We examined the nutrient profile of akokono in two “unmixed” forms: raw and roasted. Additionally, three “mixed” akokono products were developed at the Kwame Nkrumah University of Science and Technology (KNUST): akokono flour, akokono-yam biscuit, and akokono-groundnut paste. The akokono-groundnut paste was chosen by the lead food technologist as the most promising food product for further testing and development as a potential child complementary food. Macronutrient composition, amino acid profile, fatty acid profile, mineral, and vitamin tests were conducted on these three forms of akokono as described below. Additionally, we measured the macronutrient and mineral composition of raw akokono fed two different types of feed materials, pito mash and palm pith, as well as that of the feed materials themselves.
Macronutrient composition, amino acid profile, fatty acid profile, and mineral tests (except for iron) were conducted at KNUST in Ghana (described below). All tests conducted at KNUST represent the mean of duplicate analysis. Eurofins Scientific performed all the vitamin tests and examined the iron content of raw akokono, roasted akokono, and akokono-groundnut paste; the Eurofins results represent the mean of triplicate analysis (described below). KNUST used identical macronutrient and mineral testing procedures on the akokono and the feed materials (pito mash, palm pith).
The akokono were bred in a warehouse in the Ashanti Region, fed with palm pith and sugar water, and procured from the Aspire Food Group facility in Kumasi. At the time of testing, akokono were between 28 and 35 days old (same development stage). Roasted samples were prepared by first pan-frying the akokono for 5 min followed by dry roasting at low heat for 15 min. Using a recipe created at the KNUST food technology laboratory, the akokono-groundnut paste was produced by mixing and then milling dry-roasted akokono (30%) together with local groundnuts (70%) and adding a small amount of canola oil (2 mL oil per 100 g paste) to improve the organoleptic properties of the final product. Finally, KNUST tested the akokono-groundnut paste to assess shelf-life stability, microbial safety, and chemical deterioration of the product at 0, 7, and 14 days as required by the Ghana Food and Drugs Authority (FDA). The safety data satisfied the Ghana Standards Authority (GSA) requirements for similar groundnut-based products (data not presented herein) . To test the feed materials, palm pith was harvested from purchased felled palm trees in the Ashanti Region, and the pito mash, a by-product of local pito (beer) brewing, was purchased from a local market.
Proximate (macronutrient) analysis
AOAC standard methods  were used to determine the proximate composition (moisture, ash, crude fat, protein, and fiber) of the akokono samples in duplicate. Significant differences between the macronutrient contents of the three forms of akokono were analyzed using one-way ANOVAs.
Amino acid profile
The amino acid content was determined by first digesting the sample for 24 h at 110 °C using 37% HCl. Pre-column derivatization was done using o-phthalaldehyde 3-mercaptopropionic acid and 9-fluroenylmethyl chloroformate according to the method described by Schwarz, Roberts, & Pasquali . An aliquot of 100 μL was injected into a Shimadzu high-performance liquid chromatography (HPLC) fitted with a Shimadzu 10AxL Fluorescence detector.
Mobile phase A was composed of 40 mM CH3COONa at pH 7.8 and mobile phase B composed of CH3CN: CH3OH: H2O (45:45:10 v/v/v) at a flow rate of 1 ml/min, passed through a Phenomenex column (3.5 μm, 4.6 mm ID, 15 cm) at 40 °C with run time of 60 min at the following wavelengths: Exitation: 340 nm; Emission: 450 nm.
A gradient elution program was used as described herein: 0 to 10 min (20% mobile phase B), 10 to 20 min (50% mobile phase B), 20 to 30 min (60% mobile phase B), 30 to 35 min (80% mobile phase B), 35 to 40 min (100% mobile phase B), and 40 to 60 min (20% mobile phase B). Samples were analyzed in duplicate, and results expressed as g amino acid per 100 g total amino acids.
Fatty acid profiles
The triglyceride profile was determined by HPLC. The analysis was carried out using a HPLC (Infinity Series 1260, Aligent Technologies, Germany) fitted with an auto sampler and a Refractive Index Detector. The Hypersil ODS C-18 column at ambient temperature was used with mobile phase of Acetonitrile: Acetone (37.5:62.5) at flow rate of 1.5. The elution program was isocratic. The purpose of this analysis was to characterize the fatty acid composition. Using mole ratio, the concentration of each component fatty acid was calculated for each triglyceride . All samples were analyzed in duplicate.
The mineral analysis of iron, calcium, magnesium, zinc, and copper was conducted with atomic absorption spectroscopy . The samples were dry-ashed in a muffle furnace at 550 °C for 6 h. The minerals were extracted from ash with 20 mL of 2.50% HCl and heated in a steam bath to reduce the volume to 8.0 mL, which was transferred quantitatively to a 50 mL volumetric flask and diluted to volume using deionized water. The extracts were stored in dry, clean plastic sample bottles, and the mineral concentrations were determined using an atomic absorption spectrophotometer. Potassium was determined with the flame photometric method  using a low temperature direct reading single channel emission flame photometer. Significant differences between the mineral contents of the three forms of akokono were analyzed using one-way ANOVAs.
Vitamin A was released from the sample by alkaline hydrolysis using ethanolic potassium hydroxide solution and extracted three times with hexane:ethylacetate (85:15 volume/volume %). The determination was carried out by normal-phase chromatography (NP-HPLC) with ultraviolet/diode array detector (DAD) at 325 nm (EN 12823–1:2014) .
Vitamin B1 (thiamine)
Thiamine was extracted from the sample in an autoclave using acid hydrolysis and quantified by reverse phase chromatography (RP-HPLC) coupled with a fluorescence detector (Ex.: 368 nm, Em.: 440 nm) after post-column oxidation to thiochrome (EN 14122:2006 with modification) .
Vitamin B2 (riboflavin)
Riboflavin was extracted from the sample in an autoclave using acid hydrolysis and quantified by RP-HPLC coupled with a fluorescence detector (Ex.: 468 nm, Em 520 nm) (EN 14152:2006 with modification) .
Vitamin B3 (niacin)
Niacin was calculated as the sum of nicotinic acid and nicotinamide. The two compounds were extracted from the sample in a mild hydrochloric solution at 100 °C, the pH of the extract was adjusted to pH 4.5 with sodium acetate followed by a filtration.
To determine vitamin B6, the European Union reference method for the determination of vitamin B6 in foodstuff (EN 14164–2008) was used with modification . Briefly, the sample was hydrolysed in an autoclave followed by enzymatic dephosphorylation, reacting with glyoxylic acid in the presence of Fe2+ as a catalyst to transform pyridoxamine into pyridoxal, which is then reduced to pyridoxine by the action of sodium borohydride in alkaline medium. Total pyridoxine was quantified by RP-HPLC with a fluorescence detector (Ex: 290 nm, Em: 395 nm).
Folate was extracted from the sample in an autoclave using a buffer solution, followed by an enzymatic digestion with human plasma and pancreas V, and finally by a second autoclave treatment. After dilution with basal medium containing all required growth nutrients, except folic acid, the growth response of Lactobacillus rhamnosus (ATCC 7469) to extracted folate was measured turbidimetrically and compared to calibration solutions with known concentrations .
Vitamin B12 was extracted from the sample in an autoclave using a buffered solution. After dilution with basal medium (containing all required growth nutrients except cobalamin) the growth response of Lactobacillus leichmanii (ATCC 7830) to extracted cobalamin was measured turbidimetrically. This was then compared to calibration solutions with known concentrations .
The samples were saponified in alcoholic potassium hydroxide solution and extracted with hexane: ethylacetate (85:15 v/v%). The extract was concentrated and cleaned up by solid phase extraction (EN 12821:2009) . The amount of vitamin D3 was determined by RP-HPLC with DAD (265 nm).
Vitamin E was released from the sample by alkaline hydrolysis using ethanolic potassium hydroxide solution and extracted three times with hexane:ethylacetate (85:15 v/v%). Quantification was carried out by NP-HPLC with a fluorescence detector (Ex/EM 290 nm/327 nm) (EN 12822:2014) .
Significant differences between the vitamin contents of the three forms of akokono were analyzed using one-way ANOVAs.
Ethics approval and consent to participate
This study was deemed not human subjects research by the PATH Research Determination Committee.