Effect of pretreatments on the microbial and sensory quality of weaning food produced from blends of sorghum and soybean

This study investigated the effect of pre-treatment on the microbial and sensory quality of weaning foods produced from blends of sorghum and soybean. Sorghum and soybean were fermented, roasted and fermented/roasted, then milled into flours. Untreated sorghum and soybean flours were also produced separately and serve as the control. A portion of sorghum was malted and milled to obtain malted sorghum flour of which 5% was added to each sample except the control samples. Weaning foods were prepared in the following ratios: (75:20:5), (65:30:5) and (55:40:5) of sorghum: soybean: malt for the treated samples and the control samples contained (80:20), (70:30) and (60:40) of untreated sorghum : soybean. The microbial and sensory qualities of the samples were determined. The result of the microbial analysis of the formulated weaning food blends showed that all samples indicated a safety for consumption. The appearance of both treated and untreated were not significantly different (p<0.05) while the means scores of the blends in texture, taste, aroma and overall acceptability were significantly different (p<0.05). Fermentation and roasting used in this study showed that the processing methods had marked effect on the microbial and sensory quality of the weaning foods produced, thus, its recommendation for domestic processing of weaning foods.


INTRODUCTION
Weaning is the gradual introduction of foods other than breast milk into a baby"s diet from the age of six months.Weaning foods are foods widely used during the transition from consuming solely human milk or infant formulas to the introduction of a mixed diet.The foods are solely introduced to complement breast milk, progressively replace it and eventually adopt the child to adult diets.Usually, the breast milk which is the baby"s first food is inadequate to maintain the rapid growth and development of the baby after six months of age of the *Corresponding author.E-mail: esthermusadurkwa@gmail.com.Tel: +2348163917067.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License child.Hence, there is the need to introduce appropriate weaning foods to the child which will supply the additional safe sources of energy and protein to complement breast milk and fully aid the growth and development of the child.Failure to feed the baby with appropriate food could lead to malnutrition, a problem that is common with most children in the developing countries of which Nigeria is one (Asma et al., 2006;Eshun et al., 2011).
Local foods and fortifying ingredients such as maize, millet, sorghum, soybean and cowpea have been utilized creatively with acceptability in mind (Mosha and Vicent, 2004;Mugula and Lyimo, 2000).In Nigeria, traditional weaning food consist of monocereal grains prepared from either millet, sorghum and maize is referred to as "Ogi" or "Akamu" which is of poor nutritional value (Abdulkadir and Danjuma, 2015;Rombouts and Nouts, 1995).Cereals are the most important staple food being the major sources of carbohydrates.Compositionally cereals consist of 12 to 14% water, 65 to 75% carbohydrates; 2 to 6%, lipids and 7 to 12% protein on dry weight basis.In their natural form, whole grain cereals are also significant contributor of vitamins, minerals like manganese, zinc, copper and magnesium and considerable iron but its bioavailability is low which results in incidence of iron deficiency anemia.However, processes like fermentation has improved the chemical bio-availability of iron (Oyarekua, 2011).Abdulkadir and Danjuma (2015) reported that the major problem associated with infant during transitional phase of weaning is protein energy malnutrition (PEM) which results into condition such as marasmus or kwashiorkor.The formulation and development of nutritious weaning food from the combination of commonly used cereals with plant protein source like legumes can be used to prevent malnutrition during weaning, since cereals are deficient in lysine which are limited in legumes, whereas legumes are highly rich in lysine.The effects of the fortification are highly beneficial since nutritive value of products is also improved (Wang and Daun, 2006).The use of legumes seeds may be the beginning of a series of formulation which will lead to a substantial drop in dependency of animal source on nutritious foods.Unfortunately, legumes seeds contain anti-nutritional factors like enzymes inhibitors phytates, oxalate, saponin and polyphenolic compound, all of which limit their utilization (Abdulkadir and Danjuma, 2015).Although, remarkable improvement in the nutritive value and quality of legumes seeds have been achieved through de-hulling, heat treatment, germination, fermentation, soaking and partial hydrolysis of proteolytic enzymes (Akinrele and Bassir, 1967).
Soybeans and sorghum are typically processed prior to human consumptions.It is important that the antinutritional components are denatured prior to their consumptions.Numerous methods of eliminating antinutritional factors have been developed and tested (Newkirk, 2010).Most of the toxic and anti-nutrient effects could be removed by several processing methods such as soaking, germination, boiling, autoclaving, fermentation, genetic manipulation and other processing methods (Soetan, 2008).Fermented cereals are particularly important as weaning foods for infants and as dietary staples for adults.Ikemefuna et al. (1991) reported that a combination of cooking and fermentation improved the nutrient quality and drastically reduced the anti-nutritional factors to safe levels much greater than any of the processing methods tested.Ikemefuna et al. (1991) also reported that soaking and fermentation decreased the tannins content because these processes produce enzymes that break down complexes to release free tannins, thus, the free tannins were leached out.Roasting which is the application of heat in measured amounts, denatures the trypsin inhibitors, hemagglutinins (lecithins) and possibly allergenic proteins without damaging the quality and digestibility of the protein in the meal (Newkirk, 2010).Roasting also create a convenient method of increasing fat content in the diet without the need to physically handle a liquid oil product.
The aim of this study is to examine the effects of pretreatments (fermentation and roasting) on the microbial and sensory quality of weaning food produced from blends of sorghum and soybean.

MATERIALS AND METHODS
Sorghum and soybean grains were purchased from Gombe Main Market, Gombe State, Nigeria.All samples were kept in a moisture free environment until when needed.The chemicals and reagents used were of analytical grade and they were obtained from the laboratories of Food Science and Technology, Modibbo Adama University of Technology, Yola, Adamawa State and Home Economics Education Department, Federal College of Education (Technical), Gombe, Gombe State, Nigeria.

Preparation of samples
The sorghum and soybean grains were manually cleaned (separately) to remove sand, foreign seeds, broken and infested seeds, dirt and other contaminants.

Fermented sorghum
One kilogram (1 kg) of sorghum was dehulled after tempering using the commercial grains huller at Gombe Main Market.After dehulling, the grains were winnowed and washed to remove the hulls and germs.The grains were steeped at room temperature (32 ± 2°C) for 72 h and the steep water decanted.The fermented grains were then dried in hot air oven (Model: TO008GA-34, AKAI-TOKOYO, JAPAN) set at 60°C for 10 h to halt the fermentation process.It was milled to produce the fermented sorghum flour which was packaged in a clean polyethylene bag (Adebayo-Oyetoro et al., 2012).

Fermented soybean
One kilogram (1 kg) of soybean was soaked for 12 h in 3 L of clean water until the seed coat became soaked and wet to facilitate dehulling.Mortar and pestle were used for dehulling.The dehulled soybean was washed to remove the seed coat.The soybean was allowed to ferment naturally in a clean covered plastic bucket for 72 h and the steep water decanted.The fermented grain was then dried in hot air oven (Model: TO008GA-34, AKAI-TOKOYO, JAPAN) at 60°C for 10 h to halt the fermentation process.It was then milled to produce the fermented soybean flour which was packaged in clean polyethylene bag until needed.

Roasted sorghum
Whole grains of sorghum weighing 1 kg were dehulled using the commercial grains huller at Gombe Main Market.After dehulling the grains, they were winnowed, washed, drained and then partially sun dried.The sorghum was then traditionally roasted using an open thick aluminum pot.Commercial grinding machine was used to mill the grains into flour.A local sieve of about 1 mm in diameter was used to sieve the flour in order to obtain a fine particle size.The flour was packaged in a plastic container and sealed until needed.

Roasted soybean
Whole grains of soybean weighing 1 kg were soaked for 12 h in 3 L of clean water until the seed coat became soaked and wet to facilitate dehulling.Mortar and pestle were used for dehulling.The dehulled soybean was washed to remove the seed coat, drained and then sun dried.The soybean was then traditionally roasted using an open thick aluminum pot.An attrition mill was used to mill the dehulled grain into fine flour and to pass through a sieve of about 1 mm mesh screen.The roasted soybean flour was packaged in a plastic container until when required.

Fermented/roasted sorghum and soybean flour
One kilogram (1 kg) of sorghum grain was dehulled and dry cleaned.This was then fermented in a plastic bucket for 72 h.Thereafter, it was sun dried and roasted traditionally using an open thick aluminum pot.It was then milled into flour using attrition mill and stored in a plastic container until needed.These procedures were used for the preparation of fermented/roasted soybean.

Malting of sorghum
The sorghum was malted as described by Badau et al. (2006).The grains were steeped at room temperature (32 ± 2°C) for 12 h.The steeped liquor was changed after 6 h.One air rest period of 1 h was applied after 6 h of steeping.After steeping, the grains were immersed in 0.1% (v/v) solution of commercial bleach (hypo), that is, 5 ml of 3.5% sodium hypochlorite.After sterilization, the grains were wrapped in a wet piece of cotton cloth and placed on a wet jute bag.Another wet jute bag was used to cover the grain wrapped in the wet cloth.The sorghum grains were allowed to germinate at room temperature (32 ± 2°C) for 72 h.During germination, small and the grains were turned by moving a clean wooden rod inside the germinating grains.For the first 48 h of malting, the sample was quantity of water (15 ml) was sprayed on the germinating grains moistened twice a day at 08:00 and 16:00 hour by spraying a mist of water on it for about 5 s and then it was turned over.On the seventy-two hour of germination, the sample was spread in the morning (8 h) and turned over in the afternoon (16 h).
At the end of the germination, the germinated grains were dried to moisture content of 5.45 ± 0.48% in an oven set at 50°C for 24 h.The dried germinated grains were polished by removing shoots and rootlets.Rootlets and shoots were separated from the kernels by rubbing between the palms in a local sieve of about 1 mm mesh size.This allows the rootlets and shoots to escape but retain the kernels.The polished malt was then milled into fine flour in a commercial milling machine and sieved using a local sieve of about 1 mm mesh screen.The malted sorghum flour was packaged in an air tight plastic container until when it was needed.

Formulation of weaning foods
The weaning foods were formulated in ratios shown in Table 1.

Microbial analysis
Materials that were used were sterilized by autoclaving at pressure of 1 kg/cm 2 (121°C) for 15 min as described by Jideani and Jideani (2006).Glass wares were sterilized by dry-heat at temperature of 160°C for 60 min.The work-place was aseptically cleaned (with antiseptic and careful control) to ensure microbial reduction inhibiting the place.Media after preparation were sterilized at 1 kg/cm 2 (121°C) for 15 min and placed in water bath at 100°C to avoid gelling before use.Besides, Bunsen burner was used to "heat fix" smears, sterilize wire loop and other equipment.Inoculation methods such as streaking and poor plating were used to culture and subculture.Preliminary observations and tests were carried out followed by secondary observation.

Serial dilution
A prepared 1 g of sample was transferred into a bottle containing 9 ml of distilled water to form the stock solution as described by Jideani and Jideani (2006).From the stock solution, 1 ml was aseptically transferred into subsequent bottles containing sterile distilled water using sterile pipette, that is, from first to second, second to third and third to fourth until after required serial dilution was made.

Standard plate count method (SPCM)
From the prepared sample, 1 ml of the diluent from a test tube was transferred aseptically into cleaned and sterilized labeled plate (pour plate count) using sterile pipette.The lid of each was aseptically opened to introduce about 15 ml molten medium (Nutrient agar or MacConkey agar) into the plate (Petri dish) to ensure total bottom covering of the plate.It was swirled gently, cooled and solidified before incubating it at 35°C for 24 h as described by Jideani and Jideani (2006).The number of microorganism per gram of the original sample (colony forming unit per gram [Cfu/gram]) was obtained using: (1) Colony forming unit per gram (Cfu/g) =        ×

Sensory evaluation of weaning food gruel
The gruel of the developed weaning food was served to 20 weaning mothers who were asked to rank the gruel on the basis of some quality attributes (taste, texture, appearance, aroma and overall acceptability) using 9 points Hedonic scale, where 1 = "dislike extremely" and 9 = "like extremely".Weaning food gruels were served to panelist in transparent plastic cups and they were asked to rinse their mouth with fresh room temperature water provided, before next serving.The containers with the samples were coded and kept far apart to avoid crowding and for independent judgment.The panelists were selected based on the basic requirements of a panelist, such as availability for the entire period of evaluation, interest, willingness to serve, good health (not suffering from colds), not allergic or sensitive to the product emulated (Badau et al., 2005).

Statistical analysis
All experiments were performed in triplicate, and the results were expressed as means ± standard error (SE).Analysis of variance (ANOVA) was carried out to determine any significant differences in measurements using the SPSS statistical software (SPSS 20.0 for Windows; SPSS Inc., Chicago, IL, USA) and considering the confidence level of 95%.The significance of the difference between the means was determined using the Duncan"s multiple range test, and the differences were considered to be significant at p< 0.05 (Hussein et al., 2016).

Effect of pretreatments on the safety of the formulated weaning foods from blends of sorghum and soybean
The result of microbial analysis as presented in Table 2 showed that pretreatments (fermentation, roasting and the combination of fermentation and roasting) have positive effect on the microbial load of the weaning foods samples as compared to the untreated samples.The total plate count (TPC) ranged from 1.30 × 10 3 to 2.83 × 10 3 (cfu/g) with sample F55:40:5 having the highest value while sample R55:40:5 had the lowest value.In the fermented samples, there was a significant decrease in the TPC in all the blends as the percentage inclusion of soybean increased.The percentage reduction in TPC of fermented sample was 4.59%.There was also a significant decrease in the TPC of all the roasted blends as the percentage inclusion of soybean increased.The same trends of decrease were observed for fermented and roasted samples.The percentage reduction in TPC of fermented sample was 3.70% and fermented/roasted was 4.75%.While the untreated samples shows a significant increase in the TPC of all the blends as the percentage inclusion of soybean increased.The higher TPC of fermented samples to fermented/roasted, roasted and untreated samples were observed to be due to the multiplication of microorganism during fermentation process.The TPC of all the roasted and fermented/ roasted samples were very low.This was observed to be due to high temperature of roasting as most microorganisms cannot survive higher temperature.
There was no detectable yeast and moulds growth in fermented, roasted and fermented/roasted samples.The untreated samples showed very low growth of 5.00 × 10 1 cfu/g, 4.80 × 10 1 cfu/g and 4.60 × 10 1 cfu/g for samples U60:40, U70:30 and U80:20, respectively.These results are much lower than the FAO/WHO limits of 10 4 to 10 6 cfu/g for bacteria and 10 2 to 10 4 cfu/g for moulds in weaning foods (Abdulkadir and Danjuma, 2015).The yeast and moulds growth in the untreated samples were observed to be present as contaminants and do not appear after the pretreatments.The expected decrease or elimination was also reported by Mbata et al. (2009) for fermentation of maize flour fortified with Bambara groundnut.This shows clearly the importance of these pretreatments used in the aspect of weaning food processing.The microbial analysis of the formulated weaning food blends reveals that all the formulation indicated a safety of the products for consumption and this was due to higher standard of personal hygiene and quality maintenance of manufacturing practice observed during the preparation.Nwokoro and Chukwu (2012) highlighted the importance of adequate hygiene during the preparation of food and also link between infection Table 2. Microbial load of the weaning foods prepared from the blends of sorghum and soybean (cfu/g).Table 3. Sensory qualities of the weaning foods prepared from the blends of sorghum and soybean.

Sample blends Appearance
(na)  and nutrition.Also, the indication of very low microbial contents of the products can be attributed to the low moisture content of the products, which is an indication of low water activity preventing microbial growth.Similar results were reported by Amankwah et al. (2009) for formulation of weaning food from fermented maize, rice, soybeans and fish meal.

Effect of pretreatments on sensory evaluation of the formulated weaning foods from blends of sorghum and soybean
Table 3 shows the results obtained for sensory qualities of the weaning foods prepared from the blends of sorghum and soybean.All the weaning mothers were conversant with the factors governing the qualities of the weaning foods.The sensory attributes of the weaning blends show that there were significant differences (p<0.05)among the means scores of the blends in texture, taste, aroma and overall acceptability, while there were no significant difference (p>0.05)among the means scores in appearance.In terms of appearance, the scores ranged from 6.60 to 7.80 with the sample FR55:40:5, fermented/roasted having the lowest score, while sample R55:40:5 roasted had the highest score, thereby the most preferred by the panelists.The appearance of both treated and untreated were significantly preferred (p<0.05)despite no addition of 5% malt to untreated samples.Similar result was reported by Badau et al. (2005) for weaning food gruel produced from pearl millet and legumes.
The scores for texture ranged from 4.85 to 7.60 with the sample FR75:20:5, fermented/roasted having the lowest score while the most preferred by the panelists was sample U70:30, untreated.This is due the viscous nature of the untreated blend upon cooling as observed by the panelist.In terms of taste, the scores ranged from 4.35 to 8.15 with sample F55:40:5 fermented having the lowest score while sample R55:40:5, roasted, had the highest score, thereby the most preferred by the panelists.With regards to the aroma, the scores ranged from 4.05 to 7.70 with the sample F55:40:5, fermented, being the least preferred by the panelists and sample R55:40:5, roasted, was the most preferred by the panelists.The taste and aroma of the roasted samples preferred by the panelists is due to the flavoring agent and the least preferred taste and aroma of fermented blends were observed to be due to higher acidity resulting from the prolonged fermentation time.The weaning food blends from roasted samples had significantly (p<0.05)higher mean score in appearance, taste, aroma and overall acceptability followed by fermented and roasted samples.These relatively higher mean scores of the roasted samples followed by fermented and roasted samples could be probably due to the roasted flavour and aroma imparted on soybean during roasting coupled with astringency and aromatic compounds conferred on the sorghum by the fermentation process.Similar results were reported by Adedeji et al. (2015) that fermentation imparts desirable flavors, esters, ketones, aldehydes and aromatic compounds as well as characteristic astringency on products.But in this study, relatively lower mean sensory scores were obtained for fermented samples in terms of taste.These lower mean scores were due to beanny flavour imparted by the soybean supplementation.Both treated and untreated samples when reconstituted were smooth and homogeneous, without noticeable lumps.This was the reason why untreated samples were accepted coupled with the fact that the weaning mothers are used to these untreated ones.The overall acceptability scores ranged from 5.25 to 7.85 with the sample F75:20:5 fermented having the lowest score while sample R55:40:5, roasted, had the highest score thereby the most preferred by the panelists.

Conclusion
Weaning foods from blends of fermented (sorghum and soybean) and roasted (sorghum and soybean) grains were produced.The microbial analysis of the formulated weaning food blends revealed that all the formulations are safe for consumption.From the results of the sensory evaluation, it was concluded that fermentation and roasting processes improved the degree of acceptability of the various weaning foods blends.Therefore, every raw material to be used for the production of weaning foods should be pretreated for safety and enhancement of organoleptic quality.

Table 1 .
Formulation of the weaning foods.