples collected from Adjame Bromokoute 1 and Adjame Bromokoute 2 had the highest number of fungi occurrence followed by Iwo Odoori, Iwo BHS, Ejigbo Ore meji collection with Ejigbo Beulah having the least growth. However studying these samples, the rate of occurrence of aflatoxigenic fungi (Aspergillus sp.) was higher in the order; Iwo Odoori>Iwo BHS>Adjame Bromokoute 2>Adjame Bromokoute 1>Ejigbo Ore meji>Ejigbo Beulah. In a contrasting situation, the occurrence of non- aflatoxic fungus was observed in the samples (Table 2).

Table 2: Occurrences of ‘attieke’ biodeteriorating fungi in different locations.

The result shown in Table 3 shows the mean Aflatoxin concentrations of AFB₁, AFB₂ and AFG₁in the studied Attiéké samples which are significantly different across each location (P<0.01), but AFBI found in samples from Ejigbo Beulah, Iwo BHS, Adjame Bromokoute 1 and Iwo Odo ori were not significantly different from one another, while the least AFB₁ was observed in sample from Adjame Bromokoute 2 (Table 3).

Table 3: Aflatoxin concentrations in different ‘Attiéké’ samples.

There were also no significant differences between the AFB₂ found in sample from Adjame Bromokoute 1 and Iwo Odoori but significantly different from sample from Ejigbo Beulah and Adjame Bromokoute 2 which are not significantly different from each other. The least AFB₂ concentration of Attiéké was obtained from sample collected from Adjame Bromokoute2. However, there were differences in all the AFG₁ concentration in the sample, the highest AFG₁ was obtained from sample Ejigbo Beulah, while the least AFG₁ was found in sample Adjame Bromokoute 2.

Results (Table 4) also show the correlation between all the proximate compositions in wet Attiéké. The crude protein is negative and strongly correlated with the crude fat (r=-0.80) and positively associated (r=0.50) with the ash content. There was negative and some correlation between the sample and Crude Fat (r=-0.80) while the sample is positive and strongly correlated with Crude Protein. Crude Fat shows a negative correlation and highly correlated with the sample while the ash content is positive and strongly correlated (r=0.84) with the moisture content of the sample.

Table 4: Correlation matrix of the nutritional composition of wet attiéké sample.

Results obtained on proximate compositions of food correlates with previous observations on ‘Attiéké’ [24]. A slight progression in moisture was recorded as the storage period increases as similarly reported, they opined that cassava dough and yam chips absorbed moisture during storage, and enhances the growth of fungi and other spoilage organisms [25-27]. The effect of moisture absorbed increases the water activity as a result of degrading activity of these fungi.

Furthermore, percentage crude fat, crude fibre observed decreased with storage period. This might be as a result of microbial degradation of nutritive substances in this food by certain fungi that coursed for their growth and development.

Lack of growth not until the third day could be the reductive effect of steam (heat) on the vegetative microorganisms as similarly reported, and not until then that emergence of spores that growth was recorded [28].

The variation in occurrence of fungi biomass across the samples could be as a result of different methods of handling which include processing and storage. Penicillium chrysogenum had been reported to be associated with damp or wet material which could be an indication of unhygienic water source [29]. Mucor hiemalis and Candida albicans observed in this study were also reported to be found from Cassava products such as; Attiéké, Fufu and Lafun. A. flavus and A. niger also characterized in this study were reported to be aflatoxigenic fungi found in Garri Aadun and in suya spices [30-34].

The aflatoxin concentrations across different locations were so high, though some authors reported a much higher level in dried cassava chips [35]. This could be due to deteriorated tubers predisposed to aflatoxin producing fungi which was similarly confirmed [36]. Also, the storage method of Cassava dough through refrigeration, left for days before final steaming (though the flavour, taste and color used to adjudge its quality were still pristine) also makes the food susceptible to fungal contamination and aflatoxin production. Studies in these locations further revealed that most of the production process involved in the making of Attiéké follows the usual traditional technologies. Sack container used in packing and pressing could be a reservoir of different fungal growth. The drying of Cassava dough on ground surfaces predisposes these to contamination from dust, fungi and other lamentable materials. Some reported that such practice enhanced association between the products and the soil which is the primary habitat of fungi [37]. Maize cobs were reported to have a much higher aflatoxin level when dried on ground though, this is worse in peeled cassava because the inherent protection in tuber will have been removed by peeling. Inherent protection in grain husks had been reported to safeguard rice and maize from aflatoxin contamination, fungi and weevils infestation [38-41].

The results of this study showed that major spoilage (biodeteriorating) fungi of Attiéké from Ejigbo, Iwo and Adjame in West Africa were mostly molds with Aspergillus niger and A. flavus having highest occurrence and Candida albicans and their percentage occurrence has direct effect on its food values. Obviously this food is distributed to other villages, towns, cities and countries notably Ife-Odan, Osogbo, Sekondi-Takoradi, this is an indication that if not properly managed consumers of this delicacy will be at risk of aflatoxicosis. Thus, modern technologies for hygienic storage mechanisms and proper sanitary measures are needed to be put in place. Adequate information concerning the level of fungi and aflatoxin contamination of this food and how to reduce the risks of exposure to aflatoxin during its processing and storage are needed to be conveyed to all level of society.