Close this search box.

FT-IR Studies on the Influence of Germination on Compositional Differences between Protein Flour and Isolate Samples from Two Varieties (DAS and BS) of Nigerian Cultivated Solojo Cowpea (Vigna Unguiculata L. Walp)

Research Article

FT-IR Studies on the Influence of Germination on Compositional Differences between Protein Flour and Isolate Samples from Two Varieties (DAS and BS) of Nigerian Cultivated Solojo Cowpea (Vigna Unguiculata L. Walp)

  • Olubamike A. Adeyoju 1
  • Henry O. Chibudike 2*
  • Bolanle O. Oluwole 3
  • Kayode O. Adebowale 4
  • Bamidele I. Olu-Owolabi 5
  • Chinedum E. Chibudike 6

1Department of Analytical and Laboratory Management, Federal Institute of Industrial Research, Oshodi, Nigeria

2*Department of Chemical, Fiber and Environmental Technology, Federal Institute of Industrial Research, Oshodi, Nigeria

3Department of Food Technology, Federal Institute of Industrial Research Oshodi, FIIRO, Nigeria

4Department of Chemistry, Industrial Unit, University of Ibadan, Nigeria

5Department of Chemistry, Analytical Unit, University of Ibadan, Nigeria

6Department of Planning, Tech. Transfer and Information Management, Federal Institute of Industrial Research, FIIRO. Nigeria

Citation: OA Adeyoju, HO Chibudike, BO Oluwole, KO Adebowale, BI Olu-Owolabi, CE Chibudike. (2024). FT-IR Studies on the Influence of Germination on Compositional Differences between Protein Flour and Isolate Samples from Two Varieties (DAS and BS) of Nigerian Cultivated Solojo Cowpea (Vigna Unguiculata L. Walp). Chronicles of Clinical Reviews and Case Reports. The Geek Chronicles. 1(1): 1-10

Received: April 12, 2024 | Accepted: May 19, 2024 | Published: July 2, 2024


Effect of germination on nutritional and functional properties of flour derived from two varieties of Solojo Cowpea (DAS Dark-Ash Solojo and BS-Brown Solojo) were studied before and after dehulling of the germinated seeds while the un-germinated portion of the seeds represent the control experiment. The two Solojo Cowpea samples under investigation were immersed in distilled water and germinated at different duration i.e., 0, 6, 24, 36, 48 and 72hrs. Infrared spectroscopy analysis (FT-IR) was carried out to investigate possible differences in composition between the protein flour and isolate samples brought about by germination. Isoelectric precipitation method was used to obtain protein isolates from the treated samples which was subsequently followed by proximate and anti-nutritional analyses. Functional groups were determined for protein isolates by Fourier Transform Infrared (FTIR) spectrometry. Data generated were analyzed using descriptive statistics (ANOVA at α<0.05). It was noticed that Germination generally increase the OAC when compared with the control for full fat, defatted flours and protein isolates. The values of OAC obtained for DAS and BS cowpea at 36 hours were recorded at 2.26 g/g and 2.64 g/g respectively and were observed to be higher than those of the mung bean at same 36 hours which is 0.75 mL/g. Likewise at treatment for 24 hours, DAS and BS cowpea sprouted gave a value of 1.89 g/g and 2.08 g/g respectively contrary to 1.25 mL/g for Mung bean. The isolates had the bulk density varying between 0.36 to 0.50 g/cm3 for the LBD of DAS and 0.47 to 0.59 g/cm3 for PBD; while the BS isolate had for LBD 0.35 to 0.47 g/cm3 and 0.45 to 0.59 g/cm3 for PBD. It was observed that full fat and defatted flours had their water absorption capacity (WAC) increasing with time of germination, and same was observed for the isolates of DAS and BS, with the germinated flour having better WAC than the non-germinated flour (0.95 to 1.94 g/g for FFDAS and 0.94 to 2.10 g/g for DFDAS; 1.41±0.02 to 2.14±0.02 g/g for FFBS; 1.46±0.02 to 2.33±0.01 g/g for DFBS; 2.27±0.06 to 3.41±0.03 g/g for DAS isolate and 2.21±0.09 to 4.15±4.15 g/g for BS isolate).

Keywords: Solojo Cowpea; Under-utilized legumes; BS, DA; FT-IR, Un-germinated, Water Absorption Capacity


Legumes represent one of the main plant sources of proteins in human diet. They are also generally rich in dietary fibre (Rochfort and Panozzo 2007) [10]. Minor compounds of legumes are lipids, polyphenols, and bioactive peptides (Pastor-Cavada et al., 2009) [8]. Legumes provide a good source of protein (18-35%). Plant food diets increase the level of fibre intake which reduces the risk of bowel diseases, including cancer and also reduction in osteoporosis incidence. High protein (18-35%) and carbohydrates (50 60%) contents together with amino acid pattern complementary to that of cereal grains; however, make cowpea a potentially important nutritional component in the human diet. Cowpea (Vignaungiculata L.) provides more than half the plant protein in human diets. (Prinyawiwatkul Legumes et al., 1997) [9]. Most folks in the developing countries rely upon grain legumes as major sources of dietary protein, because, animal proteins are expensive (Shimelis et al., 2006) [11]. Various research efforts are now on going on the application of non-animal proteins for the evolution of innovative nutritional produce or substitute against high- priced animal proteins. (Khalid et al., 2003) [4]. Collaborative efforts towards exploiting the capacity of legumes to curtail the complication of malnutrition (protein) in Africa and to reduce the pressure on the commonly consumed legumes is on-going (Adebowale and Lawal, 2003 [1]. Legumes not only possess significant protein content but also essential protein character, research has also shown their capacity to oppose the action of malnutrition especially in emerging nations by including them in the everyday regime (Butt and Batool 2010) [3]. Functional properties are the physical and chemical characteristics of the specific protein influencing its behavior in food system during processing, storage, cooking and consumption. Examples of functional properties include bulk density, protein solubility, water and oil absorption capacity, emulsifying and foaming properties. The factors that affect the functional behavior of proteins in foods are their size, shape, amino acid composition and sequence, net charge, hydrophobicity, structure, molecular rigidity in response to external environment (pH, temperature, salt concentration) or interaction with other food constituents (Aluko and Yada, 1997) [2]. Formation of an emulsion and its stability is very important for any food systems (Tounkara et al., 2013) [12]. Protein has been found to possess the ability to form and stabilize emulsion. According to modern nutrition recommendations, human beings ought to depend majorly on proteins of vegetable and legume origin for their dietary protein needs (Oreopoulou and Tzia, 2007; Sibt-e-Abbas et al., 2015) [7, 6]. Pulses have been found to play very essential role in achieving the required nutritional recommendations, particularly in emerging and third world countries where the consumption of mammalian protein is low because of the high cost. Apart from the high cost, large amounts of saturated fat and cholesterol are other problems associated with animal protein sources (Klupšaitė, and Juodeikienė, 2015) [5]. Legumes will therefore continue to play important part in diets in the foreseeable future. This work therefore is designed to evaluate the ability of biochemical modification in enhancing the functional properties, and nutritive quality of Solojo protein. Solojo an underutilized legume commonly grown in the South-West region of Nigeria, will be biochemically modified for its possible industrial application through its functional properties.

Materials and Methods

Two varieties of the underutilized cowpea (V. unguculata) found in South west region of Nigeria where it is called ‘solojo’ were used (Figure 1: Brown Solojo Cowpea.  and Figure 2: Dark-Ash Solojo Cowpea).

Figure 1: Brown Solojo Cowpea

Figure 2: Dark-Ash Solojo Cowpea

Seeds obtained from Bodija market in Ibadan, Western Nigeria, were screened to get rid of every irrelevant material and unwholesome seeds. The beans were then portioned into six (6). The solojo seeds for germination were sterilized by soaking in 0.07% sodium hypochlorite for 30 min, then, it was rinsed thoroughly. The solojo seeds were then immersed for 6 h in distilled water at ambient temperature (1:10 w/v) (~25℃), then placed in a colander and germinated under subdued light in an open laboratory for, 24 h, 36 h, 48 h and 72 h (Figure 3).

Figure 3: Preparation of Beans Flour/ Schematic representation

Preparation of flours

Raw flour: The grains were segregated to remove the spoilt ones; then dry dehulled with a mechanical dry dehuller (fabricated in FIIRO), dried at 40℃ and later milled dry to powder then sifted using 80 µm mesh. The flour was stored in flexible bags and preserved at 4℃ preceding utilizations in a refrigerator freezer.

6 h Soaked flour: The seeds were segregated to remove the unwholesome ones, then immersed for 6 h in the ratio (1:10 w/v) (seed/water). The grains were then frozen to prevent germination from setting in, then the hull was removed manually, dried for 48 h at 40℃ later milled dry to smooth powder prior to sieving using 80 µm mesh screen. The resulting flour was packaged in plastic pack and preserved in a fridge freezer at 4℃ pending utilizations.

Figure 4: Germinated Brown Solojo Cowpea

Figure 5: Germinated Dark-Ash Solojo Cowpea

Germination of seed: This was implemented by the method of Mubarak AE with minor adjustment. The seeds for germination were disinfected by soaking in 0.07% sodium hypochlorite for30 mins, then, it was rinsed painstakingly. The solojo seeds were then immersed for 6 hours in distilled water at ambient temperature (1:10w/v) (~25℃), then placed in a colander and germinated under subdued light in an open laboratory for, 24 h, 36 h, 48 h and 72 h (Figure 3). The process of germination was terminated by freezing; the seeds were manually dehulled, dried in a draught oven at 40℃ for 48 h, cooled, milled and packaged in an air tight plastic bag in the refrigerator pending analysis.

Fourier Transform Infrared Spectroscopy

Fourier transform infrared (FT-IR) spectra were recorded with a spectrophotometer (Pelkin Elmer Spectrum BX, FT-IR system) within the spectrum 400–4,000 cm−1, utilizing a resolution of 4.000 cm−1 and four scans. The solid sample (1 mg) was blended with KBr in a ratio of 1:100. Pellets were formed at 6000 psi pressure in a manually operated hydraulic press (International Crystal Laboratories, 12 Ton E-Z Press). The spectra were documented in the transmission method from 4000 to 400 cm-1 with a resolution of 2 cm-1(López-Franco et al., 2013) [13].

Results and Discussion

All experiment was replicated, one-way analysis of variance (ANOVA) was carried out to calculate significant differences in treatments. Differences in mean values were determined using Duncan’s multiple range test at (p< 0.05) (95% confidence level) was used to separate means (SAS1999).

According to Butt and Batoola, 2010, determination of the quality of materials, overall acceptability of the product by consumers and nutritional value establishment are all hinged on proximate analysis of the product. The nutritional chemical analysis of both raw and germinated seed flours of full fat and defatted dark-ash and brown solojo cowpea (FFDAS, FFBS, DFDAS and DFBS) varieties, as well as that for DAS and BS isolates were obtained and already published in previous journals.

The FT-IR analysis showed bands of absorption characteristic of proteins: C=O stretching vibrations characteristic of amide I at 1640 cm-1, the coupling of N-H in-plane bending and C-N stretching modes causes absorption of amide II at 1540 cm-1 while amide III band in 1350- 1190 cm-1 region caused by the C-N stretching coupled to the in-plane N-H bending mode.

Black—-Raw; Blue—–6 h; Red——24 h; Green—36 h; Dark green—48 h; Pink—-72 h

Figure 7. Fourier Transform Infrared (FTIR) spectrometry of FFDAS flours

From the FT-IR spectra analysis study in figures 7 to 12, appreciable increase in protein quantity could be observed in sprouted Solojo Cowpea and this could be ascribed to increased formation of some amino acids from protein degradation during sprouting and significant increase in protein content could also be ascribed to improved water activity as a result of activation of hydrolytic enzymes. It could also be due to hormonal changes. The increase could be as a result of formation of enzyme proteins. This observed increase in protein quantity may be associated with loss in dry matter, especially carbohydrates due to respiration during malting. However, a reduction in protein content was observed after germinating for 120 h.

Black—-Raw; Blue—–6 h. Red——24 h. Green—36 h. Dark green—48 h. Pink—-72 h

Figure 8. Fourier Transform Infrared (FTIR) spectrometry of DFDAS flours

Black—-Raw; Blue—–6 h; Red——24 h; Green—36 h; Dark green—48 h; Pink—-72 h

Figure 9. Fourier Transform Infrared (FTIR) spectrometry of FFBS flours

Black—-Raw; Blue—–6 h; Red——24 h; Green—36 h; Dark green—48 h; Pink—-72h

Figure 10. Fourier Transform Infrared (FTIR) spectrometry of DFBS flours

Amide I band which typically occurs in the region 1700–1600 cm−1 has been shown for all protein extracts to give information about the secondary structure of proteins. The band around 1159.6cm-1 showed the -C-O group from carbohydrates which was absent in the protein isolates. This was corroborated by other researchers. Germination did not bring about the formation of additional functional groups, it only brought about increase in intensity of the bands, by increase in width area.

Black—-Raw; Blue—–6 h; Red——24 h; Green—36 h; Dark green—48 h; Pink—-72 h

Figure 11. Fourier Transform Infrared (FTIR) Spectrometry of DAS Protein Isolates

Black—-Raw; Blue—–6 h; Red——24 h; Green—36 h; Dark green—h; Pink—-72 h

Figure 12. Fourier Transform Infrared (FTIR) Spectrometry of DAS Protein Isolates

The first upper spectra – FFDAS; The second upper spectra- FFBS; The rest are some common cowpea in the market

Figure 13. Fourier Transform Infrared (FTIR) Spectrometry Comparison between Solojo samples and some common market cowpea of DAS Protein Isolates

The DSC curves exhibited the existence of only one broad endothermic peak between 63 and 74oC which coincide with the denaturation temperature (Td) of the PIs analyzed in the powder form. The only peak is that of total protein and consist of various donors as numerous protein portions are expected in the isolates. This was observed to be within the range obtained for the solojo cowpea. Fontanari et al. (2012) also observed that the biggest value of ΔH 335.5 J/g, that is, the energy needed to totally denature the protein isolate (PI), was obtained for the PI isolated at neutral pH. The PIs isolated at pH 10.0 and 11.0 without NaCl, exhibited reduced values of ΔH, 277.2 and 250.3 J/g respectively. pH in the alkaline region gives rise to lesser values of ΔH, indicating that at high pH situation, the protein arrangement is reformed, provoking its unfolding. These outcomes imply that, in protein isolated at close to iso-electric precipitation point pH and with enthalpy close to pH (7), the proteins continue in their natural form, retaining an entwined formation (Fontanari et al., 2012). As reported by Fontanari et al. (2008), in the DSC study of protein isolated from Psidium guajava seeds, it was observed that the temperature peak shifted from Tp of 88.1°C to 107.1°C, suggesting that the presence of ascobic acid in the guava could have induced a reduction in denatured protein during the isolation process and an increment in the thermal constancy of the protein.


The amount of the crude protein of the full fat ranged between 24.82 and 31.00% for FFDAS and 24.90 to 30.14% for FFBS while that of DFDAS and DFBS was between 25.86 to 34.62%, and 25.64 to 31.80% respectively. This is expected because the removal of oil due to defatting reduces the competition of the oil with protein in the flour during analysis. The protein content of the isolates too was observed to increase with germination. The NFE of the FFDAS was also found to be higher than that of DFDAS; this was due to the removal of the fat.

Fat, a major component, which is also an avenue of production of nutritional and biologically active compounds such as fatty acids of the mono- and polyunsaturated class, tocopherols and phytosterols, reduced with germination time for both the flour and the isolate. This degradation of fat is as a result of the germination process. The decrease in fat content is equally very good for shell life stability. The germinated flour and isolate will be able to last longer on the shelf than the un-germinated samples.

Energy for germination is obtained through the oxidation of fatty acids to carbon dioxide and water. This reduction in oil content on malting, may be connected to its utilization as a source of energy in malting process

Ash content generally reduced with germination, only the 48-h protein isolate of DAS and the 6 h isolate of BS had values greater than the control. The reduction in content of ash may be as a result of mineral loss in water during washing in order to minimize the acerbic smell produced over the period of sprouting. The reduction in ash content observed in this project may be as a result of the leaching of both the macro and micro elements as a result of soaking.

The crude fiber of germinated FFDAS, FFBS and DFDAS generally reduced with germination, except for 72 h for all of them and 24 h FFDAS. While DFBS had its crude fiber increasing with germination except that of 36 h which reduced. The experienced reduction is probably due to degradation of fiber into simple sugars brought about by endogenous enzymes.

The total carbohydrate quantity as Nitrogen free extractive was calculated by difference and was found to reduce with rise in germination time for the DAS flour and isolate, while the NFE of the BS variety of both flour and isolate increased with germination.


The high bulk density of germinated flours and protein isolate investigated in this research study shows that the flour and protein isolate will be very useful for infant and geriatric food formulation. This will allow for higher ease of dispersion and also reduce paste thickness, which is a very important attribute in this class of food product.

The solubility of protein at both high and low pH observed with germination in this research study is an advantage because protein solubility is a useful guide for the conduct of protein in the food system.

This research work also shows that biochemical modification (germination/malting/sprouting) had an enormous impact on the nutritional composition, functional properties, mineral bioavailability, and amino assay of solojo bean; thus, it could be used as protein supplement in infant, young children and geriatric foods. More efforts should be geared towards promoting the cultivation of this legume plant. Also, the consumption and industrial application of this under-utilized legume should be encouraged by the government, especially in the south-western region where it can survive the rain fall level. Large scale production of this legume should be encouraged in order to fight the menace of malnutrition in developing countries where animal protein price is exorbitant. This will ensure food security and also create jobs in different aspects of the production process thereby reducing the rate of unemployment and also prevent the crop from going into extinction.


Copyright: © 2024 Henry O. Chibudike, this is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.