Nutritional and functional attributes of mungbean (Vigna radiata [L] Wilczek) flour as affected by sprouting time

Sprouting of grains improves their nutritional value and functionality, but information on the appropriate sprouting time required to obtain an optimum quality of mungbean flour is limited. This study evaluated the attributes of mungbean flour as influenced by sprouting time. Mungbean seeds were cleaned, sorted, surface‐sterilised, rinsed and sprouted (28°C and 26% R.H) for 24 to 120 hr. Proximate, amino acids (AA), vitamins, mineral, anti‐nutritional (phytate, tannin, oxalate, trypsin inhibitor, raffinose and stachyose) composition, functional properties (viscosity, bulk density and swelling index), microbial quality (total plate and mould counts) and energy of the flours obtained from the sprouted seeds were analysed. Data were subjected to analysis of variance and the means separated by Duncan's Multiple Range Test. Significant (P < 0.05) differences were observed in the energy contents, chemical and functional properties of mungbean flour. There was no fungal growth in the samples until after 72 hr. Leucine, followed by lysine, was the dominant essential AA while methionine was the least. In conclusion, increase in sprouting period improved the nutrient composition but reduced the anti‐nutrients of mungbean flour. Samples sprouted for 24 hr had the highest total essential and conditionally essential AA.

. According to Shah et al. (2011) the protein, crude fibre, ash, and vitamin C contents of mungbean increased while the fat, carbohydrate and phytic acid reduced throughout the 96 hr of sprouting. However, Afam et al. (2016) revealed that all the other nutrients (protein, fibre, calcium, iron, magnesium and potassium) except fat, ash, carbohydrate, phosphorus, sodium, flavonoids and antinutrients increased during a 72-hr sprouting of mungbean. Duration is significant in assessing the impact of sprouting on the properties of grains, and enzymatic activities have been reported to reduce when sprouting went beyond 96 hr (Nkhata et al., 2018). According to El-Adawy et al. (2003) and Elkhalifa et al. (2010) sprouting of cereal grains for 120 hr has desirable influence on their functional properties. Furthermore, Helland et al. (2002) who germinated maize grain for 7 days reported increase in germination period led to increased production of α-amylase and reduced viscosity. Little or no information exists on the influence of sprouting on the functional, microbial and amino acid profile of flour obtained from Nigerian mungbean.
This study therefore determined the nutrient and functional attributes of mungbean flour as influenced by sprouting.

| Procurement and preparation of sample
Mungbean seeds were obtained from a local market in Enugu, Nigeria.
The seeds were sorted, sterilised, washed and sprouted in duplicate for 24, 48, 72, 96 and 120 hr. They were dry-milled and sieved with 60 mesh size screen to obtain the flour (Figure 1).

| Chemicals and reagents
The chemicals, which were all of analytical grade, and the standards of β-carotene, ascorbic acid, vitamins (B1 and B2) were purchased obtained from Sigma-Aldrich.
About 2 g of mung bean flour was put in a flat bottom reflux flask, followed by the addition of 10 ml of distilled water. The content was shaken with care to form a suspension. About 25 ml of 10% KOH in methanol (v/v) solution was added, and with the mounting of a reflux condenser, the flask and its content were heated in a water bath (70-80 C) for 1 hr. The flask was shaken frequently during the heating. On rapid cooling of the flask, 30 ml of water was added. The flask's content was transferred into a separating funnel and extracted three times with 250 ml of chloroform. About 2 g of anhydrous sodium sulphate was employed to remove traces of water. Thereafter, the flask's content was filtered (using Whatman filter paper No. 42) into a 100-ml volumetric flask, and chloroform was added until the 100 ml was reached. Standard solutions of β-carotene (0-50 μg/ml) were prepared. The absorbance of each of the standard solutions was F I G U R E 1 Preparation of mungbean flour (modified method of Imtiaz et al., 2011) taken, from which a standard graph of absorbance against concentration was constructed, and the slope was calculated as the ratio of the absorbance to concentration. The absorbance of the sample solution was read on a Methrohm Spectronic 21 D Spectrophotometer (Gallenkamp, UK) at 328 nm. β À carotene μg 100 g À 1 ð Þ ¼ absorbance of sample Â gradient factor Ã Â dilution factor weight of sample:

| Ascorbic acid, vitamin B1 and vitamin B2
Ascorbic acid was determined by titrimetry as described by Onwuka (2005). A known weight of the sample was mixed with 3% meta phosphoric acid, filtered using a Whatman filter No. 3, and titrated with a standardised solution of 2,6-dichlorophenolindophenol to a faint pink endpoint.
Vitamin B1 was determined according to the modified method of Pearson (1976). About 25 ml of 0.1 M H 2 SO 4 was added to a 100-ml volumetric flask containing 1-g sample. Additional 25 ml of 0.1 M H 2 SO 4 was used to wash down adhering particles on the flask. The flask was put on a boiling water bath to ensure a complete dissolution of the sample in the acid. The flask was shaken frequently in the first 5 min and subsequently every 5 min for 3 hr. The flask was then cooled under running water to less than 50 C. The flask was stoppered and kept at 45-50 C for 2 hr. About 5 ml of taka-diastase in 0.5 M C 2 H 3 NaO 2 solution was added. Thereafter, the flask and its content were made up to 100 ml with water in the dark after mixing thoroughly. The mixture was filtered (Whatman filter No. 42), and 10 ml of the filtrate was transferred into a 50-ml volumetric flask.
Five millilitres of acidic potassium chloride solution (8.5 ml of conc. HCl diluted with 25% [w/v] potassium chloride solution) was then added, shaking thoroughly to mix well. Standard thiamine solutions of range 10-50 mg ml À1 were prepared. The absorbance of both the standard and samples was using at 285 nm using the spectrophotometer.
Vitamin B1 ¼ absorbance of sample Â average gradient Â dilution factor weight of sample For vitamin B2 the method described by Onwuka (2005) was used with modification. About 1 g of sample was put in a 250-ml volumetric flask. Five millilitres each of 5 N HCl and dichloroethene were added sequentially. On shaking the mixture, 90 ml of deionised water was added. The mixture was thoroughly mixed and heated on a water bath for 30 min so as to extract all the riboflavin. This was followed by filtration and cooling. The absorbance of sample and standard was read on a fluorescence Spectrophotometer (DS-11 FX Series, DeNovix Inc) at 460-nm wavelength.
Vitamin B2 ¼ metre reading Â standard equivalence Â dilution factor weight of sample

| Minerals
For mineral determination method 975.03 of AOAC (1990) was used.
The sample was dried by initially drying in an oven at 70-80 C for 2 hr, and then at 105 C until weight was constant. Wet ashing of the sample was done by adding 10-ml nitric-perchloric acid (2:1, v/v) to a flask containing 5-g sample. The flask was heated until a clear digest was obtained. This was followed by cooling and filtration through a For the determination of phosphorus, 25 ml distilled water added to a flask containing 5 ml of the digest. Within 5 min, 10 ml of vanado molybdate reagent was transferred to the flask, followed by mixing.
The P content was obtained from the standard curve obtained from plotting absorbance against concentration.

| Plate count
The plate count was done using the method described by Jideani and Jideani (2006) while the fungal and coliform counts were carried out using the method of Harrigan and McCance (1976). For each microbial determination, serial dilutions (10 À1 to 10 À4 ) of the sample were made with Ringers solution. About 1 ml of each dilution was added to a Petri dish containing 15 ml of the appropriate media (nutrient agar for plate count, Sabroud Dextrose agar for fungal count and Mac-Conkey for coliform count). The petri dish was shaken in a circular movement for 10 s. The plates were then allowed to set and incubated (inverted) in incubator (Gallenkamp, SG-94/02/853) for 24 hr at 38 C for plate count, 72 hr at 38 C for fungal count and 48 hr at 38 C for coliform count. The colonies formed were counted and recorded as colony forming unit (cfu) per gram.
Number of colonies ¼ average count Â dilution factor 2.3.6 | Viscosity, bulk density and swelling power The methods described by Onwuka (2005) were used to determine the viscosity and bulk density while the swelling power was determined by the method described by Ikegwu et al. (2010). The viscosity using a viscometer (Brookfield DV-E, RVDVE230) was measured by mixing sample of mung bean flour with water at a ratio of 1:1.
Viscosity at 30 C ml=s ð Þ¼ Volume of flow to maximum time at 30 C maximum timeused at 30 C A previously weighed measuring cylinder was filled to the 10 ml mark with the sample. The bottom of the cylinder was tapped gently but repeatedly on a laboratory bench until there was no further reduction of the sample level at the 10-ml mark. The cylinder with the sample was weighed. The bulk density of the samples was determined by using the formula: where BD = bulk density in g/cm 3 ; W1 = weight of empty cylinder (g); W2 = weight of cylinder + sample (g); V = Volume of cylinder occupied by the sample (ml).
One gram of the flour sample was mixed with 10-ml distilled water in a centrifuge tube and heated at 80 C for 30 min under continued shaking. After heating, the suspension was centrifuged (Gallemkamp, 90-1) at 1000 Â g for 15 min. The supernatant was decanted and the weight of the paste taken. The swelling power was calculated as follows: Swelling power g=g ð Þ¼ weight of the paste weight of dry flour 2.3.7 | Phytic acid, oxalate, trypsin inhibitor activity and tannin The phytic acid, oxalate, trypsin inhibitor activity, tannin and the oligosaccharides were determined following the methods described by Maga (1983), Onwuka (2005), Kakade et al. (1974), Swain (1979) and Tanaka et al. (1975) The determination of trypsin inhibitor involved the centrifugation of the suspension of the sample and phosphate buffer; digestion with 2% casein solution; termination of the digestion with 5% trichloroacetic acid; and measurement of the absorbance at a wavelength of 280 nm on a spectrophotometer.The Folin-Denis spectrophotometric method was used for the determination of tannins. A known weight of sample was measured into a 50-ml beaker of 50% methanol, covered with paraffin and placed in a water bath at 77-80 C for 1 hr.
It was shaking thoroughly to ensure a uniform mixing. The extract was quantitatively filtered using a double layered Whatman No. 41 filter paper into a 100-ml volumetric flask, 20-ml water added, 2.5-ml Folin-Denis reagent and 10 ml of 17% Na 2 CO 3 were added and mixed properly. The mixture was made up to the 100-ml mark with water, mixed well and allowed to stand for 20 min. The absorbance of the samples was read a Spectronic 21D spectrophotometer at a wavelength of 760 nm.

| Stachyose and raffinose
For the determination of starchyose and raffinose, a known weight of the sample was suspended in 80% ethanol, refluxed for 1 hr,

| Statistical analysis
Triplicate data were analysed using one-way analysis of variance.
Duncan's Multiple Range Test of the SPSS version computer software 20 was used to separate the means of the data. The significance of the determinations was accepted at P < 0.05.

| RESULTS AND DISCUSSION
3.1 | Effect of sprouting on the proximate composition of mungbean flour Except for carbohydrate and fat, the proximate composition of mungbean flour increased as the sprouting period increased (Table 1).  was also reported by Kalimbira et al. (2004), may be due to its use for the metabolic activities of the young shoot. This low energy makes mungbean sprouts beneficial for individuals with obesity and diabetes (Zheng, 1999).

| Sprouting effect on the vitamin content of mungbean
Sprouting leads to the activation of several enzyme systems which brings about notable changes in the chemical constituents of legumes.
As presented in Table 2, there were significant (P < 0.05) increases in T A B L E 1 Effect of sprouting time on the proximate composition and energy content of the mungbean flour the β-carotene, vitamins C, B1 and B2 contents of the sample as the sprouting period increased, and this agrees with previous reports (Fernandez & Berry, 1988;Riddoch et al., 1998;Shah et al., 2011;Uppal & Bains, 2012;Vidal-Valverde et al., 2002;Yang et al., 2001).
Germination has been reported to result in the synthesis of water soluble vitamins (Bibi et al., 2008;Nkhata et al., 2018). 3.3 | Effect of sprouting on the mineral content of mungbean flour Table 3 shows that there were significant increases in the mineral content (Ca, Zn, P, Mg, Fe and K) of the flour samples. Previous researchers (Afam et al., 2016;Dave et al., 2008;Devi et al., 2015;Tizazu et al., 2011) also reported increases in mineral contents of legumes during sprouting, and this may be due to increase in the activity of phytase, which breaks down protein-enzyme-mineral bond to release the minerals (Abdelrahaman et al., 2007;Elemo et al., 2011). Nout and Motarjemi (1997) attributed the increase in mineral contents to the decrease in the antinutrients during sprouting.
On the other hand, sprouting had been reported to have no significant influence on the iron content of cowpea (Bains et al., 2011;Devi et al., 2015).  (Gupta et al., 2015;Shah et al., 2011).

| Effect of sprouting on the antinutrient composition of mungbean flour
According to Murugkar et al. (2013), oxalic acid is broken down to carbon (IV) oxide and hydrogen peroxide during sprouting and subsequent release of calcium. The reduction in the trypsin inhibitor may be due to the proteolytic activity of enzymes during sprouting (Chauhan & Chauhan, 2007). Several other workers (Chopra & Sankhalla, 2004;Devi et al., 2015;Modgil et al., 2009;Uppal & Bains, 2012) also reported that antinutrients of legumes decreased as sprouting period increased. Murugkar and Jha (2009)

| Effect of sprouting on the functional properties of mungbean flour
There was a decrease in the viscosity of the flour samples as the sprouting time increased (Table 5), and this may be due to the loss of water holding capacity of starch granules resulting from their degradation by αand β-amylases that are formed during germination (Helland et al., 2002). The bulk density, which is a measure of heavi-  et al. (2015). This reduction may be attributed to the fact that starch and proteins are broken down during sprouting (Ocheme et al., 2015).
There was a decrease in the swelling power of the flour samples as the sprouting time increased. Similar decrease was reported by Gernah et al. (2011) during the sprouting of maize, and this may be due to the dextrinization of the starches.

| Effect of sprouting on the microbial count of mungbean flour
The total plate and fungal counts of the samples increased with increase in sprouting time (Table 6). Yang et al. (2001) andDziki et al. (2015) also observed a similar occurrence. Aydin et al. (2009) reported that although flour has a low water activity, the indigenous microbial population is diverse and their activities are triggered by the warm and humid conditions characteristic of sprouting.

| Influence of sprouting on the amino acid composition of mungbean flour
Protein quality is influenced by amino acid pattern (Ayalew et al., 2017). There was an increase in the total essential amino acids (TEAA; valine, tryptophan, histidine, isoleucine, leucine, methionine, phenylalanine, lysine and threonine) as sprouting time increased (Table 7). This agrees with Mubarak (2005). This increase may be due to the breakdown of protease-resistant prolamin protein, releasing some amount of amino acids (Afify et al., 2012). Leucine in the range from 6.48 to 8.33 g/100 g was the dominant essential amino acid while methionine (0.85 to 1.85 g/100 g) was the least essential amino acid in all the samples. Among the conditionally essential amino acid, arginine (5.33 to 7.74 g/100 g) was the most abundant amino acid while cystine (0.61 to 1.45 g/100 g) was the least concentration in all the samples. Regarding the non-essential amino acids, glutamic acid (10.75 to 17.50 g/100 g) was dominant while alanine (3.41 to 4.48 g/100 g) was the least amino acid.
The TEAA ranged from 30.13 to 35.67 g/100 g protein with the flour sample obtained from 24-hr sprouted seed having the highest.
Mubarak (2005)  The results of the conditionally essential and non-essential amino acid concentration in this study compares favourably with most vegetable proteins (Mune et al., 2011). Mungbean flour sprouted for 24 hr had the highest TEAA and total conditionally essential amino acid, although flour sample sprouted for at least 96 hr was richer in protein, while 120-hr sprouted sample had the highest contents of fibre, ash, ascorbic acid and minerals elements.

CONFLICT OF INTEREST
All the authors declare that there are no conflicts of interest.

DATA AVAILABILITY STATEMENT
The corresponding author declares the availability of data upon reasonable request.
T A B L E 7 Effect of sprouting time on the amino acid profile of mungbean flour Note: TEAA = total essential amino acid; TCEA = total conditionally essential amino acid; TNEA = total non-essential amino acid; TAA = total amino acid. Means in a row having same alphabets are not significantly different at P > 0.05.