Proximate and antioxidant activities of bio-preserved ogi flour with garlic and ginger

Materials

Chemicals and materials used in this study were of analytical grade namely; 1,1-diphenyl-2-picrylhydrazyl radical (Sigma Aldrich, Germany, D9132), Gallic acid (Sigma Aldrich, Germany, G7384), Quercetin (Sigma Aldrich, Germany), Folin Ciocalteau phenol reagent (Sigma Aldrich, Germany, F9252), Aluminum chloride (BDH, England,101084), potassium acetate (Sigma Aldrich, Germany, 791733-500G), sulfuric acid (38308-1EA), Ethanol (BDH, England, BDH1156-4LP), phenolphthalein (Sigma Aldrich, 74760-100ML), sodium hydroxide (Sigma Aldrich, 38227 1EA), hexane (Sigma Aldrich, Germany, C100307-2.5L) Boric acid (Sigma Aldrich, Germany,38750-1EA), Hydrochloric (Sigma Aldrich, Germany, 382801EA), Whatman No.1 filter paper (28413923) supplied by Finlab Nigeria Limited and Equilab Business solution Limited Nigeria.

Powdered garlic and ginger preparation

Two hundred and fifty (250) grams of garlic bulbs and ginger rhizomes that were freshly harvested were washed, drained, peeled, diced into cubes, and dried at 65 °C for 12 h using hot air oven (Gallenkamp, UK). They were then ground using a grinder (Marlex Appliances PVT, Mumbai, India). Powdered garlic and ginger passed through a sieve (60 µm) (BS mesh sieves, Dual manufacturing Co. Chicago, USA) for removal of residues¹³.

Ogi preparation co fermented with garlic and ginger

Quality protein maize (ART/98/SW06/OB/W) was obtained from the Institute of Agricultural Research and Training (I.A.R.T.), Ibadan, Nigeria, while sorghum was procured from a local market in Ile – Ife, Osun State, Nigeria. 15 kilogram of grains were examined, winnowed, and steeped separately for 3 days. Milling into smooth paste after draining of the grains was done with an attrition mill (No 1 Premier mill, England). Powdered garlic and ginger were added for co-fermentation to smooth paste of maize/sorghum at 2 and 4% (w/w) garlic or ginger singly and in combination (ginger-garlic), which resulted into 7 conditions. The samples were labeled A-H as follows: A: control samples (without garlic/ginger); B: Ogi + 2% Garlic; C: Ogi + 4% Garlic; D: Ogi + 2% Ginger; E: Ogi + 4% Ginger; F: Ogi +2% Garlic + 2% Ginger; G: Ogi +2% Garlic + 4% Ginger; H: Ogi + 4% Garlic + 2% Ginger. The slurry was evenly homogenized, then allowed to ferment spontaneously (naturally) at ambient temperature (27± 2°C) for 24 h. After fermentation the water was pressed in muslin cloth to form an ogi cake¹⁷.

Preparation of biopreserved ogi flour

Ogi cakes were dried for 48 h at 42± 2°C with a cabinet dryer (Gallenkamp, UK), which was then grounded into flour, cooled for 5 min at room temperature, then packaged in a pouch and sealed. The packaged samples were stored at room temperature for 16 weeks during which samples were obtained for analysis at 4 week intervals (monthly). Ogi flour samples were then placed on a shelf for further analysis¹⁸.

Determination of titratable acidity and pH

The total titratable acidity (TTA) of ogi flour was determined for all samples to quantify the acid produced during sample storage. 1g ogi flour was reconstituted in 10 ml of distilled water. Three drops of phenolphthalein was added as indicator; then titrated against 0.1M NaOH while gently swirling the content in the conical flask until pink colour appeared. Each ml of 0.1N NaOH used was equivalent to 90.08 mg of lactic acid. Titration readings were taken in triplicate and mean values of the readings were calculated. Total titratable acidity of lactic acid (g/ml) was calculated. A pH meter (Corning Scholar 425, UL Laboratories, Shenzhen, China) was used to determine pH values of the 5g of reconstituted flour in 50 ml of distilled water. Buffer at pH 4.0 and 7.0 were used to calibrate the pH meter. The pH of all the samples were read after stabilization of the value on the apparatus screen, the pH values were recorded in triplicate and mean values of the reading was calculated¹⁹.

Determination of proximate composition and mineral content

Moisture content determination. 5 g of each sample was weighed in triplicate into pre-weighed moisture content cans. The samples were dried for 3 h at 105 °C in the Gallenkamp hot-air oven (Gallenkamp, UK) and the weight was taken. The drying continued until their weights were constant. The samples were cooled to room temperature in a desiccator and weighed. The final weight of each sample was determined¹⁹. The moisture content was calculated from weight loss equation below

Moisture content = $\frac{w1-w2}{w1}\times 100$ (%)

w₁= Weight of sample before drying (g)

w₂= Weight of sample after drying (g)

Crude protein determination. 2 g of ogi sample was weighed into a digestion flask. Kjeltec catalyst 31835-2501AE (0.8 g) and 15 ml of concentrated sulphuric acid was added to each flask. Each flask was heated on pre heated digester set (K12, Behr LaborTechnik, Germany) at 420 °C in a fume cupboard, and digested until a clear homogenous mixture was obtained. After digestion, the flask was removed from the heater, cooled, and the content was diluted with 50 ml of distilled water. The flask was then placed in micro-kjedahl analyser (Kjelmaster K-375, Buchi, Switzerland) where it received 50 ml of NaOH automatically. The mixture was subsequently heated up to release ammonia which was distilled into a conical flask containing 25 ml of 2% (w/v) boric acid as an indicator for 4 min, the ammonia reacted with boric acid to form ammonium borate which was titrated against 0.1M hydrochloric (HCl) acid until the purplish – grey end point was attained. The percentage nitrogen content of the samples was calculated using the equation below:

Nitrogen = $\frac{A\times M\times 0.014}{weightofsample(g)}\times 100$ (% g)

where A= 0.1 HCl (ml)

Crude protein content was estimated by multiplying with the factor 6.25 (The protein content in food is estimated by multiplying the determined nitrogen content by a nitrogen-to-protein conversion factor 6.25 as the standard. AOAC, 2010). The experiment was carried out in triplicate and the means for each sample were recorded

Crude fat content determination. Fat content of all the ogi samples was determined by a continuous extraction liquid – solid method using soxhlet extractor with a reflux condenser and a distillation flask (E914, Buchi, Switzerland). Each sample (2 g) was weighed into a fat free thimble plugged with cotton wool and placed in the appropriate chamber of the extractor. The distillation flask was filled to two third capacities with n-hexane (60–80 boiling points); the flask was boiled on a heating mantle; the distillate was collected. Thereafter, n-hexane was recovered into a clean container until almost all had been distilled. The remaining solvent in the mixture was evaporated in a Gallenkamp hot-air oven (Gallenkamp, UK) set at 70 °C. The flask was allowed to cool subsequently in a desiccator (PYREX, Corning, Inc USA after which the final weight of the flask was determined. The difference in the final and initial weight of the distillation flask represented the oil extracted from the sample¹⁹.

The percentage of crude fat was obtained using the equation below:

Fat = $\frac{Finalweightofflask-initialweightofflask}{weightofsample(g)}\times 100$ (%)

The experiment was carried out in triplicate for each sample.

Crude fibre determination. The crude fibre was determined using the weighed samples resulting from fat extraction. Each sample was transferred into conical flask and 100 ml boiling 1.25% H₂SO₄ added. Each beaker was heated for 30 min with periodical rotation to prevent adherence of solids to the sides of the beakers. The solution was filtered using Whatman No.1 filter paper (28413923) and rinsed with 50 ml portions boiling water; repeated trice then dried. Boiling 1.25% (w/v) NaOH solution (200 ml) was added and the mixture was boiled for 30 min after which the contents of each beaker was removed and filtered; washed with 25 ml boiling 1% sulphuric acid, three portions of 50 ml boiling water and 25 ml ethanol. The residue was dried at 100 °C to a constant weight followed by cooling in a desiccator at room temperature and weighed. The weighed residue was ignited at 600 °C in a Gallenkamp muffle furnace (Gallenkamp, UK) for 30 min, cooled in a desiccator and reweighed¹⁹.

The percentage crude fibre in each sample was calculated as:

Crude fibre = $\frac{w2-w3}{w1}\times 100$ (%)

W₁ = Weight of sample (g)

W₂ = Weight of crucible + sample (g)

W₃ = Weight of crucible + Ash (g)

The experiment was carried out in triplicate for each sample and the average calculated for each sample.

Total ash determination. The total ash (inorganic residue from the incineration of organic matter) was determined by dry ashing procedure. The samples (2 g) were weighed into a preweighed dry porcelain crucible. The samples were incinerated in a Gallenkamp muffle furnace (Gallenkamp, UK) at 550 °C for 6 h. After ashing, the remains were removed from the furnace, cooled to room temperature in a desiccator and weighed¹⁹. The porcelain crucible was weighed and the % total ash weight was obtained by using the equation below:

Total ash = $\frac{weightofash(g)}{weightofsample}\times 100$ (%)

Carbohydrate determination. The carbohydrate content was determined by difference. The sum of the moisture, ash, crude fiber, fat and protein of the respective samples was subtracted from 100 to obtain percentage carbohydrate¹⁹.

Determination of mineral content

The amount of minerals present in the sample was determined as described by AOAC¹⁹. The ash of the sample obtained was dissolved in 10ml of 2M HNO₃ and boiled for 5 min, filtered through Whatman No.1 filter paper into volumetric flask. The filtrate was made up with distilled water to 50 ml and used for determination of minerals content. Zinc (Zn), Manganese (Mn), Magnesium (Mg) and Iron (Fe) were determined by using Atomic Absorption Spectrophotometer (AAS 220GF, Buck). The standard curve for each mineral was prepared from known concentrations of mineral and the mineral content of the samples was estimated from the standard curve, while sodium and potassium content were determined using Jenway flame photometer PFP7 (Cole-palmer, UK).

Estimation of total phenolic content

Total phenolic content was determined using the modified procedure from Olaniran et al.¹⁶. Extracts (0.1 ml) of ogi flour samples were pippeted into 5.9 ml distilled water; afterward, 1.0 ml Folin Ciocalteau reagent was added to 1.0 ml of the diluted extract in test tubes. The mixture was left for 5 min before addition of 2 ml of 20% (w/v) Na₂CO_3. After 30 min of rigorous mixing was done with a vortex mixer, absorbance was taken at 725 nm using a spectrophotometer (Model SP9, PyeUnican UK). The results were expressed as Gallic acid equivalent (GAE) using a calibration curve with Gallic acid as standard (100 mg/ ml) y = 0.0022x - 0.0292, R² = 0.9962.

1, 1-diphenyl-2-picrylhyrazyl (DPPH) radical scavenging activity of ogi flour extract

The free radical scavenging ability of ogi flour extracts using α, diphenyl-β-picrylhydrazyl (DPPH) were carried out following Pownall et al.²⁰. 1 mL of 0.3mM DPPH dissolved in ethanol in different test tubes was added to different concentrations of 1 mL of the aqueous extracts of each of the samples. The tubes were then shaken vigorously and allowed to stand for 30 min at room temperature in the dark. A control was also prepared as mention previously without the addition of the sample. Absorbance of the samples was measured at 517 nm using a UV-VIS spectrophotometer (Model SP9, PyeUnican UK) to record the changes. Free radical scavenging ability was expressed as 50% maximal radical inhibition concentration (DPPH IC₅₀).

Determination of total flavonoid content

The total flavonoid content (TFC) of ogi flour extract was determined following Lamien et al.²¹. Ogi flour extracts (0.5 ml) had ethanol (0.5 ml), 50 μl of aluminum chloride (10%), potassium acetate (50 μl), and water (1.4 ml) added to them, and then were incubated for 30 min at room temperature. The absorbance of the reaction mixture was read at 415 nm using spectrophotometer (Model SP9, PyeUnican UK). 0.01 g quercetin dissolved in 20 ml of ethanol was used to prepared standard quercetin solutions (y = 0.001x + 0.0018, R² = 0.992). The quantity of flavonoids present in the extracts was expressed as quercetin equivalent (QE). The quercetin solution without sample solution was used as positive control due to it’s a polyphenol content. All determinations were carried out in triplicate.

Statistical analysis

The means were calculated and separated using MS Excel 2010 and SAS 9.4 version (2014) respectively. Means were separated with Duncan Multiple Range Test (DMRT) at 5% level of probability.