Physicochemical and Functional Properties of Flour and Protein Isolates from Two (2) Solojo Cowpea (Vigna Unguiculata L. Walp) Varieties in Nigeria

: Protein isolates from dehulled defatted solojo cowpea seeds were prepared using isoelectric (CPIA) procedure. Two varieties of cowpea, Dark-Ash Solojo (DAS) and Brown Solojo (BS) were cleaned and divided into six portions. Both varieties of cowpea (DAS and BS) investigated were soaked in distilled water and germinated at varying periods i.e. 0, 6, 24, 36, 48 and 72hrs. Protein isolates were obtained from the treated and processed samples by isoelectric precipitation method which was subsequently followed by proximate and anti-nutritional analyses. Functional properties were also analysed which include Water Absorption Capacity (WAC) and Oil Absorption Capacity (OAC) of the protein isolates using standard methods. Amino acids and molecular weight of the protein isolates were determined by amino acid analyser and sodium-dodecyl-sulphate-polyacrylamide-gel-electrophoresis. Surface morphology, functional group and thermal properties were determined for protein isolates by scanning electron microscopy, Fourier Transform Infrared (FTIR) spectrometry and differential scanning calorimetry, respectively. Data were analysed using design expert software and Analysis of Variance (ANOVA) was carried out at α 0.05 . The moisture content, crude protein, crude fat, crude fibre and total ash of DAS ranged from 9.00-11.40, 24.82-31.00, 1.56-2.66, 1.43-1.67, and 3.20-4.14%, respectively; while those of BS flours ranged from 7.10-9.50, 24.90-30.14, 1.17-2.37, 1.06-1.52 and 3.05-3.93%, respectively. The protein contents for DAS were 81.57±0.53, 86.44±0.84, 89.39±1.51, 90.23±0.53, 91.81±0.77 and 94.85±0.86, while for BS were 84.39±0.39, 85.44±0.56, 90.05±0.10, 90.47±0.89, 92.78±0.28 and 95.81±0.19% for 0, 6, 24, 36, 48 and 72 hrs, respectively.


Introduction
The demand for protein has become so great and is still rapidly increasing due to world population explosion and growth of the food industry, cumulating in increasing demand for animal protein thereby putting pressure on the conventional animal sources. In the developing countries, animal protein such as meat, egg, fish and milk are out of the reach of many because of shortage in supply which has eventually led to increase in cost which makes adequate quantity consumption impossible [1,2].
Consequently, it is undeniable that conventional animal protein sources are not adequate to meet with the demand, and on the long run unsustainable. It has therefore become imperative to look into new sources of protein to reduce the pressure on the existing sources [3][4][5].
According to modern nutrition recommendations, human beings ought to depend majorly on proteins of vegetable and legume origin for their dietary protein needs [6,7]. 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 [8]. Apart from the high cost, large amounts of saturated fat and cholesterol are other problems associated with animal protein sources [9].
Concerns about high-cholesterol, has necessitated the recommendation of regular consumption of vegetable protein as opposed to animal protein by nutritionists. This has led to a renewed interest in legume protein because of their high level of protein which ranged between 20 and 60% [10]. They also have good protein quality in respect to their digestible and nutritional characters. Apart from this, the level of fibre in the body also increases with increased consumption of more plant food that helps in reducing the danger of bowel diseases, as well as cancer of the colon and prevalence of osteoporosis [11]. Compared to cereal grains, legume grains are also very excellent source of weight reduction fibers.
Several efforts by researchers have gone into methods of improving the functionality of protein by different modification methods. Physical modification of food proteins to enhance their capabilities, example, gel formation, adhesiveness, emulsification and foaming has been studied [12]. Instances of the physical modification of proteins include altering the preparation parameters such as temperature and pH. This is done by bringing about partial denaturation of proteins using heat (dry or moist). Denaturation is believed to result in limited unraveling of the tightly crammed structure of the storage globulin proteins or in the regulated unravelling of the poly-peptides which brings about increased availability of sensitive areas of the molecules previously buried, thereby improving the protein functionality [13]. Physical modification using heating, freezing, or extrusion has also been carried out, and this has been found to denature protein structure causing reduced solubility and functionality [14]. Protein modification by high-pressure homogenization, causes insolubilization of proteins [15]. Other modifications that have been investigated include genetic modification [16] enzymatic modification and chemical modification [17].
Another modification method researcher has found is enzymatic modification, which is an uncomplicated and beneficial way to improve sensory and nutrition of plant proteins. Partial hydrolysis by enzymes has been found to improve the foam, gel and emulsification abilities of protein by enhancing its solvation [18]. Emulsification abilities is improved by exposing buried oil-loving groups, improving its surface hydrophobicity and decreasing its molecular mass; these improvements allow for better imbibition along water-oil border. The reason being that such enzymatic polymerization of proteins could be regulated to boost the functionality to the requested level for desired time [19]. Partial enzymatic hydrolysis has also been identified as somewhat easy and beneficial procedure for better palatability and nutritious value of plant proteins [20].
Enzymes, in particular, mammal and microbe transglutaminases also, have been harnessed to alter proteins range of capabilities [21], discovered that this modification considerably enhanced within a large spectrum of pH, the emulsifying capacity and ability to form foam characteristics of the protein polymers of the two beans Cajanus cajan and Lablab purpureus were found to be better than the native protein [22]. As good as this is, it is very expensive.
Biochemical modification (Germination) as a means of improving functionality has not yet been fully exploited. 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
The raw material investigated in this research study is Solojo Cowpea (Vigna unguiculata L.) which occur in two varieties i.e. Dark-ash solojo (DAS) and Brown Solojo (BS). These two underutilized varieties found in South-West region of Nigeria where they are called 'Solojo' were obtained from Bodija market in Ibadan, Western Nigeria. They were stored in polyethylene bags at room temperature (25-26°C).

Methods
The dehulled cowpea seeds were cleaned and screened to get rid of every irrelevant materials and unwholesome seeds. The Solojo seeds (DAS and BS) for germination were sterilized by soaking in 0.07% Sodium hypochlorite [23] for 30 min, then rinsed thoroughly. The Solojo seeds were then immersed for 6 h in distilled water at ambient temperature (1:10 w/v) (~25°C), then placed in a colander and germinated under subdued light in an open laboratory [24] for 0, 6, 24, 36, 48 and 72hrs. Other treated portions of the Solojo seeds (DAS and BS) were dehusked, dried, milled into flour and defatted. Protein was isolated by isoelectric precipitation method. Proximate, antinutritional analysis and functional properties [Water Absorption Capacity (WAC), Oil Absorption Capacity (OAC)] of the flours and protein isolates were determined by standard methods. Amino acids and molecular weight of the protein isolates were determined by amino acid analyzer and sodium-dodecyl-sulphate-polyacrylamide-gel-electrophoresis. Surface morphology, functional group and thermal properties were determined for protein isolates by scanning electron microscopy, Fourier Transform Infrared (FTIR) spectrometry and differential scanning calorimetry, respectively. Data were analysed by descriptive statistics and ANOVA at α 0.05.
Protein isolates (CPIA and CPIB) showed 75% and 76% protein content and a decrease in carbohydrate content from 59.78% to 13%. The nutritional chemical analysis of both raw and germinated seed flours of full fat and defatted darkash and brown solojo cowpea. (FFDAS, FFBS, DFDAS and DFBS) varieties, as well as that for DAS and BS isolates are as shown in Tables 1, 2, 3, 4, 5 and 6.   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 [26]. 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 are as shown in Tables 1-6. Storage stabilit of any food is established by determining the moisture content directly or by ascertaining the dry matter quantity of the sample indirectly. Germination caused the moisture content to decrease with time from Raw (Control) to germinated beans, this observation has similarity to that expressed by Nahid, et al. [27] for Lima beans, where the amount of moisture ranged between 4 to 13%. Ghavidel and Prakash also reported similar observation for lentil, cowpea and chickpea germinated seed flours with decrease in moisture content after removing the hulls.
In 2001, Abdalla et al., [28] in his study, on chickpea flours, also observed an initial increase in moisture content with soaking which later reduced on germinating from 7.69% to 7.30%. Moisture content reduced significantly (p<0.05) with germination. This might be as a result of utilisation of the water for metabolic processes initiated by soaking. On the contrary, observation of increase in moisture content of two varieties of tigernut with germination from 7.14 to 9.98% was made by Chinma et al., 2008 [29]. Likewise, D'souza, made similar observation of increase in moisture for field bean with germination, going from 5.23-11.35 after 60 h of germination, this was adduced to increased number of cells for hydration and low dry matter content.  Observation of moisture content range from 9.19% to 11.83% for five lima bean varieties was made by Yellavila et al., with 'Koloenu brown' having the least moisture content while 'Nsawam black and white' recorded uppermost moisture quantity. Moisture contents of the present study fall within the approved range for flours (<14%). The defatted samples were also noticed to have larger moisture content than the full fat, as was also expressed by Rumiyati et al., [30]. In all, low moisture content of less than 14% is recommended for greater resistance to microbial growth, better storability and prevention of the development of hydrolytic rancidity.
The appreciable increase in protein quantity observed in sprouted Solojo Cowpea could be ascribed to increased formation of some amino acids from protein degradation during sprouting. Chinma et al., also observed a similar increase in protein content of sprouted brown tiger nut from 10.6-12.4% after 48 h sprouting. In 2013, Myrene [31] deduced that significant increase in protein content could be ascribed to improved water activity as a result of activation of hydrolytic enzymes. It could also be due to hormonal changes, according to Nenogaki et al., or a component change resulting from the degradation of other constituents. While Kavitha and Parimalavalli [32] also surmised the increase could be as a result of formation of enzyme proteins.  A comparable increase in protein content was also reported in Ugba (African oil bean), Mungbean; Field beans and Australian sweet lupin upon germination. In 2015, Devi et al., [33] also detected notable rise in crude protein after malting in all the three accerssions of cowpea they worked upon. This observed increase in protein quantity may, according to them, be associated with loss in dry matter, especially carbohydrates due to respiration during malting. Similarly, Zhang et al. [34], observed significant increase in protein content of buckwheat after germination for 72 h, this is probably due to higher rate of protein synthesis compared to proteolysis. On the contrary, El-Adawy et al. [35], observed a reduction in protein content after germinating for 120 h, mung bean, going from 26.40 to 22.52%; Pea from 34.70 to 30.73%; and lentil from 31.41 to 28.37%. Murugkar et al., [36] alluded the rise in protein observed in the nutrient of their mixes to compensative increment in free amino acids and peptides. 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. This assumption was similar to that obtained by Moses et al., for Lima bean (Phaseolus Lunatus).
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. Several researchers have reported the degradation of fat as a result of germination process. Comparable results were obtained for Soya bean, Mungbean, Sesame and three-genotypes of Cowpea on germination [37]. This reduction in oil content on malting, may be connected to its utilisation as a source of energy in malting process [38] energy for germination is obtained through the oxidation of fatty acids to carbon dioxide and water [39]; could also be as a result of enhanced lipolytic enzymes activities during germination [40]. 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 ungerminated samples.
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. This reduction in ash content was parallel to observation in Soybean; Mung bean and Sesame [41]. The reduction in content of ash may be as a result of mineral loss in water during washing in order to minimise the acerbic smell produced over the period of sprouting. On the converse, Chinma et al, observed an increment in ash content for varieties of tigernut also observed weighty rise in the ash content after germination in each varieties of cowpea improved genotype (PL-1, PL-2 and PL-3) used by them, which they surmised as probably due to loss of carbohydrate. 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 indigestible plant material capable of lowering the level of blood cholesterol, preventing cancer, reducing the hazard of developing hypertension, diabetes and hypercholesterolemia is the crude fiber [42]. The crude fibre 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 fibre increasing with germination except that of 36 h which reduced. The experienced reduction is probably due to degradation of fibre into simple sugars brought about by endogenous enzymes. This is collaborated by other research work on chickpea, mungbean, kidney beans [43]. Enujiugha [46] during the study of germination effect on crude fibre for four legumes, observed, that the crude fibre of Kidney bean and Mung bean both decreased with germination, while that of soya bean and peanut increased with germination. Thus, it could be concluded that the effect of germination on crude fibre depended on the nature of the legume. Victor N. Enujiugha et al. in 2003, [47], observed decrease in fiber in soaked peanut, mung bean, wheat, and barley, but contrariwise increased in soaked soy bean and rice. This led to the conclusion that, fiber level is actually affected during the soaking period rather than at the germination proper.
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 observed reduction was similar to the observation of D'souza for field beans, Devi et al. for three genotypes of cowpea. This decrease could be ascribed to the use of carbohydrate to give energy to the growing embryo for germination. The complex carbohydrate is fragmented to smaller sugar molecules such as glucose and fructose needed by the growing seed by the increased activity of α-amylase at early stage of germination [48].

Conclusion
This research work shows that biochemical modification (Germination/Malting/ Sprouting) had an enormous impact on the nutritional composition, functional properties, mineral bioavailability, anti-nutrient content and amino assay of Solojo bean, thus, it could be used as protein supplement in infant, young children and geriatric foods.
Efforts should be increased to promote the cultivation, encourage the consumption and industrial application of this under-utilized legume by the Government, especially in the south-western region where it can survive the rain fall level. Large scale production of this legume which is gradually going into extinction 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 creation of jobs, because people can engage in different aspects of the production process and thereby reducing the rate of unemployment.