IntroductionProteins are large molecules consisting of amino acids which are needed for the proper functioning of cells. The body functions, the regulation of the body cells, tissues and organs cannot exist without proteins. But unfortunately people in most developing countries including Nigeria still live with nutritional deficiencies especially protein and vitamins. The protein malnutrition experienced in the developing world, (Nigeria inclusive), is due to poverty level because higher proportion of the populace cannot afford the high cost of animal products such as meat, fish and eggs [1,2]. Hence, there is need to look for a safe, cheap and yet affordable protein sources Legumes have been the major sources of low-cost proteins which are consumed worldwide, when combined with cereals or grains, a complete protein is achieved. Plant proteins have been used to fortify cereal-based diets [2]. However, the existing problems of food security and malnutrition coupled with escalating population, uncertain crop yield and high cost of animal based food supplies has necessitated the need to identify and incorporate unconventional protein sources to enrich the traditional formulation [3].
Protein isolates are refined form of protein that contains a greater amount of protein with better digestibility. It is currently a major source of cheap proteins for sportsmen and vegetarians [4]. Protein isolates are the acceptable ingredients for dairy application due to their fine particle size, dispersibility, emulsification properties, color and flavor which are critical in dairy applications. Isolates (especially soy proteins) are being used to fortify all types of pasta products such as macaroni, spaghetti, to improve the nutritional value. They are also utilized in meat and baking applications due to their water and fat absorption properties [4].
Soy protein has also been combined with other food ingredients such as vitamins; minerals and flavor in the preparation of soy protein shake powder [5]. Protein isolates has the potential of combating the problem of malnutrition. Technological advancement has enhanced the commercialization and marketing system of soy bean protein isolates leading to the neglect of other legumes. Other underutilized plant sources apart from soybean can also be exploited for protein extraction and also make available for use as food supplement. Pigeon pea (Cajanus cajan) is a perennial legume, a member of the family fabaceae. It is widely cultivated in all tropical and subtropical region of the world [6]. Pigeon peas are used both as food crop and as forage/ cover crop. In combination with cereals, pigeon pea makes a wellbalanced human food. It is reported to contain 20 - 22% protein [6].
Lima bean (Phaseolus lunatus) is one of the most widely cultivated pulse crops both in the temperate and subtropical regions. Lima bean like other legumes is a fat-free source of highquality protein which compares favorably with other legumes. [7]. While studies on functional properties and proximate were done on the seed flour of lima bean by [7], the present research is focused on investigating the chemical composition and functional properties as well as the quality evaluation of the protein isolates extracted from these underutilized legumes. This assessment may provide answers to possibility of their utilization in food
formulations and food products.
Materials and Methods
Materials
Mature lima bean seeds (Phaseolus lunatus) and pigeon pea seeds (Cajanus cajan) were obtained from Oja-oba market in Ado-Ekiti, Ekiti state, Nigeria. The samples were authenticated by a seed scientist in the Department of Crop, Soil and Pest management of The Federal University of Technology, Akure, Ondo State, Nigeria. All chemicals used were of analytical grade.
Sample Preparation
The samples were sorted manually to remove bad seeds and other extraneous materials. The cleaned healthy seeds of both samples were soaked at room temperature separately in water for six hours to facilitate the removal of the hulls. Dehulling was done manually and dried in the oven at 50 degree C for 48 h. The dried cotyledons were milled into powdered form. The milled samples were defatted using n-hexane for nine hours in a soxhlet extractor after which the defatted samples were air-dried at room temperature and set aside for protein isolation.
Production of the Protein Isolates
In preparation of the protein isolate, defatted seed flour was dispersed in distilled water at a meal ratio of 1:20 w/v (flour/ water). The mixture was stirred with a magnetic stirrer for 30 minutes after which the pH of the slurry was adjusted to the pH at which the protein in the flour is most soluble (predetermined for each sample) using 0.1M HCl or 0.1M NaOH drop wisely. The solution was further stirred for 2 hours at 30 +/- 2 degree C using a Gallenhamp magnetic stirrer to enhance high degree of protein solubility. The solution was centrifuged at 4,000rpm for 30 minutes at 4 degree C. The residue obtained after decanting the supernatant was re-extracted with half the volume of the same solvent under similar conditions. The pH of the combined supernatants was adjusted to the pH at which the protein in the flour is least soluble (the isoelectric point which has been predetermined for each sample) with 0.1M HCl to precipitate the protein. The isolate was recovered by centrifugation for 30 minutes at 4 degree C after which it was dispersed in distilled water, poured in dialysis tube and dialyzed against distilled water for 18 hours. The dialyzing water was replaced at intervals of 3 hours during the period of dialysis. The dialysate was freeze dried and then stored in air-tight container in the deep freezer for further analysis (Figure 1).
Chemical Analyses
The proximate composition was determined using the method of [8].the carbohydrate was calculated by difference between 100 and the summation of other proximate parameters. Determination of Phytic acid: The method of [9] as described by [10] was used to determine Phytin. Two grams (2g) of finely ground samples was soaked in 100 ml of 2 % HCl for 3 hours and then filtered. The filtrate (25 ml) was placed in a 100 mL conical flask and 5 ml of 0.03 % NH4SCN solution was then added as indicator. 50 ml of distilled water was then added and was titrated with ferric chloride solution which contained 0.005 mg of Fe3+ per ml of FeCl3 used, the equivalent was obtained and from this, the phytate content in mg/100g was calculated.
Iron equivalent = titration value (v) x1.95 x1.19 = phytin P mg/g. {1}
Phytic acid = titration value (v) x 1.95 x 1.19 x 3.5mg/phytic acid {2}
Source: [9]
Determination of tannin: The tannin content of the seed flour was determined by modifying the procedure of [11]. Finely ground sample (0.2g) was weighed into a 50 ml sample bottle. About 10 ml of 70 % aqueous acetone was added and properly covered. The bottles were put in an ice bath shaker and shaken for 2 hours at 300C. Each solution was then centrifuged and the supernatant stored in ice, 0.2 ml of each supernatant was pipetted Sanchezinto test tubes and 0.8 ml of distilled water was added. Standard tannic acid solution was prepared from a 0.5 mg/ml stock and the solution made up to 1ml with distilled water. About 0.5 ml of Folin reagent (colour developer) was added to both sample and standard, followed by 2.5 ml of 20 % Na2CO3. The solutions were then allowed to incubate for 40 mins at room temperature after which absorbance was read at 725nm against a reagent blank. Concentration of the sample was obtained from a standard tannic acid curve.
Determination of Amino Acid profile: The amino acid profile of the seed flour and protein isolates were determined. The sample was defatted, hydrolysed and evaporated in a rotary evaporator and then injected into the Technicon sequential multisampling amino acid analyser (Technicon CO. Ltd., United Kingdom), as described by [12-14].
Evaluation of protein quality: The amino acids obtained were used to evaluate the protein quality of the seed flours.
Predicted Biological value (BV) was calculated using the regression equation of [15] as reported by [16, 17]
The predicted protein efficiency ratio (PER) was calculated using one of the equations developed by [17] as stated below.
PER = -0.468 + 0.454 (Leu) - 0.105 (Tyr) {7}
Determination of In-vitro Multienzyme Protein Digestibility (IVPD)
The in-vitro multienzyme protein digestibility of all the samples was determined following the procedure of [18] as reported by [19]. The enzymes used were purchased from Rovet Scientific, Benin City, Edo State, Nigeria. They are porcine pancreatic trypsin, bovine pancreatic chymotrypsin and porcine protease type xxiv peptidase all from sigma USA. The activity of the enzymes was initially determined before use by using them to digest casein. All the samples were ground into fine powder. Each of the samples was dispersed in 50ml distilled water to give a sample suspension of 6.25mg protein per ml. The pH of each sample suspension was adjusted to pH8.0 with 0.1M HCl and 0.1M NaOH and incubated in water bath at 37 degree C with constant stirring. Fresh multienzyme solution was prepared to contain 1.6mg trypsin, 3.1mg chymotrypsin and 1.3 mg peptidase per ml. The pH of the multienzyme solution was also maintained at 8.0. Five milliliters (5 ml) of the multienzyme solution was added to each sample suspension with constant stirring at 37 degree C. The pH of each sample suspension was recorded at10mins after the addition of the enzyme solution. The IVPD was calculated using the regression equation.
Y = 210.464 - 18.103x {8}
Y = invitro protein digestibility (%)
X = pH of sample suspension after 10mins.
Functional properties of the protein isolates.
Determination of water and oil absorption capacity (WAC and OAC): The WAC was determined using the procedure of [20].
The foam capacity and stability (FS) were determined after 2 h using the method [21]. The least gelation concentration (LGC) was determined as described by [22]. The emulsion capacity and stability were determined as described by [23]. The variation of protein solubility with changing pH was determined as described by [24].
Data analysis
All data used were means of triplicate analysis (n=3) determinations. The coefficient of variation (CV) between the different products were also determined
Results and Discussion
The result of the chemical composition and invitro multienzyme digestibilities of the protein isolates is shown in (Table 1). Lima bean protein isolate had 93.50 +/- 0.01% protein while pigeon pea isolate contained 92.54+/-0.01% protein. The values were higher than those reported for some legume protein; mung bean isolate, 87.9% [25], chickpea isolate 88.1% [26] and Canarvalia einsformis, 73.3% [27]. The variations in protein contents of the different legume protein isolate were attributed to genetic makeup of legumes along with some environmental factors [28]. The moisture content of both isolates was very low. A higher ash content was observed in pigeon pea isolate (3.56+/-0.06%) while the percentage crude fiber in both isolate were low. The values obtained for the ash content in lima(1.13+/-0.02%) and pigeon pea(3.56+/-0.06%) were much higher than those reported for green pea (0.50 - 0.93%) and grass pea (0.20 - 1.20%) but these results were in agreement with those reported for mung pea and field pea [29,30]. The protein isolates had reduced content of phytate in lima bean (1.40+/-0.01%) than 5.32+/-0.04% in pigeon pea while tannin contents were 0.14+/-0.01 and 0.23+/-0.05% in lima bean and pigeon pea respectively. Thus, indicating that these compounds were largely removed under alkaline condition employed for the preparation of the protein isolates. Polyphenols react with proteins yielding dark protein isolates and decreases the bioavailability of several amino acids. Low levels of polyphenols are therefore desirable. Both protein isolates had high invitro digestibilities of 94.26+/-0.01% .This result is comparable with those observed in other legume protein isolates like 90% for rapeseed [31] and 90-94% for Chickpea protein isolate (Sanchezinto Vogue et al., 1999). Digestibility is limited by the presence of trypsin and chymotrypsin inhibitors and the globular structure of protein. The removal of protease inhibitor in protein isolates during extraction (since they are albumin) increases the invitro digestibility of the isolates [32]. The reduced level of phytic acid during extraction can also increase digestibility of the proteins. The isolates could, therefore, be used in new food formulation based on its high digestibility and low phenolic compound. (Table 2) show the amino acid composition of lima bean and pigeon pea protein isolates. It was observed that glutamic and aspartic acid were the predominant amino acid in both proteins while methionine and cystine were the least. This observation is consistent with majority of other plant proteins. Glutamic acid was the highest predominant amino acid in Zonocerus variegates (133.7 mg/g) [33]. Adedeye & Omotayo [34] also reported glutamic as the most concentrated amino acid in pumpkin seed kernels and T. occidentalis (90.8mg/g). From the result the most concentrated essential amino acid was leucine with a higher value of 72.42+/-0.03mg/g (lima bean) and 53.00+/-0.02mg/g for pigeon pea. Essential amino acid like leucine, lysine, methionine, phenylalanine and Arginine in lima bean protein were better than in pigeon pea
Total essential amino acid of the lima bean and pigeon pea protein (Table 3) were 466.75+/-0.39mg/g and 418.23+/-0.22mg/g
respectively. Lima bean protein will satisfy the amino acid requirement for infant (460mg/g) [35]. The total sulphur amino acid for both were lower than the 56mg/g crude protein recommended for infants [36]. The aromatic amino acid range suggested for ideal infant protein is 68-118mg/g crude protein [36]. The protein isolates from both legumes will satisfy the requirement and thus indicating that these isolates can be used to supplement weaning foods. The percentage ratios of essential amino acids to the total amino acids (which signifies the optimal nitrogen retention and utilization) in both isolates were 51.38+/-0.17 and 47.83+/-0.01% respectively. These values were well above the 39% considered to be adequate for ideal protein food for infants, 26% for children and 11% for adults [35]. The percentage of essential total amino acids in the lima bean was comparative to that of egg. The values obtained for pigeon pea isolate was comparable with 43.08-44.4% reported for beach pea protein isolate [37].
The amino acid score (Table 4) showed that amino acid composition was more balanced in lima bean protein isolate than pigeon pea protein isolate. However, methionine, cysteine, valine and threonine were the limiting amino in the isolates. The predicted biological value (P-BV)( which is one of the quality parameters used for protein evaluation) for pigeon pea protein isolate was higher than the suggested biological value for plantbased proteins [35] while that of lima bean was comparable to the beach pea protein isolate (36.5 - 40.13)reported by [15]. The predicted protein efficiency ratio (P-PER) of 2.57(lima bean) was higher than 1.79 (pigeon pea) indicating that lima bean protein isolate was of better quality.
The functional properties of lima bean and pigeon pea are presented in (Table 5). The potential uses of protein isolates depend largely on their functional properties. Significant differences in water absorption, foaming and emulsification properties were observed between the isolates. Lima bean and Pigeon pea protein isolates had low water absorption (0.31+/-0.12%; .024+/-0.1) respectively and low oil absorption capacities (0.20+/-0.12%; 0.29+/-0.06%) while lima bean had a better foaming (36.08+/-0.31%) and emulsification (46.60+/-0.04%) properties than pigeon pea isolate 16.40+/-0.08 and 34.00+/-0.15% respectively. Both protein isolates had the same least gelation concentration of 4%. These values for water absorption capacity for both isolates were lower than those reported for beach pea protein isolate (257-288%) [16], woodstone pea protein isolate (278-293%) reported by [29]. The low values in the water and fat absorption capacities of the protein isolates may be due to the low level of starch and fiber compared to fiber products or whole flour, where flour is milled from whole seeds containing hulls. However, water binding capacity of protein is a function of several parameters including size, shape, steric factors, conformational characteristics and hydrophilic -hydrophobic balance of amino acids in the protein molecules. The higher foaming properties observed in Lima bean protein could be attributed to the solubility of the protein which facilitated adsorption of protein at the air-water interface. This implies that the isolates can be used in food products such as cake, breads and ice creams. The high emulsion properties also implied that lima bean protein can be used in desserts and toppings. Both protein isolates had low least gelation concentration capacity and this suggest good gelation property. Protein gelation is vital in the preparation and acceptability of many foods, including vegetables and other products [37].
The effect of pH on the protein solubilities of both isolates were investigated (Figure 2). The solubilities decreased as the pH increased up to the isoelectric point at pH5 after which it increased at the alkaline region. The proteins of both legumes were observed to be more soluble at the alkaline region. This implies that they could have promising food applications.
Conclusion
Judging from the result of chemical composition of the protein isolates, it was revealed that they had improved protein contents; they also had high levels of amino acids when compared to recommended levels for infants. The predicted biological value and protein efficiency showed that the protein isolates (especially lima beans) were of better quality. They had low polyphenolic compounds which increased the digestibilities The result of the functional properties showed that they had low water and oil absorption; however, they have appreciable emulsion capacity and gelation properties which implies that the isolates could be potentially useful in food formulations.
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