2.1 Plant material and Starter Organism
Cassava roots, both peeled and unpeeled, were collected from Sapele, Songhai Delta Amukpe, Nigeria. Skins were removed from tubers after thorough washing to achieve tubers without peels. Peeled and unpeeled tubers were cut into bits separately and dried to a uniform weight. The dried materials were milled and kept (37 °C) until additional assays were performed. The strains of R. oligosporus (produced by Aneka Fermentasi Industri. PT - Ragi dalam BANDUNG) were obtained from the Harmony Path Limited, Sapele Delta State. Solid state fermentation was carried out in petri plates for 72 hours at room temperature under acidic and basic conditions (pH 3 - 9) using 50 mM phosphate and citrate buffers.
2.2 Substrate Preparation for Solid State Fermentation
One gram (1 g) of R. oligosporus (1.4 x 102 CFU) (The fungus's colony forming unit (CFU) per gram was calculated using the method published by Ofuya and Nwajiuba (1990), was homogenized in 10 ml of prepared citrate and phosphate buffers ranging from pH 3 - 9 in seven different petri dishes which were labeled according to the corresponding pH. In the homogenization phase, 10 g of the ground peeled and unpeeled cassava roots were utilized; the they were allowed to ferment for 72 hours at 25 oC. An unfermented control (containing dried and grounded peeled and unpeeled cassava, devoid of any presence of molds; with buffer only, and without any cells) was prepared alongside the test samples. Following fermentation, 6 g of the mixture was removed from each of the petri dishes at the various pH levels; 40 ml of distilled water was added prior to homogenization using mortar and pestle; and 10 ml of homogenate was centrifuged at 3500 rpm for 10 minutes to get supernatant. The supernatant served as the crude extract or sample for the different tests, which were performed in triplicate.
2.3 Determination of antioxidant activities inhibition of 2,2-diphenyl-1- (DPPH) radical of peeled and unpeeled cassava tubers
The antioxidant activities of peeled and unpeeled cassava tubers were evaluated using the DPPH Assay. This was computed using the method provided by Hatano et al. (1988). A 2.8 ml methanolic solution of DPPH radical (6 × 10−5 mol/l) was put to 0.3 ml of extract. To get consistent absorption readings, the mixture was rapidly agitated and placed in the dim for 60 minutes. The absorbance at 517 nm was utilized to calculate the DPPH radical's reduction. Ascorbic acid was utilized as a control. The radical scavenging activity was calculated by the formula:
%RSA = ((ADPPH – AS)/ ADPPH) x 100.
Where %RSA = % DPPH discolouration; A absorbance of DPPH solution, and AS absorbance of the solution after a certain amount of sample was added
2.4 Determination of Total Phenol Content
This was done in line with the protocol outlined by Singleton and Rossi (1965). One milliliter of Folin C reagent was added to one milliliter of material. After 3 minutes, 1 ml of saturated Na2CO3 solution was added, followed by 10 ml of distilled water. For 90 minutes, the reaction mixture was maintained in the dark. At 725 nm, the absorbance was measured. Catechin was used as standard.
2.5 Determination of Total Flavonoid Contents
Colorimetric determination of total flavonoid contentswas done using the method of Jia et al. (1999). 250 µl of extract was combined with 1.25 ml of distilled water and 75 µl of 5% NaNO2. After 5 minutes, 150 l of 10% AlCl3.H2O, 500 µl of 1 M NaOH, and 275 µl of distilled water were added. The solution was thoroughly mixed, and the mixture's color intensity was measured at 510 nm. Catechin served as the standard.
2.6 Determination of Antigenotoxic activities
2.6.1 Preparation of Extracts
400g of fermented peeled and unpeeled cassava flour was measured at room temperature and placed in a basin with 120cl of valve water. This was left to saturate for 5 minutes before being agitated and the extract pressed and sieved through a cheese cloth. The leftover particles were thrown away.
2.6.2 Allium cepa Assay
In Sapele, Delta State, Nigeria, onion bulbs (Allium cepa L., 2n=16) of average size (15-22 mm diameter) were purchased locally. After six weeks of sun drying, to uncover the nascent meristematic tissues, with a fine razor blade, the dry roots at the base of the onion bulbs were meticulously scraped out. To keep the primodial cells from dehydrating, the bulbs were immersed in newly produced purified water. Taking into consideration the quantity of bulbs in the population are essentially slow or poor growers, each test sample and control received seven duplicate bulbs, and the best five bulbs were examined concurrently (Rank & Nielsen1993). Blotting paper was used to dry the bulbs.
To determine root growth inhibition, newly obtained stock extracts were watered into five concentrations of 20, 10, 5, 2.5, and 1%. For each concentration of each extract and the control, seven onion bulbs were used (tap water). For 72 hours in the dark, the bases of each bulb were hung on the extracts in 100mL beakers. The test extracts were refreshed on a regular basis. After the exposure time, the roots of the five onion bulbs that grew the quickest at each concentration were detached with forceps and their lengths (in cm) were measured using a metre rule. The percentage root growth inhibition in comparison to the negative control and the EC50 (effective concentration at which root growth equals 50% of the controls) for each extract were computed using weighted averages for each concentration and the control (Fiskesjo, 1985). The effect of each sample on the morphology of growing roots was also studied.
To test chromosomal aberration induction, 5 onion bulbs were suspended for 48 hours on 10, 5, 2.5, and 1 percent (v/v) concentrations of each extract and the control. After 48 hours, the root tips of these bulbs were stored in a solution of ethanol:glacial acetic acid (3:1, v/v). These were hydrolyzed in 1N HCl for five minutes at 60oC before being rinsed in distilled water. After pressing two root tips onto each slide for 10 minutes, they were coloured with acetocarmine and cover slips were carefully attached to exclude air bubbles. Grant (1982) suggested using clear fingernail paint to seal the cover slips to the slides. This is done to keep the preparation from drying out as a result of the heat generated by the microscope (Sharma, 1983).
Six slides were created for each concentration and the control, with five (at 1000 cells per slide) examined for chromosomal aberration induction at 1000 magnification. The mitotic index was calculated by dividing the total number of cells by the number of cells identified per 1000. (Fiskesjo, 1985; Fiskesjo, 1997) The proportion of abnormal cells was calculated by dividing the total number of cells examined at each concentration of each extract by the number of abnormal cells (Bakare et al., 2000).
2.7 Statistical Analysis
All statistical analyses were performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Values were reported as Mean ± Standard deviation and the experimental results were analyzed using analysis of variance (ANOVA) and also a Fischer test of least significance (LSD) was carried out to compare the various group means. The results were considered significant at p-values of less than 0.05, that is, at 95% confidence level (p< 0.05).