Assessment of the nutritional quality of weaning food prepared from fermented blends of sorghum (Ogi) and soybean

This study investigated the nutritional quality of weaning food prepared from fermented blends of sorghum (ogi) and soybean. The raw samples were processed into flour and were mixed in ratio of 60:40 and 70:30 respectively. Lactic acid bacteria (LAB) were isolated from fermented ogi and were accessed for their technological properties. The pH and total titratable acidity of the ogi was accessed, proximate composition and shelf-life of the weaning blends were determined by standard methods. A total of 10 LAB isolates were obtained, characterized and identified as L. plantarum (50%), L. fermentum (30%), L. delbrueckii (10%), and L. brevi (10%). Lactic acid, diacetyl and hydrogen peroxide produced by the LAB isolates ranged from 1.203 ± 0.006 - 3.410 ± 0.010, 1.21 ± 0.02 - 3.63 ± 0.05 and 0.650 ± 0.060 - 1.910 ± 0.040 (mg/mL) respectively. The fermentation of the ogi resulted in pH reduction from 4.5 ± 0.01-2.40 ± 0.01 and increased titratable acidity 12.49 ± 0.01 - 23.00±0.01 g/L. The 60:40 weaning blend of sorghum ogi flour and soybean improved protein, ash, crude fiber, moisture, fat, vitamin A, ascorbic acid, riboflavin, and beta carotene content compared to a 70:30 blend. The blend also showed improvements in mineral content, with a significantly higher overall nutritional value compared to the 70:30 blend. Coliforms were not observed during the shelf-life monitoring. This study concluded that these blends meet daily nutritional requirements, provide health benefits, and meet recommended standards for weaning.


Introduction
Weaning involves gradually introducing foods other than breast milk into a baby's diet to provide basic nutrients and support the transition to adult diets.Breast milk is often insufficient for rapid growth after six months, so appropriate weaning foods are introduced to supplement it and support the child's development [1,2].Complementary feeding is recommended between 6-24 months of age [3], containing balanced nutrients like 25-50% protein, copper, riboflavin, thiamine, calcium, manganese, and 75-100% phosphorus, zinc, and iron [4,5].
Commercial complementary foods, often associated with exclusivity and luxury, are popular among high-income groups, perpetuating inequality and marginalizing lower-income individuals, hindering true food equality for all by focusing on brand names and price tags [6].To address this, weaning foods from local, readily available raw materials can be adopted to meet nutritional requirements and promote food equality.
Ogi is a popular meal commonly used as a complementary food for infant in Nigeria.It is prepared from cereal grains such as (Sorghum bicolor), millet (Pennisetum americanum) and maize (Zea mays).The traditional preparation of ogi involves soaking grains in water for 72 h at 28 ± 2°C and milling into a smooth paste.The slurry obtained is then sieved using a muslin cloth to remove the bran, germ and hull.The filtrate undergoes secondary fermentation for 24-72 hours to develop its sour taste [7,8], aided by lactic acid bacteria and yeasts, which improves nutritional value, organoleptic properties, shelf life, and food preservation [9].However, ogi is low in nutritional quality due to the absence of essential amino acid such as lysine, threonine, and tryptophan, crucial for infant growth and development [10].This loss is attributed to processing techniques during preparation.[11].To improve its nutritional value, studies have focused on enriching ogi gruel with legumes like soy-beans, as a strategy to enhance the cereal meal's nutritional requirements [12,13,14].
Soyabean, a popular protein source, is expected to become the primary protein source in the future to address Protein Calorie Malnutrition (PCM), as animal-based protein is too expensive for many people, and to bridge the growing gap between protein demand and availability [15].Soybean, a nutritious staple food, is gaining popularity due to its health benefits, including plant-based protein, essential amino acids, fiber, antioxidants, and phytochemicals, making it a beneficial addition to a balanced diet used in milk, oil production, livestock feeds, and industrial applications [16,17].
Sorghum and soybean combination prevents malnutrition during weaning, as cereals lack essential nutrients.Sorghum provides carbohydrates and energy, creating a balanced diet for children transitioning from breastfeeding to solid foods, establishing healthy eating habits early and reducing long-term malnutrition risk [18].The study aimed to develop affordable, nutritious complementary foods using processed sorghum and soybeans through fermentation and roasting techniques, enhancing nutrient availability and making these foods easily prepared in rural areas.

Collection of samples
Sorghum (Sorghum bicolor L), and brown soybean (Glycine max) were purchased from Oja-Oba market, Ibadan, Oyo state, Nigeria.The samples were collected in a separate sterile polythene bag and transported to the Microbiology and Biotechnology Laboratory of First Technical University, Ibadan.

Preparation of soybean
Two hundred grams (200 g) of soybean were roasted in an oven (3606 Thermo Oven Lab-line Vacuum) at 100 o C for two hours.The roasted soybean seeds were dehulled by hand to remove the seed coat and milled into powder using a clean electric grinder (VTCL Excella Grinder-1000W), sieved through a fine mesh of 0.5mm pore size, and stored at room temperature in an airtight plastic bucket (Figure 1) [19]

Preparation of sorghum
Two hundred grams (200g) of sorghum was steeped in 5 liters of distilled water at room temperature (28 ± 2 o C) for 96 hours.The steeped sorghum was wet-milled to remove bran, hulls, and germ (Electric Grain Grinder 3000W) and sieved with a clean muslin and allow to ferment for 48 hours at (28 ± 2 o C).The steep corn water was decanted, and the sorghum ogi was packed in a clean muslin bag, tied to release residual water, and left to drain for about 18-24 hours.The drained sorghum ogi was collected and oven dried at 55 o C for 72 hours (3606 Thermo Oven Lab-line Vacuum), dried milled into ogi powder using an electric grinder (VTCL Excella Grinder-1000W), sieved through a sieve a fine mesh of 0.5mm pore size, and stored in an airtight plastic bucket at temperature 4 o C (Figure 1) [19].

pH and titratable acidity (TTA) of the fermented sorghum ogi
The pH and titratable acidity were determined using the AOAC [20] method, with samples taken at intervals of 24 hours to 96 hours using a digital pH meter (HANNA INSTRUMENT 8021).The total titratable acidity of 25 mL of ogi samples was determined by titrating them with 0.1N NaOH until a pink color appeared with three drops of phenolphthalein (1 % w/v) as an indicator.

Isolation of lactic acid bacteria
One gramme (1 g) of the sorghum ogi slurry was aseptically taken and subjected to microbiological analysis added to 90 ml sterile 0.1% peptone water (Oxoid, UK) and homogenized by using vortex machine (CM-101 Remi Cyclo Mixture, 1000 RPM) for 10 seconds.The homogenate was further diluted to ten-fold serial dilution.Dilution 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 were pour-plated in sterile De Man Rogosa Sharpe (MRS) agar plates (Oxoid, UK) in duplicates and incubated anaerobically in Gas Pak jars (GasPak System, BBL) at 30 °C for 48 hours.Representative colonies were randomly picked and purified by repeated streaking on MRS agar plates.Pure cultures were grown on MRS agar slants and kept at 4 o C for further use [21].

Lactic acid production
The AOAC [20] method was used to estimate lactic acid production by titrating 25 ml of 24-hour-old broth cultures with 0.1 N NaOH, using phenolphthalein as an indicator.The titratable acidity was calculated using the provided formula.

Diacetyl production
The LAB isolates' diacetyl production was estimated using the AOAC [20] method.A 7.5ml hydroxyl amine solution was added to 25ml of 24-hour old MRS broth culture, and the flasks were titrated with 0.1N HCl to a green-yellow end-point using bromophenol blue as an indicator.Diacetyl was calculated using the formula as provided: AK=Percentage of diacetyl, B=ml of 0.1N HCl consumed in titration of the sample, E=Equivalent factor of 1 mL of 0.1N HCl to diacetyl=21.52mg,W=Volume of sample used, S= Volume of ml 0.1N HCl consumed in titration of 7.5 mL Hydroxyl amine.

Hydrogen peroxide production
Twenty milliliters (20 mL) of 0.1 M H2SO4 were added to 25 mL of the MRS broth cultures of the test isolates (24 h).Titration was carried out with 0.1 N potassium permanganate.Each mL of 0.1 M H2SO4 is equivalent to 1.70 mg of Hydrogen peroxide and decolorization of the sample was regarded as an endpoint [20].

Growth at different pH and salt concentration (4% NaCl)
De Man Rogosa Sharpe (MRS, Oxoid) broth was adjusted to pH 3, 7 and 12 using 1N HCl and 5N NaOH and 4 % NaCl, and sterilized by autoclaving at 121 °C for 15 minutes.The LAB isolates were inoculated into the MRS broth and incubated anaerobically in Gas Pak jars (GasPak System, BBL) at 30 °C for 48 hours The medium's turbidity was measured at 600nm, while uninoculated broth served as a control.

Formulation of the weaning foods
Two composite weaning blends were formulated using varying ratios of sorghum ogi flour and soy-bean flour in 60:40 and 70:30 (Figure 1).This combination was chosen based on each ingredient's predicted nutrient content, protein content, and energy density in order to meet the acceptable WHO-recommended nutrient intake for infants.The moisture content of the weaning blend samples was determined by drying clean crucibles in a hot air oven at 100 °C for 1 hour, then cooling in a desiccator.Two grams of each sample were then weighed and dried at 100°C until a constant weight was obtained [28].
W1 = Initial weight of the empty crucible; W2 = weight of dish + sample before drying; W3 = weight of dish + sample after drying

Determination of fat
The Soxhlet extraction method was used to determine fat content of the weaning blend samples, following AOAC's guidelines [28].A Soxhlet extractor with a reflux condenser and a 500 mL round bottom flask was used.Two grams of sample were weighed into a labelled thimble, and 300 ml of petroleum ether was filled into the flask.The extractor thimble was sealed, refluxed for 6 hours, and collected.Petroleum ether was dried at 105 o C for 1 hour and oven-cooled before weighing.

Determination of crude protein
The micro-Kjeldahl method was used to determine protein percentage in the weaning blend samples.A gram of each sample was weighed into a Kjeldahl flask, and 2.5 g of anhydrous Na2S04, 0.5 g of CUSO4, and 5 mL concentrated H2S04 were added.The flask was heated in a flame chamber, then transferred to a volumetric flask.The digest was distilled, mixed with 5 mL boric acid indicator and 3 drops methyl red, and titrated against 0.01 N HC1, resulting in a purplecolored endpoint.[28].The percentage protein was calculated using the following expression.The AOAC [28] procedure was used to determine ash content in the weaning blend samples.Two grams of each sample were weighed, ignited, and cooled before being transferred to a muffle furnace at 550°C.After 8 hours, the sample was moistened, dried, and re-ashed at 550 °C for an additional hour.The percentage of ash was calculated using the following expression.

Determination of crude fiber
The AOAC [28] method was used to determine crude fiber in the weaning blend samples by boiling two grams of each sample in 200 mL 1.25 % H2SO4 for 30 minutes, filtering through cloth, washing with water, returning to 200 mL NaOH, washing with 1% HCl, draining, and drying.The residue was then transferred to a silica ash crucible, dried, and cooled.Percent crude fiber was calculated using this method.

Determination of carbohydrate
The total carbohydrate content was estimated as the difference between 100 and the total sum of moisture, fat, protein, crude fiber, and ash as described by AOAC [28]

Determination of minerals and Vitamins
The mineral contents including sodium, potassium, iron, zinc and phosphorus content of the weaning blends were analyzed using an X-ray spectrometer (XRS) using a MiniPal 4 Version PW 4030 X-Ray Spectrometer (Perkin Elmer, Inc., USA).The samples were dried at 110°C, weighed, and mixed with a binder (PVC dissolved in Toluene) to absorb the primary beam.A turbulent mixer was used to homogenize the samples.Pellets of 1.5g were produced using a turbulent mixer at a pressure of 7182 mmHg.The pellets were loaded into the X-ray spectrometer chamber at a maximum voltage of 30 kV and a maximum current of 1 mA.A preset time of 10 minutes was used for each sample, and an inbuilt Si (Li) detector was used to count secondary X-rays.The X-ray spectrum was analyzed using a computer running a dedicated MiniPal Analytical Software.Riboflavin, thiamine, niacin and ascorbic acid were determined using standard procedures as described by AOAC [28].

Shelf-life evaluation of the weaning blends
Shelf-life monitoring was evaluated for three months, with one gram (1g) of each weaning blend taken aseptically and subjected to microbiological analysis added to 9 mL sterile 0.1 % peptone water.Total bacteria and coliform count were examined by inoculating the dilution 10 -2, 10 -4 and 10 -6 into Nutrient agar and MacConkey agar plate respectively, and incubated at 37 o C for 24-48 hours.The plates were observed and colonies were enumerated and expressed as cfu/mL.

Statistical analysis
Results were obtained in triplicates and presented as means with standard deviation and was subjected to one-way analysis of variance (ANOVA) using Statistical Package for Social Sciences (SPSS version 16.0].Significant differences between means were determined at 95 % confident limit (p< 0.05) and were compared using Duncan multiple range test.

Identification and Technological properties of lactic acid bacteria isolates
Ten (10) lactic acid bacteria isolates were obtained, characterized and identified as L. plantarum (50%), L. fermentum (30%), L. delbrueckii (10%), and L. brevis (10%) (Figure 2).L. plantarum was found to be the predominant isolates obtained in this study.The predominance of L. plantarum among the LAB species has also been reported in previous studies on spontaneously fermenting of cereals (Adeyemo and Onilude, 2014;Sharma et al., 2020).This may be due to the limited nutritional requirement of L. plantarum, which is advantageous for metabolism of several substrates compared to most other species of Lactobacillus [29].
The highest amounts of lactic acid, diacetyl, and hydrogen peroxide were produced by L. plantarum SS3 and L. plantarum SS6.The production of lactic acid lowers the pH of the fermenting medium and produces bioactive inhibitors like diacetyl and hydrogen peroxide, which are primarily responsible for the antimicrobial activity [30].Tannock [31] linked production level and proportion to strains, medium compounds, and physical parameters, so the different antimicrobial compound concentrations observed could be the result of different LAB strains producing them.

pH and TTA of the fermented sorghum ogi
The fermentation of sorghum ogi over 96 hours resulted in a pH reduction of 4.50 ± 0.01 -2.40 ± 0.01 and an increase in Total Titratable Acidity (TTA) from 12.49 ± 0.01-23.00± 0.01 (Figure 3).This decrease in pH values is likely due to the production of organic acids by lactic acid bacteria [34), which eliminate undesirable microorganisms; a desirable quality of a weaning food.The degree of acidification observed is comparable to the values reported by Adeyemo et al. (19).

Proximate, vitamins, and mineral composition weaning blend samples
The proximate composition of the weaning blends is presented in Table 3.The weaning blend of sorghum ogi flour combined with soybean (60:40) had an improved proximate value of protein, ash, crude fiber, moisture content and fat (p<0.05) of 26.882 ± 0.044, 2.5.3±0.577,0.590 ± 0.001, 9.333 ± 0.256 (%) respectively.The increased crude protein content of weaning blends is due to a combined ratio of soybeans and sorghum, which meet the Codex Alimentarius Standards [12] for complementary/weaning foods of 14 -30g between 6 months and 5 years for infant diets.Protein supplementation can improve the quality of cereals protein, as observed by traditional evaluation methods.[35,36,37,38,39,40).The values of crude fiber obtained for the weaning blends are within the Protein-Calorie Advisory Group's recommended range (PAG) (5 -8%) [41].It is advised that infants consume a low-fiber diet because fiber-rich foods can be filling and may hinder nutrient absorption CDC [42].The moisture content of the weaning blends is within the recommended values for weaning/complementary foods Less than 10% moisture content is recommended for better keeping quality.Moisture content is an important determinant of shelf-stability of foods.Low moisture content implies low water activity, which prevents micro-organism from acting freely on formulations [43].The weaning blends' carbohydrate contents for the 60:40 and 70:30 ratios were 55.695 ± 0.780 and 61.344 ± 0.810 (%) respectively.The carbohydrate content may be attributed to the sorghum grain's amylose and amylopectin components [44].
Carbohydrate is most significant and easily accessible source of energy.Additionally, they play a crucial role in maintaining healthy immune, nervous, digestive, and heart systems [45].The results obtained in this study are consistent with the findings of Wakil and Onilude [46], Elemo et al. [47].Weaning blends of sorghum ogi combined with soybean (60:40) had improved vitamin A, ascorbic acid, riboflavin, and beta carotene contents (P<0.05) of 0.471 ± 0.001, 6.100 ± 0.755, 0.213 ± 0.015 and 76.663 ± 2.887 respectively; compared to weaning blends of sorghum ogi combined with soybean (70:30) (Table 3).Vitamins are antioxidants and foods from plant sources are good sources of vitamins [48].The mineral content of the weaning blend of sorghum ogi combined with soybean (60:40) had an improved phosphorus, iron, potassium, sodium, calcium and zinc (P < 0.005) of 665.333 ± 12.097 13.500 ± 0.619, 778.400 ± 7.638, 688.952 ± 6.512, 130 ± 5.000 and 0.56 ± 0.100 respectively (Table 3).The increase in temperature after fermentation may be due to the accumulation of proteolytic enzymes produced by natural bacteria, which break down grain cell walls resulting in an increase in mineral content [49].The findings are consistent with the findings of Day and Morawicki [50], which attribute the increase in mineral content to the loss of dry matter during fermentation as microbes degrade carbohydrates and protein, as well as the use of pretreatment.

Shelf-life monitoring of the weaning blends
The shelf-life of the weaning blends was monitored over a three-month period at room temperature (28 ± 2 o C), relative humidity at 65% (Table 4).The total bacteria count ranged from 0.11 ×10 -0.23 ×10 which was within the tolerable limit recommended by FDA's Bacteriological Analytical Manual (BAM).However, no coliform counts were discovered.This is due to the weaning blends' moisture content, which prevents the proliferation and growth of spoilage organisms [43].Moisture is an indicator of a food's shelf life.Moisture Content less than 10% has been reported to be responsible for food preservation [51].

Conclusion
The West Africa Sub-region's abundant cereals and legumes can be used to provide adequate weaning diets for infants, aiming to achieve the UN's Sustainable Development Goals by providing affordable, simple, and probiotic-rich weaning foods.These blends meet daily nutritional requirements, provide health benefits, and meet recommended standards for weaning.Future research should optimize weaning food formulations to enhance nutritional value, sensory appeal, and infant acceptance, while also exploring the incorporation of locally available fruits and vegetables for improved health benefits.

Compliance with ethical standards
Disclosure of conflict of interest )  ×    ×        … .  Normality of NaOH= 0.1, Lactic acid equivalent = 90.08mg,Volume of sample used = 25mL

Figure 2
Figure 2 Percentage occurrences of the LAB isolates

Table 1
Technological properties of the LAB isolates (Antimicrobial productions (mg/mL))

Table 2
Optical density at 600nm of the LAB isolates of different pH and 4% NaCl

Table 3
Proximate, minerals and vitamins of the weaning blends

Table 4
Shelf-life monitoring of the weaning blends No growth 0.15×10 3 No growth 0.10 ×10 3 No growth