Material
The biological material used in this study was purchased at the “Abattoir” market in the city of Maroua, the regional capital of the Far North region of Cameroon. Pearl millet grains have been identified by the IRAD (Institute of Agricultural Research for Development) as Pennisetum glaucum species. Sweet potatoes (Ipomea babatas) used was of the yellow variety (TiB 1).
Preparation of sweet potatoes mash
Sweet potatoes were washed with tap water and then cooked in boiling water for 30 minutes. Once cooled, they were peeled and cut into small pieces. To 1 kg of sweet potatoes, 1.5 litres of water was added, followed by grinding in a domestic blender (SONASH, Dubai, UAE) until a smooth mash was obtained.
Preparation of the gruel
The Pearl Millet grains were washed with tap water and dried in the sun. After drying, they were sorted and then crushed using an IMEX brand mill, type Y132S1-2. Two hundred grams of this flour and 400g of peanut paste were mixed in 1L of water and the homogeneous mixture obtained was sieved using a 250µm diameter mesh sieve. The mixture was cooked for 20 min while homogenising and 220 g of sugar was added at the end of cooking. The gruel obtained was kept for the preparation of the various acidified samples.
Food-to-Food Fortification
The gruel was fortified with mashed sweet potato at the incorporation rate of 20, 30 and 40% (volume/volume). A part of fortified gruels was used for sensory evaluation and the other one kept for further analyses.
Sensory evaluation of fortified gruels
A sensory test was carried out with a panel of 30 housewives who were regular consumers of local cereals gruels. Each panellist had to rate the texture, colour, taste, and overall acceptability of the samples using a five-point hedonic scale, where 1 = very unpleasant, 2 = unpleasant, 3 = fair, 4 = pleasant, and 5 = very pleasant [25].
Chemical Analyses
Determination of carotenoids
Carotenoids contents were determined using AOAC [26] standard method. Briefly, 30 mL of a hexane-acetone 30/70 (v/v) solution were added to 1 g of sample dried at 45°C for 24 hours. The mixture was then heated for one hour and filtered after cooling. The filtrate was washed with distilled water in a separatory funnel; the lipid phase was transferred to a 25 mL volumetric flask and the volume was adjusted to the mark with hexane. The obtained solution was diluted 10 times with hexane and maximum absorbance was read between 430 and 450 nm using a spectrophotometer.
Determination of phytates
The phytate contents were evaluated according to the method of Gao et al. [27]. In a centrifuge tube, 0.5g of sample was introduced and mixed with 2.4% HCl to 10 mL. After shaking at 200 rpm for 16 h, the mixture was centrifuged at 1000 rpm at 10°C for 20 min. The crude extract was transferred to tubes containing a pinch of NaCl, shaked at 350 rpm for 20 min to dissolve the salt, and then incubated at 4°C for 60 min. The mixture was centrifuged at 1000 rpm at 10°C for 20 min. One milliliter of the clear supernatant was diluted 25 times with distilled water. To 3 mL of diluted sample, 1 mL of Wade's reagent (0.03% FeCl3.6H2O + 0.3% sulfosalicylic acid) was added then, mbefore mixing vigorously and centrifuging at 1000rpm at 10° C for 10 min. Absorbance was read at 500 nm.
Determination of polyphenols
Polyphenols were determined using the method described by Makkar et al. [28]. Dry samples (0.1 g) were placed in a 25 ml beaker and 10 ml of 70% acetone were added. The mixture was placed in a boiling water bath and stirred for 20 mLminutes. The contents of the beaker were centrifuged for 10 min at 3000 g and 4°C. in a tube, 200 µL of each supernatant was made up to 500 µL with distilled water. 250 µL of Folin-Ciocalteu reagent and 1.25 mL of 20% sodium carbonate solution were added to each tube. The extracts were shaked and stored in the dark for 40 min. The absorbance was read at 725 nm.
Determination of total ash, total iron and total zinc contents of fortified gruels
The total ash content of the gruels was quantified using the method proposed by AFNOR [29].
Total iron and total zinc contents were determined using Atomic Absorption Spectrophotometry [30]. Briefly, concentrated HNO3 acid was added to 0.5 g of finely ground sample, followed by digest for 4 hours on a block digester. The digestate was allowed to cool down and was dilute to 50 mL with deionised water. The supernatant was carefully transferred into centrifuge tubes for analysis on the Flame Atomic Absorption Spectrophotometry (Buck Scientific 200 Serie AA, East Norwalk, Connecticut, USA).
Preparation of simulated digestive fluids
The simulated digestive fluids (SSF: Simulated salivary fluid, SGF: Simulated gastric fluid, SIF: Simulated intestinal fluid) were as shown on Table 1, using Minekus et al. method, with slight modifications [31].
Table 1
Composition of simulated digestive fluids
| SSF | SGF | SIF |
pH 7 | pH 3 | pH 7 |
Constituents | Concentration (mmol.L− 1) | Volume in the fluid (mL) | Volume in the fluid (mL) | Volume in the fluid (mL) |
KCl | 0.5 | 30.2 | 20.7 | 13.6 |
KH2PO4 | 0.5 | 7.4 | 2.7 | 1.6 |
NaHCO3 | 1 | 13.6 | 37.5 | 85 |
NaCl | 2 | - | 35.4 | 19.2 |
Mg2SO4 | 0.15 | 1 | 1.2 | 2.2 |
(NH4)2SO4 | 0.5 | 0.12 | 1.5 | - |
Oral digestion
Five grams of gruel was mixed with 5 mL of salivary α-amylase solution (150 U.mL− 1) obtained by mixing 1 g of α-amylase (86250 Sigma-Aldrich Chemie Gmbh) with 0,25 mL of 0.3 M CaCl2 and SSF electrolyte solution to reach a final volume of 10 mL. The mixture was homogenised for 2 minutes at 37°C.
Gastric digestion
Ten milliliters of oral digestate was mixed with 7.5 mL of SGF electrolyte solution, 1.6 mL pepsin solution of 25 000 U.mL− 1 made up by mixing 0,1 g of pepsin (P7125 Sigma-Aldrich Chemie Gmbh) with 5 mL of 0.3 M CaCl2, and 0.695 mL of distilled water. The mixture was acidified with HCl 1 M to reach pH 3.0 and incubated during 2 hours at 37°C under gentle regular shaking.
Intestinal digestion
Twenty milliliters of gastric digestate was mixed with 11 mL of SIF electrolyte solution, 5.0 mL of a pancreatin solution 800 U.mL− 1 obtained by mixing 0,42 g pancreatin (P7545 Sigma-Aldrich Chemie Gmbh), 1,25 g bile extract (B8631 Sigma-Aldrich Chemie Gmbh), 40 µL of 0.3 M CaCl2 and 50 mL of SIF. One molar NaOH solution was added to reach pH 7.0 and the mixture was incubated during 2 hours at 37°C under gentle regular shaking.
The final digestates obtained were placed in ice until cooling to stop enzyme activities, and then filtered using Macherey Nagel MN 640d filter paper. The filtrates were then used for determining the minerals contents using Flame Atomic Absorption Spectrophotometry.
Statistical analyses
The results obtained were subjected to analysis of variance (ANOVA) using IBM SPSS Statistics software version 19.0.1. Differences between means were tested by Duncan's multiple range comparison with a significance level of 5%.