## 2 Materials and Methods
### Methods
#### 2.2.2 Determination of Crude Protein, In Vitro Protein Digestibility (IVPD), and Vitamin C  
The crude protein was determined using the AOAC [18] Official Method 991.20. A 2 g sample was added to the digestion flask. A total of 1 g of copper sulfate, along with sodium sulfate (catalyst) in the ratio 1:10, respectively, and 5 mL of concentrated sulfuric acid were also added to the digestion flask. The flask was positioned into the digestion block in the fume cupboard and heated until frothing ceased, giving a clear and light blue-green colouration. The mixture was then allowed to cool and diluted with 30 mL of distilled water, and 30 mL of 40%NaOH was added. The distillation apparatus was connected. The ammonia released by boric acid was then treated with 0.01 mL of hydrochloric acid until the green colour changed to purple. The percentage of nitrogen in the sample was calculated using the following formula:  
\[\%\text{Nitrogen}(\text{N})\ =\ \frac{\text{VHCl}\times\text{NHCl}\times 14}{ \text{ample weight}}\times 100\]  
Note: volume of HCl consumed to the endpoint of titration (V), normality of HCl used is about 0.01 N(N), and molecular weight is that of nitrogen [16]. Conversion of nitrogen percentage into % protein = F\({}^{\circ}\)N, where F is the conversion factor (in most cases, 6.25).  
The IVPD was determined using the method of [PERSON] et al. [19]. About 100 mg of the fermented cassava root flour was measured into a 50 mL centrifuge tube. A total of 35 mL of 0.1 M phosphate buffer containing freshly prepared repsin at a concentration of 1.5 mg/mL was introduced into the tube. The sample was then incubated in a shaking water bath at 37 \({}^{\circ}\)C for 2 h to allow enzymatic digestion. Following the incubation, digestion was halted by the addition of a 2 M NaOH solution. The resulting suspension underwent centrifugation at 4800 rpm for 20 min at 4 \({}^{\circ}\)C. The supernatant was removed, and the remaining pellet was rinsed with 15 mL of 0.1 M phosphate buffer (pH 7). Another round of centrifugation was performed, after which the pellet was separated by filtration using Whatman No. 3 filter paper, with the filtrate being discarded. The residual undigested protein on the filter paper was then dried at 80 \({}^{\circ}\)C for 2 h, and its protein content was quantified using the Dumas combustion method.  
The quantification of vitamin C (L-ascorbic acid) was conducted using the dye-titration technique, as outlined in AOAC 967.21. The ascorbic acid content in fermented cassava root flour samples was assessed following the AOAC [18] protocol. Approximately 20 mg of cassava root flour was diluted to a final volume of 50 mL using 0.1 M oxalic acid, which acted as a metal chelator, and the mixture was subsequently filtered. A 5 mL aliquot of the filtrate was transferred into a beaker using a pipette and titrated against a standardized 2,6-dichlorophenol indophenol dye solution. The endpoint of the titration was indicated by a color change from orange to pink. The titration procedure was performed in triplicate, and the obtained titer values were used to calculate the ascorbic acid concentration, expressed as mg per 100 g of fermented cassava root flour.  
Determination of antinutritional factors of the cassava root flour. The antinutrients phytate, hydrogen cyanide (915.03), and trypsin inhibitors in the cassava root flour were determined using the AOAC rapid test method [18].