Evaluation of sugar and free amino acid during fermentation of ogi from maize, acha and sorghum

This research investigated effect of fermentation time and cereal type on the total reducing sugar (TRS), total sugar (TSS), and total free amino acid (TFA) during the production of ogi. The result showed that TFA generally increased with increase in fermentation time (7.916 – 17.596 mg/g). Maize, acha and sorghum ogi had the lowest total reducing sugar (TRS) at 0 h (16.927glucose mg/g), 12 h (16.655 glucose mg/g) and 48 h (18.212 glucose mg/g) respectively and TSS was lowest in acha ogi from 12 h to 48 h (33.191 34.370 glucose mg/g). Principal component analysis and Agglomerative hierarchical clustering were used to evaluate the variability in sugar and amino acid contents and ranked the contributions of the variables. The factors were divided into four principal components with cumulative variance contribution rate of 87.47%. The result showed that acha and sorghum ogi had lower sugar content than maize ogi during fermentation. This research suggested that maize, acha and sorghum can be used in the production of cereal based ogi for weaning food at 48 h due to high free amino acid content, and also advanced the use of acha in production of ogi for diabetic patients due to its low total sugar content.


Introduction
Ogi is a fermented cereal gruel or porridge made from maize (Zea mays) or corn; sorghum (Sorghum vulgare) or millet (Pennisetum typhoideum). The choice of grain depends on preference and ethnicity of the producer (Ohenhen and Ikennebomeh 2007). Ogi, usually called pap, akamu and koko by the people of West Africa can be processed into a slurry paste by heating in boiling water under constant stirring (Adeshokan et al. 2010). It is produced by steeping cereal in water for 72 h at ambient temperature and wet milling (Ohenhen and Ikennebomeh 2007). Fermentation and fermentation period after wet milling depend on individual preference. Ogi slurry usually has a smooth texture, a sour flavour resembling that of yoghurt and a characteristic aroma that differentiates it from starch and flour (Omemu 2011). Acha (Digitaria exilis) also known as Fonio, Findi, Funde, Pom, and Kabug is a highly nutritious cereal crop of West African origin belonging to the family Graminaea (Oyetayo and Agbaje 2012;Malomo et al. 2018) is a lesser-known cereal rich in vitamins, minerals, fiber, carbohydrate, protein, amino acids. It is important because it is high in methionine and cysteine lacking in wheat, rice, maize and other cereal crops and also have low glycemic index which could be an advantage in type II diabetic condition (Alegbejo et al. 2011;Ukeyima 2019). Fermentation of cereals has been reported to increase acidity, total free amino acids and their derivatives by proteolysis and/or by metabolic synthesis. This process reduces the pH thereby inhibiting pathogenic organisms (Kohajdová and Karovičová 2007;Malomo et al. 2018) and also improves the rheological properties, acidification, taste and flavour of fermented foods (Malomo et al. 2019). Starch digestibility also increases during fermentation and this could be due to enzymatic properties of fermenting microflora that brings about the breakdown of starch into simple sugar (Mugula et al, 2003;Malomo et al, 2019). The microorganisms involved in fermentation of food increases palatability and improve the quality of food by increasing the availability of proteins and vitamins (Ogodo et al, 2019). Many researchers have worked on ogi (Omemu et al, 2011;Olaniran et al, 2019) but there is dearth of information on effect of fermentation on sugars and free amino acid. Fermentation may have direct effect on the taste, sugars, total free amino acid and other properties of ogi. Ogi intended for weaning food should be high in free amino acid and those intended for diabetic patient supposed to have lower sugar content. Therefore, this present study was carried out to determine the effect fermentation time and different cereal "maize (Zea mays), acha (Digitaria exilis) and sorghum (Sorghum bicolor)" on the total sugar, total reducing sugar and total free amino acid of ogi.

Materials and Methods
Procurement of materials. Quality protein maize (ART/98/SW06/OB/W) was obtained from the Institute of Agricultural Research and Training (I.A.R.T.), Ibadan, Nigeria. Sorghum (red variety) was purchased from a local market in Ile-Ife, Osun State, Nigeria. Acha grain was obtained from a local market in Zaria, Nigeria and the identity were ascertained at the herbarium of the Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria. Chemicals used for analysis were obtained from Sigma-Aldrich and were of analytical grade.
Production of ogi. The cereals (maize, acha and sorghum) were sorted, weighed and steeped separately for 72 h. The grains were drained and wet-milled into slurry using an attrition mill. The ogi (100 ml) obtained was dispensed into sterile plastics and fermented for 48 h (Olaniran et al. 2019).

Extraction of sugar from Ogi samples.
Ogi samples from maize, acha and sorghum were picked at six-hour interval for extraction. The samples were dried in Gallenkamp oven at 45°C for 10 h, ground in a blender (USHA MG 2053 N, India) and sieved using 50 µm. Ogi sample (5 g) was weighed and thoroughly mixed in a conical flask containing 50 ml of 80 % ethanol v/v and 10 ml of petroleum ether was added. The ethanol-petroleum ether suspension was stirred at room temperature for 30 min in magnetic stirrer (Lab-line, Model No 1580-1, U.S.A.) and mixture was transferred into centrifuge tubes and centrifuged (Bosch Model No TDL-5, Germany) at 5000 rpm for 30 min. The petroleum ether phase was discarded and the clear ethanol phase was kept in the refrigerator for further analysis (Malomo et al. 2019).

Determination of total reducing sugar.
Ethanolic extract (1 ml) was measured into each test tube; 2 ml of Dinitrosalicyclic acid reagent was added and boiled for 5 min at 100°C in Gallenkamp water bath (Gallenkomp, HH-S6, England) and cooled thoroughly under running water. Distilled water (7 ml) was added and the absorbance was read against reagent blank at 540 nm in a UV Spectrophotometer (Spectrumlab 752S, YM1206PHB2, China). The amount of reducing sugar in the samples was extrapolated from a standard curve of known concentrations of glucose (0-1000 μg/ml) (Adepoju et al. 2016).
Determination of total sugar. Total sugar was determined using the anthrone reagent method of Morris (1948) described by Malomo et al. (2019). Ethanolic extract (1 ml) was added to 4 ml of anthrone reagent, heated in boiling water bath (Gallenkomp, HH-S6, England) for 10 min and rapidly cooled. Absorbance was read at 620 nm against a reference blank in spectrophotometer (Spectrumlab 752S, YM1206PHB2, China) and the amount of sugar liberated was obtained from the standard curve based on known concentrations of glucose (10-100mg/l) Determination of total free amino acid. Ogi samples (5 g) were weighed into 250 ml conical flask and 50 ml of 80 % ethanol v/v was added. The suspension was mixed properly and 10 ml of petroleum ether was added. The ethanol-petroleum ether suspension was stirred at room temperature for 30 min using a magnetic stirrer (Lab-line, Model No 1580-1, U.S.A) and centrifuged at 5000 rpm for 30 min. The petroleum ether phase was discarded and the clear ethanol phase was used as the sample extract (Malomo et al. 2019). The ninhydrin method described by Rosen (1957) was used for determination of free amino acid. Cyanide acetate buffer (0.5 ml at pH 5.4) and 0.5 ml of 3.0 % ninhydrin solution (3 g of ninhydrin in 100 ml of 2methyl ethanol) was added to the extract (1.0 ml) in test tube and heated in boiling water bath (Gallenkomp, HH-S6, England) for 15 min. Isopropyl-alcohol water mixture (10 ml) at ratio 1:1 was added rapidly and the solution was allowed to cool to room temperature (27±2°C). The absorbance was read at 570 nm using spectrophotometer (Spectrumlab 752S, YM1206PHB2, China). Total free amino acid in the samples was obtained from a standard curve of known concentrations of glycine (10-100μm/ml) (concentrations of glucose (10-100mg/l) (Omafuvbe 2000).
Statistical analysis. Data obtained were subjected to descriptive and inferential statistics using SPSS (version, SPSS, Inc., USA). Means of samples were separated using Duncan Multiple range Test (SAS Institute 1985). Principal component and clustered analysis were carried out on the data obtained using XLSTAT 2016 (Addinsoft Inc. USA).

Results and Discussion
Total free amino acid of ogi powder at different stages of fermentation. The total free amino acid (Table 1) generally increased from the beginning of fermentation to 36 h and slightly decreased from 36 h to 48 h in maize and sorghum ogi. It was significantly higher (p < 0.05) in ogi produced from maize (9.788 -17.600 mg/g) than sorghum (8.474 -16.610mg/g) and acha (7.916 -16.607mg/g) from 0 h to 24 h but there was no significant difference (p > 0.05) from 36 h to 48 h. There was no significant difference (p > 0.05) between ogi produced from sorghum and acha from 12 h to 48 h of fermentation. The total free amino acid of ogi produced from sorghum and acha had similar total free amino acid content throughout the period of fermentation. Malomo et al. (2019) also reported increase in total free amino acid during fermentation of acha and maize and attributed it to the proteolytic activities of fermenting organisms. Total reducing sugar ogi at different stages of fermentation. The total reducing sugar was highest in maize ogi from 12 h to 36 h (20.544 -23.591 glucose mg/g) and was not significantly (p > 0.05) different from acha ogi at 48 h. It was higher in sorghum ogi than acha ogi from 0 h to 12 h while acha had significantly higher (p < 0.05) than sorghum ogi from 36 h to 48 h of fermentation. Malomo et al. (2019) also reported that the total reducing sugar of fermenting maize was higher than that of acha during fermentation. The range of result of this work is in agreement with the report of    Values are means of three replicates ± standard error. Means followed by different superscript in the same column are significantly different at p < 0.05.

Total sugar in ogi at different stages of fermentation.
The total sugar content of ogi was highest in ogi produced from acha (39.335 glucose mg/g) and lowest in ogi produced from sorghum (30.333 glucose mg/g) at the beginning of fermentation. It was lowest in achaogi between 24 h and 48 h (32.191 and 34.370 glucose mg/g). Ogi produced from maize had the highest total sugar content from the beginning of fermentation to the 36 h (37.156 -40.086 glucose mg/g). The breaking down of complex carbohydrate into simple sugar by fermenting microorganism and utilization of these sugars as carbon source could be responsible for fluctuation in total sugar (Oyarekua and Adeyeye 2009;Adepoju et al. 2016). The highest total sugar content in maize ogi could be due to high amount of digestible starch. Jideani and Podgorski (2009) and Jideani and Jideani (2011) reported the digestible starches (DS) of maize and acha to be 43.7 and 41.4 respectively. This suggest that acha could be consumed as food with low glycemic index. PCA was applied to pooled measurements in order to describe the group of physical data, to establish the relationships between the different physical variables, and to detect the most important factors of variability. The PCA with Eigenvalue more than 1 were selected. Table 4 showed that component PC1 and PC2 best represent the samples with Eigenvalues of 1.623 and 1.002 respectively. These components account for 87.470 % of the total variance with PC1 having 54.086 % and PC2 with 33.384 % (Table 4). Total reducing sugar (0.901) and Total free amino acid (0.889) were best represented in component 1 while the total sugar (0.987) was represented on component 2. Total reducing sugar (TRS) and total free amino acid have strong positive correlation as shown in Figure 1.

Figure. 1. The plot showing relationship between variables
Total sugar has weak positive correlation with Total reducing sugar and negative correlation with free amino acid. This was an indication that the decrease in the total sugar led to increase in total free amino acid.

Figure 2. The plot showing relationship between observations
Samples were grouped according to the hour of fermentation (Fig. 2). Ogi produced from maize (M0), acha (A0), sorghum (S0) are represented at the negative side of PC1 at the beginning of fermentation. Ogi produced from acha (A12) at 12 h, was also represented on the negative axis of PC2.
Ogi produced from maize at 24 h (M24), 36 h (M36), 48 h (M48) and sorghum at 36 h (S36), are grouped together on the positive axis of component 1. Acha fermented for 24h (A24) and 36 h (A36), and (A48) are grouped together at the negative axis of component 2 likewise Ogi produced from sorghum at 12 h (S12) and 24 h (S24) while ogi maize fermented for 12 h (M12) and sorghum for 48 h (S48) were grouped on the positive axis of component 2. Figure. 3 showed that maize ogi had the highest total sugar and starch but were both higher in samples fermented at 12 and 48 h than at 0, 24 and 36 h. The Biplot (Fig. 3.) showed that total sugar (TSS) was high in maize ogi at 12 h, 24, 36 and 48 h and in sorghum at 36 and 48 h. Ogi produced from acha had negative correlation with total sugar from 0 hour to 12 h of fermentation. This showed the longer the fermentation time, the lower the total sugar in acha. It also has negative correlation with sorghum at the beginning of fermentation and at 12 h indicating that TSS increased in sorghum with increase in time of fermentation. Total free amino was low in all ogi powder samples at 0 h (A0, M0 and S0) and only had weak positive correlation at 12 h in maize ogi (M12). It was highest in sorghum at 36 h, followed by maize ogi at 48 h. It also had strong correlation with achaogi fermented between 24 and 48 h and maize ogi fermented for 24 and 36 h. Increase in fermentation time led to increase in total free amino acid (TFA) in all ogi samples produced. Total reducing sugar also increased with increase in fermentation time and was highest in ogi produced from maize. Agglomerative hierarchical clustering showed that acha ogi (A0) and maize Ogi (M0) are related at the beginning of fermentation and are grouped in class one ( Figure. 4). Ogi maize fermented between 12 and 48 h was related to acha ogi at 12 h in class two. Acha ogi fermented between 24 to 48 hours is related with sorghum ogi at 24 h in class 3. Sorghum ogi fermented for 0 h and 12 h was grouped separately in class 4. This shows that ogi from acha is more related to maize ogi than sorghum ogi in terms of total reducing sugar, total sugars and total free amino acid during fermentation of the slurry from these three cereals.