EFFECT OF STORAGE PERIOD ON PHYSICAL, CHEMICAL, MICROBIAL, AND SENSORY QUALITIES OF INSTANT MASA FLOUR PRODUCED FROM BLENDS OF RICE AND BAMBARA GROUNDNUT

: Masa is a traditionally fermented meal usually made from cereals. The aim of this research was to produce an enriched meal from rice and Bambara groundnut. The colour, functional properties and microbial quality of composite blend of rice and Bambara groundnut flour, in the ratio of 100:0, 95:5, 90:10, 85:15, 80:20, were evaluated using standard methods. Sensory properties of freshly made masa and masa prepared from stored flours were also determined. The microbial load of the masa flour blend increased over the storage period. Water absorption capacity, swelling capacity and bulk densities increased, while the oil absorption capacity decreased with the storage period. The objective colour result showed a decrease in the lightness (L*) value. Sensory properties of masa were not substantially altered with Bambara groundnut inclusion, but the ratings reduced with storage. Instant masa may be prepared from flour stored for 4 weeks without considerable changes in quality.


Introduction
The knowledge of consumers on the relationship between diet and health has led to a change in the dietary pattern of the African populace. According to Samuel et al. (2015), there are swift and widespread shifts in food consumption patterns towards the western diet and lifestyle. Consumption of snacks such as masa produced from cereals contributes significantly to the calorie intake of the populace in many parts of the world, including Africa. Masa is a traditional fermented product in Nigeria from millet, maize or rice flour (Badau et al., 2018). Good quality masa is round in shape with brown and smooth surfaces (Badau et al., 2018).
The nutritional value of masa may vary with the type and variety of cereals and perhaps the processing conditions used in its preparation. For example, the protein content of rice-masa (7.59%) was found to be significantly lower than those of maize (9.56%) and millet (9.21%) (Ayo et al., 2008b). In general, masa is very low in protein but may contain up to about 7% to 11% of protein depending on the cereals used and their combinations with legumes. Nkama and Malleshi (1998) reported that the protein content of rice-masa increased by 54% when 20% of cowpea was added to enrich the masa. Other authors reported a 10% increase in protein content of maize-masa enriched with groundnut paste at 20% levels (Ayo et al., 2008a).
The addition of protein-rich leguminous crop such as Bambara groundnut (Vigna subterranea) to cereal-based snack is a promising way to enrich the snack and improve the nutritional intake of the populace. Bambara groundnut is a leguminous crop that is rich in protein (15-27%) and carbohydrate (57-67%) . It is often referred to as a complete food because of its reasonably high protein content (Adebowale et al., 2002;Arise et al., 2015;Oyeyinka et al., 2015;Sirivongpaisal, 2008) and a good balance of the essential amino acids (Yao et al., 2015). The high levels of lysine (6.5-6.8%) in Bambara groundnut and the considerable amount of methionine (1.8-2.84%), which is normally limiting in most legumes, further confirm the grain as a balanced diet (Aremu et al., 2006;Kudre et al., 2013;Ijarotimi and Esho, 2009). Bambara groundnut is a drought-tolerant crop that has potentials for cultivation during extreme conditions of drought. Bambara groundnut has similar composition to cowpea but has limited utilisation when compared to cowpea (Vigna unguiculata), presumably due to the very limited research to unlock the potential of the crop (Oyeyinka et al., 2015). The hard-to-cook defect (HTC) commonly associated with legumes including Bambara groundnut may also explain the limited utilisation of the crop. It has been found that HTC defect results in high energy utilisation and consequently reduces the nutritive value of the grain (Molina and Bressani, 1975;Paredes-López et al., 1991).
Previous studies on masa enriched with legumes such as cowpea or groundnut found significant improvement in the nutritional value of the masa (Ayo et al., 2008a;Nkama and Malleshi, 1998). Besides the improvement in the nutritional value of masa, the addition of legumes to masa may affect the physical, chemical, and sensory properties. The sensory properties of maize-based masa enriched with 20% and 25% of groundnut paste were found to be superior to those of masa without groundnut paste or to those with lower levels of the paste (Ayo et al., 2008a). So far, studies on masa have focused on nutritional properties as well as sensory properties of the snack. In this study, instant masa flour was produced from blends of rice and Bambara groundnut flours. The physical, chemical, sensory, and microbial qualities of the freshly produced and stored masa flour were investigated.
Effect of storage on some qualities of flour produced from rice and Bambara groundnut 167

Materials
Local rice, Bambara groundnut, corn starch, and polyethene bags were obtained from the Oja Oba market in Ilorin, Kwara State. Materials were transferred into the Food Processing laboratory of the Department of Home Economics and Food Science, University of Ilorin, for further processing.

The preparation of masa flour
Masa was prepared from rice as previously described by Ayo et al. (2008a), except that Bambara groundnut was used to enrich the masa instead of groundnut paste. Briefly, rice grains (500 g) were sorted to remove foreign matters, washed, and soaked in distilled water (600 ml) at 34 o C for 12 hrs. A quarter ( 1 / 4 ) of the cooked rice grains was mixed with three-quarters ( 3 / 4 ) of milled rice flour to form a batter. The resulting batter was inoculated with bakers' yeast (1% w/w of rice grain) and left to ferment at room temperature (25±2 o C) for 16 hrs, and the thick batter obtained was diluted with 10 ml of 20% sodium carbonate solution. Salt (10 g/500 g of rice), sugar (30 g/500 g of rice) and Bambara groundnut flour in varying proportions (5, 10, 15 and 20% w/w of rice grain) were added to the batter and properly mixed. Samples were dried in an oven at 60 o C for 5 hrs, cooled and milled into fine flour, mixed with 5% of corn starch, and packaged into a high-density polythene bag. The masa flours were stored for two months at room temperature (25±2 o C), and the colour and functional properties of the flour, as well as the sensory and microbial qualities of the masa, were assessed bi-weekly from the day of production.
The proximate composition of masa flour The proximate composition (ash, fat, fibre and moisture contents) of the flour was determined using standard methods (AOAC, 2000). Protein content was measured using the Kjeldahl method (6.25 × N), while the total carbohydrate was calculated by difference.
The colour of masa flour Tristimulus l* a* b* parameters of masa flour were determined after standardisation using colour Flex (A60-1014593, USA). Snapshots in triplicate were taken, and values were read directly from a digital print. The average of the readings was computed and recorded.
Functional properties of masa flour Water absorption capacity was determined by the method described by Oyeyinka et al. (2015). One gram of sample was mixed with 10 ml of distilled water. The mixture was left at room temperature (25±2 o C) for 30 mins and thereafter centrifuged (model 5810R, Eppendorf International, Frankfurt, Germany) for 30 mins. Water absorption capacity was expressed as a gram of water bound per gram of flour. The same procedure was repeated for oil absorption capacity, except that the water was replaced with soybean oil.
Swelling capacity was determined as previously described with slight modifications (Oyeyinka et al., 2019). Flour blends were filled to the 10 ml mark in a 50-ml glass measuring cylinder. Distilled water was added at room temperature to give a total volume of 50 ml. The top of the cylinder was tightly covered, and the contents mixed by inverting the cylinder. The cylinder was then left to stand for additional 4 hrs, and the final volume occupied by the sample was recorded. Swelling capacity was determined by dividing the volume of the flour in water by the initial volume of the flour blends.

The microbial analysis of masa flour
Quantitative bacteriological analysis of the samples was carried out using the total plate count on nutrient agar (NA), and potato dextrose agar (PDA) was used for the fungal count. The counts were expressed as colony-forming units per millimetre (cfu/ml) (Balogun et al., 2016).

Sensory analysis
For sensory evaluation, masa was prepared from the instant masa flour, and sensory evaluation was carried out to know the most acceptable blend of rice flour and Bambara groundnut flour from the various mixing ratios. The analysis was done using a nine-point hedonic scale questionnaire. The masa samples were placed in front of 30 panellists to decide on the most acceptable one by assessing the samples for sourness, appearance, aroma, taste, texture, and overall acceptability. The panellists were selected among the staff and students at the University of Ilorin who are regular consumers of masa. Dried masa flour was mixed with water to form a batter. The batter was deep-fried in heated soybean oil for 4 mins on one side and turned over for additional 4 mins. Fried masa samples were cooled, packaged, and stored at 4 o C until needed for analysis for a maximum of 1 week. Freshly made samples and masa from stored flours were prepared and used for sensory evaluation. Panel members were provided with water to rinse their mouths after evaluating each sample to prevent carry-over effects.
Effect of storage on some qualities of flour produced from rice and Bambara groundnut 169 Statistical analysis Duplicate samples were prepared, and analyses were done in triplicate. Data were analysed using the one-way analysis of variance (ANOVA), and means were compared using the Fisher's least significant difference (LSD) test (p ≤ 0.05) using the Statistical Package for the Social Sciences (SPSS) Version 16.0 for Windows (SPSS Inc., Chicago, USA).

Results and Discussion
Effect of storage period on the colour of rice-Bambara masa flour The objective colour parameters of the freshly made and stored rice-Bambara groundnut masa flour blends are presented in Table 1. Lightness (L) values (83.99-85.81) for the flour blends containing Bambara groundnut flour were generally lower than that of the control sample (88.11) without Bambara groundnut (Table  1). Although the L values of the enriched samples were lower than the control, the addition of Bambara groundnut up to 20% level did not significantly (p < 0.05) change the colour of the samples. The L value result suggests that the masa flour without Bambara was whiter in appearance, which could be attributed to variation in the composition of the grains. Enriched masa flour samples showed a significant decline in all the colour values (L, a and b) with an increase in the storage period (Table 1), which could be due to the Maillard reaction in the stored flour (Ward et al., 1998;Yeboah-Awudzi et al., 2018). This seems plausible since the presence of protein from Bambara grains and carbohydrate in the masa flour may facilitate the Maillard reaction. Previous studies similarly observed a decline in the colour of maize with an increase in the storage period (Paraginski et al., 2014). Effect of storage period on proximate composition of rice-Bambara masa flour The proximate composition of freshly prepared and stored masa flour from blends of rice and Bambara flour is shown in Table 2. Carbohydrate was the major nutrient in the freshly prepared masa flour (75.43-82.57%) and stored flour samples (74.13-81.19%). Other nutrients such as ash (0.50-0.87%), fat (0.41-0.95%), fibre (4.28-6.03%) and protein (6.80-9.74%) were generally low ( Table  2). Bambara groundnut flour addition to rice flour increased the ash, fibre, fat, and protein contents in the composite flour but decreased the carbohydrate content. The protein content of the composite flour increased by approximately 15%, 21%, 33% and 42% when adding 5%, 10%, 15% and 20% of Bambara groundnut flour, respectively. The increase in protein is expected since legumes, including Bambara groundnut, are known to be good sources of protein . This increase in protein represents a good strategy to improve the nutritional intake of masa-loving individuals and could further help reduce protein-energy malnutrition prevalent in Africa, including Nigeria. The storage of the flour in a high-density polythene bag at room temperature (25±2 o C) for a period of 8 weeks did not substantially alter the composition of the flours, except for the moisture values ( Table 2). The moisture content of the flours Effect of storage on some qualities of flour produced from rice and Bambara groundnut 171 increased significantly (p < .05), suggesting that the polythene bags were not impermeable to water vapour. Similar results were reported for Bambara and cowpea snacks for a period of 4 weeks . The ash, fibre, fat and protein contents were almost constant, while the carbohydrate content of the flours reduced with an increase in the storage period. The proximate results were within the range reported for rice-Bambara nut flour extruded flakes (Adebowale et al., 2016).
Effect of storage period on selected functional properties of rice-Bambara masa flour The water absorption capacity (WAC) of the flour blend increased with increasing levels of Bambara groundnut (Figure 1). This could be due to higher polar amino acid residues of proteins in the Bambara flour which presumably have more affinity for water molecules (Yusuf et al., 2008).  According to Lawal and Adebowale (2004), the major chemical compositions that enhance the WAC of flours are proteins and carbohydrates since these constituents contain hydrophobic part such as polar or charged side chains. Thus, the increased WAC of the blends may also be associated with an increase in the starch content of the flour. This seems plausible since previous studies reported Bambara groundnut to be a good starch source, contributing to increased swelling (Oyeyinka et al., 2015;Oyeyinka et al., 2017). Although the WAC increased slightly with an increase in the storage period, the increase was not significant (p ≥ 0.05). The WAC of the samples ranged between 126.08 and 157.74 % (Figure 1), which is similar to the values previously reported for Bambara groundnut during short time storage (Goudoum et al., 2016).
The oil absorption capacity (OAC) of the flour blends (Figure 2) was generally lower than their WAC (Figure 1). The storage period similarly did not significantly (p < 0.05) alter the OAC of the flour blends, though there was a slight increase ( Figure 2). In general, the OAC decreased with an increase in the percentage of Bambara groundnut flour, suggesting that Bambara groundnut has fewer hydrophobic proteins compared with rice. Although rice is a cereal, its protein has been reported to have surface hydrophobicity, which may increase with heat denaturation (Ju et al., 2001). In addition, earlier studies have indicated hydrophobic proteins have better oil binding properties (Lawal and Adebowale, 2004).  The swelling capacity of the flour blends increased with increasing levels of Bambara groundnut flour (Figure 3). However, the swelling index showed an insignificant (p > 0.05) increase with an increase in the storage period. The swelling index represents the ability of the flour samples to imbibe water and swell at room temperature. The extent of swelling is determined by the amount of water that starch and protein can absorb before the chains become separated completely Effect of storage on some qualities of flour produced from rice and Bambara groundnut 173 (Chrastil, 1990). Starch is chiefly responsible for swelling, and the degree of swelling depends on the ratio of amylose to amylopectin and the chain length distribution of the amylopectin chain length. Functional properties of cereal and legume flours are important parameters to be considered in developing food products because they contribute to the texture, mouth feel as well as consistency of the product (Bhat and Nabilah, 2014).  Effect of storage period on the microbial load of rice-Bambara masa flour The total bacterial count (TBC) and total fungal count (TFC) as measured in cfu/g in rice-Bambara masa flour samples during storage at room temperature for a period of 8 weeks are presented in Figures 4 and 5, respectively. In general, there was an increase in both TBC and TFC of the flour blends with an increase in the storage period (Figures 4 and 5). Furthermore, the TBC and TFC similarly increased with an increase in the levels of Bambara groundnut. Bambara groundnut is a good source of protein and other nutrients that are needed by microbes for growth. Balogun et al. (2016) also reported an increase in microbial growth with an increase in the storage period of gruel made from fermented maize and soybean. Bacteria seem to grow faster in the flour blends than the fungi. For example, the TBC ranged between 2.1 and 9.05 cfu/g ×10 4 , while the TFC ranged between 0 and 3.8 cfu/g ×10 4 . These values fall within the range of the maximum permissible level (>10 4 to <10 6 cfu/g) of the total aerobic colony.  Effect of storage on some qualities of flour produced from rice and Bambara groundnut 175 Effect of storage period on the sensory properties of rice-Bambara masa Masa samples were prepared from the flour blends, and the sensory quality was assessed using panel members as described above. Mean sensory scores showed that there were no significant differences (p<0.05) in sourness, appearance, aroma, and texture, but in taste and overall acceptability among the samples (Table 3). Masa flour with 100% of rice had the highest rating for appearance, aroma, taste, texture, and overall acceptability. Panel members consisted of regular consumers of masa. Thus, the higher rating recorded for masa prepared from 100% of rice flour could be due to the familiarity of the panel members with masa. The storage period significantly (p<0.05) affected the sensory properties of masa (Table 3). The ratings recorded for all the sensory properties reduced significantly with an increase in the storage period. Masa prepared from 80% of rice and 20% of Bambara groundnut flour had the highest rating in overall acceptability after the storage period of 8 weeks. Table 3. Mean sensory scores of rice-Bambara groundnut masa flour blend stored for 0 and 8 weeks.

Conclusion
The objective of the study was to determine the physical, chemical, sensory, and microbial qualities of freshly produced and stored masa flour from rice and Bambara groundnut flour blends. Bambara groundnut flour addition did not significantly alter the colour of masa samples prepared from rice but did change the proximate composition. However, a slight colour change was observed after storage for 8 weeks. There was an improvement in the nutritional value of the masa flour due to the Bambara groundnut addition. Flour functionality improved with the Bambara groundnut addition and did not change significantly with an increase in the storage period. However, the total bacterial and fungal count increased with an increase in the storage period. Although the microbial load was within the range accepted for ready-to-eat foods, better packaging may be required to reduce the proliferation of microorganisms during storage to extend the shelf-life of the masa flour. Sensory properties of masa were not substantially altered with Bambara groundnut inclusion, but the ratings reduced with an increase in the storage period. This study showed that enriched masa flour can be stored for at most 4 weeks before significant changes can be observed in the sensory properties. Conclusively, instant masa flour from the rice-Bambara groundnut blend had improved nutritional qualities, with extended shelf life and convenience in terms of handling and less preparation time.