Amino Acid Profiling and SDS-PAGE Analysis of Protein Isolates Obtained from Nonconventional Sources

Department of Food Science & Nutrition, TIMES Institute Multan, Multan, Pakistan National Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistan Department of Food Science and Technology, Texas A&M University, College Station, TX, USA College of Agriculture, School of Nutrition, Food Science and Technology, Hawassa University, Hawassa, Ethiopia Kauser Abdulla Malik School of Life Sciences, Forman Christian College University, Lahore, Pakistan


Introduction
High-quality proteins play an imperative role in maintaining better health of an individual. Purposely, the proteins obtained from animal sources are of high quality as compared to plant sources; nevertheless, they are more expensive than vegetable proteins [1]. Owing to the high cost and comparative dearth of food with animal proteins, it has become inevitable to find some new sources of better-quality proteins [2]. In addition, the increasing cost and insufficient provision of animal proteins have diverted the interest of researchers towards some nonconventional protein sources, i.e., high-protein oilseeds [3]. e sesame (Sesamum indicum L.), an imperative oilseed crop belonging to the Pedaliaceae family, contains 25.8-26.9% protein [4]. e sesame meal acquired after oil extraction exhibits a reasonable proportion of high-quality proteins that have the ability to be potentially used as a functional ingredient in numerous food commodities and nutritional supplements [5].
e flaxseed (Linum usitatissimum), belonging to the Linaceae family and commonly known as "Alsi" in IndoPak, is a multipurpose crop mainly cultivated for the production of oil, seed, and textile fiber. It also contains an appreciable amount of high-quality proteins (20%) and polyunsaturated fatty acids [6]. e defatted flaxseed meal contains about 35-40% protein, having a balanced amino acid profile, that has paved the way for its utilization in value-added food products [7].
Canola (Brassica napus L.) is a widely cultivated oilseed crop in Canada, and nowadays, it is grown throughout the world including different areas of the subcontinent. e extraction rate of canola oil is about 40%, and the resultant meal is a rich source of protein. e canola meal contains about 35-36% protein, exhibiting a balanced amino acid profile [6,8].
Proteins can be separated on a molecular weight basis by using the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique [9]. e SDS-PAGE technique gives information regarding the molecular size along with intermolecular disulfide bonds of proteins. e proteins as well as their fractions are presented on an electropherogram and characterized as fingerprints [10].
Amino acids being the important building blocks of protein play an imperative role in determining the protein quality. Defatted oilseed protein isolates contain higher amounts of leucine, glutamine, arginine, and glutamic acid, while lower quantities of sulfur-containing amino acids [11,12]. Previous studies have explicated that defatted sesame seeds contain sufficient quantities of excellentquality proteins with a balanced amino acid profile. e data for essential as well as nonessential amino acids indicated that the highest values were observed for leucine (3.86-7.54 g/100 g) and glutamic acid (12.23-18.67 g/100 g) [13]. Moreover, flaxseed protein contains somewhat high amount of arginine, glutamic acid, and aspartic acid, while lysine, cysteine, and methionine are considered as limiting amino acids. Albumin and globulin are the major types of proteins in flaxseed with globulin fraction up to 73.4% and albumin up to 26.6% of total protein [14]. Furthermore, canola protein isolates (CPI) exhibit higher quantities of leucine, arginine, glutamine, and glutamic acid while lower quantities of sulfur-containing amino acids. e lysine content of CPI mainly depends on the methods of extraction and ranged from 5.04 to 6.34% which is almost equal to infant's requirements. Similarly, CPI contains a considerably higher quantity of threonine (4.49%-5.30%) in comparison with sesame protein isolates (3.98%) [15].
Owing to the fact that animal proteins exhibit high quality as compared to plant sources, however, high cost and insufficient supply of animal proteins demands exploration of some new and nonconventional protein sources [2]. Moreover, cost-effectiveness of food is among the basic concerns for most of the population in developing economies. People demand quality food that must be less expensive and nutritionally sound. Keeping in view the abovementioned facts, the present research project was designed to evaluate the quality of proteins obtained from defatted oilseeds (inexpensive and nonconventional protein sources). ese proteins can be further utilized in numerous food formulations and can serve the purpose of fulfilling the nutritional requirements of individuals, especially infants and young children.

Preparation of the Raw Material.
Oilseeds, i.e., sesame (TS-5), flaxseed (Chandni), and canola (Faisal canola), were procured from Ayub Agriculture Research Institute (AARI), Faisalabad, Pakistan. e seeds were initially cleaned and then ground to fine powder [16]. Moreover, the conventional solvent (hexane) method was employed to extract oil from the selected samples using the Soxtec system (Model: H-2 1045 Extraction Unit, Hoganas, Sweden) [17]. e resulting defatted oilseeds were dried and stored for further processes.

Protein Isolate Preparation.
For preparation of protein isolates (Figure 1), defatted oilseeds were dissolved in distilled water (1/10) with pH 9.5. Furthermore, centrifugation was carried out at 4000 rpm for 20 min to separate the supernatant. Later, the pH of collected supernatant was set at 4.5 following recentrifugation, neutralization, and freeze-drying [18].

Gel Electrophoresis (SDS-PAGE).
Initially, 250 μL sample buffer was used to solubilize the protein isolate samples. In order to perform the electrophoresis on the Bio-Rad Mini-Protean 3 System (Bio-Rad Laboratories, Hercules, CA, USA), 12.5% and 4% stacking and separating gels were used, respectively. Purposely, samples' loading was performed at 10 μL/ lane. A constant voltage (60 V) was supplied for 2.5 hr to run loaded gels till the front dye moved far down the gel. Coomassie brilliant blue (CBB) was used to stain the gels, while methanol water mixture was used for destaining purpose [19].

Amino Acid Profile.
e amino acid profiling was conducted at the University of Veterinary and Animal Sciences (UVAS) Lahore, Pattoki campus. Purposely, a calculated volume of the prepared supernatant was injected using Biochrom 30 + Amino Acid Analyzer [20]. However, for tryptophan, samples were hydrolyzed in the presence of Ba (OH) 2 , isolated through gel filtration, and colorimetrically analyzed.

Amino Acid Score.
e amino acid score was determined by following the amino acid requirement for preschoolers [21,22].

PDCAAS Value.
e protein digestibility corrected amino acid score (PDCAAS) was calculated via true digestibility of respective protein isolates and the lowest amino acid score by the following expression [23]: PDCAAS(%) � true digestibility × lowest amino acid score. (1)

In Vitro Protein Digestibility (IVPD)
. IVPD (%) of protein isolates was determined using the procedure outlined by Aboubacar et al. [24]. For the purpose, protein isolate samples (200 mg) were weighed into Erlenmeyer flasks and mixed with 35 mL of porcine pepsin solution (1.5 g of pepsin/L in 0.1 M KH 2 PO 4 , pH 2.0). Samples were digested for 2 hr at 37°C in a shaking water bath. Digestion was stopped by adding 2 mL of 2N NaOH. Samples were centrifuged (4900 × g, 4°C) for 20 min, and the supernatant was discarded. e residues were washed and centrifuged twice with 20 mL of buffer (0.1 M KH 2 PO 4 , pH 7.0). Undigested nitrogen (N) was determined with a Technicon nitrogen analyzer. Digestibility was calculated as 2.8. Statistical Analysis. All the abovementioned parameters were analyzed in triplicate to ensure the precision and accuracy of results, and the collected data were statistically analyzed using statistical package (Costat-2003, Co-Hort, v 6.1). Accordingly, the level of significance was estimated by analysis of variance (ANOVA) using completely randomized design (CRD) as defined by Steel et al. [25].

SDS-PAGE.
Proteins of resultant isolates, i.e., sesame protein isolates (SPI), flaxseed protein isolates (FPI), and canola protein isolates (CPI), were characterized for their molecular weight using sodium dodecyl sulfate polyacrylamide gel electrophoresis. e electropherogram for sesame, flaxseed, canola protein isolates and the reference standard are illustrated in Figure 2. Respective isolates were recorded ranging from 15 to 65 kDa. e electropherogram also presented numerous fractions having low molecular weights. Moreover, SPI included several polypeptide bands ranging from 15 to 45 kDa, while FPI bands ranged between 25 and 48 kDa. Furthermore, the CPI bands ranged from 16 to 65 kDa with fewer bands than other tested protein isolates. Nonetheless, a trivial difference in the movement was observed in electrophoretic bands of SPI, FPI, and CPI. Certain variations may be attributed to structural as well as compositional changes in proteins along with their interaction with salt. e present findings are in conformity with the outcomes of Vemuri et al.; they delineated the molecular weight between 15 and 50 kDa for sesame proteins [26]. Earlier, a group of scientists explicated that the molecular weight of sesame ranged from 45 to 66.2 kDa [27]. Recently, Chen et al. observed the molecular weight of sesame proteins to be ranging from 09 to 47 kDa at different pH levels [28]. Moreover, SDS-PAGE for major fractions of flaxseed proteins was studied, and molecular weights were observed between 13 and 19 kDa [29]. Likewise, in another research exploration, the molecular weight of flaxseed proteins was demarcated in the range of 10-50 kDa [30]. Furthermore, canola protein isolates exhibited proteins with the molecular weight ranging from 12 to 16 kDa [31]. Similar results (10-29 kDa) were reported for the molecular weight of canola proteins via SDS-PAGE in another study [32]. Moreover, it has been delineated that canola protein isolates exhibit 8 major bands in the range of ∼4.5-66.4 kDa [33].

Amino Acid Profile of Defatted Oilseed Protein Isolates.
e mean values for essential and nonessential amino acids of oilseed protein isolates are given in Tables 1 and 2. ese isolates exhibit better amino acid profile as the protein quality mainly depends on essential amino acids. e maximum lysine content was recorded in CPI as 2.60 ± 0.09 g/100 g followed by FPI (1.62 ± 0.07 g/100 g), while SPI showed the lowest value of 1.48 ± 0.04 g/100 g.
Deficiency of these essential amino acids in diet prevents normal growth and metabolic activities [37]. Furthermore, essential amino acids cannot be synthesized by the body; therefore, these are mandatory to be supplied through diet. Oilseed protein isolates can be obtained with high protein contents, lack of impurities and exhibiting appropriate sensory attributes [38]. Keeping in view the aforementioned amino acid profile, oilseed protein isolates can be potentially utilized in numerous food preparations. ese proteins and their amino acids are essential components of food in order to provide better growth and maintenance to the body.

Amino Acid Score of Defatted Oilseed Protein Isolates.
e amino acid score of defatted oilseed protein isolates was associated with the reference pattern required for preschool children. e respective amino acid scores have been given in Table 3. Sesame protein isolates (SPI) revealed relatively a better essential amino acid score as than FPI and CPI. Oilseed protein isolates exhibited good-quality proteins, ensuring the provision of required amount of essential amino acids for preschoolers [22].
Lysine was found as a limiting amino acid in oilseed protein isolates, i.e., SPI, FPI, and CPI. e protein score of oilseed protein isolates was noted as 28.46, 31.15, and 50.00 for SPI, FPI, and CPI, respectively. In the present study, several essential amino acids in oilseed protein isolates explicated good-quality protein that can be recommended for human utilization [39,40].
Nutritional proficiency of proteins is estimated by their ability to fulfill human amino acid requirements. e amino acid score clearly indicates the existence of different essential amino acids in the samples as compared to the reference pattern. In a recent research investigation, the amino acid profile of different oilseeds like soybean, rapeseed, and Means having the similar letter in a row do not differ significantly. * Aromatic amino acid (phenyl alanine + tyrosine). * * Sulfur-containing amino acid (methionine + cysteine). SPI � sesame protein isolates, FPI �flaxseed protein isolates, and CPI � canola protein isolates. canola were studied to assess their nutritional performance [40]. ey delineated that the tested oilseeds were rich in lysine, leucine, and proline that can satisfy the human needs for essential amino acids. It was also described earlier that oilseeds, legumes, cereals, and their products exhibit appreciable quantities of quality protein comprising adequate essential amino acids. Moreover, the heat treatment can lower the quality of protein by affecting the lysine content during processing [41].

Protein Digestibility Corrected Amino Acid Score (PDCAAS).
e PDCAAS is estimated by the ratio between the first limiting amino acid in sample protein and the respective amino acid in the reference pattern [42]. e present results indicated significant variations among different protein isolates (Table 4) depicting variation in amino acid content and digestibility of protein isolate samples. e PDCAAS results revealed that the maximum value in CPI was 35.17 ± 1.31%, followed by FPI (22.58 ± 0.66%) and SPI (21.98 ± 1.22%).
e PDCAAS is a method to assess the quality of protein requiring description of limiting amino acid and true digestibility. Moreover, the PDCAAS method for protein quality determination is a way to assess the ability of proteins to fulfill human requirements of essential amino acids [43]. Previously, the PDCAAS value of 61% was revealed for extruded flaxseed meal (EFM) [44]. Likewise, the PDCAAS values for rapeseed and mustard protein isolates were noted as 83% and 79%, respectively [45]. Later, it was documented that animal proteins exhibit relatively better quality than plant proteins due to rich amino acid profile ranging from 92% to 100% [46]. e PDCAAS is a simple method of protein quality determination and is advantageous owing to its ease and direct association with human protein requirements. e reference pattern depicts minimum quantity of amino acids required for proper growth and maintenance of body tissues. e PDCAAS is calculated by using three different protein quality assessment parameters, i.e., profile of essential amino acids, their digestibility, and ability to fulfill children's requirements [47]. According to the authenticated technique, the PDCAAS values, i.e., 1.00 or 100%, indicate that protein provides adequate quantity of essential amino acids for children as well as adults [1]. e PDCAAS is extensively used and an approved method for protein quality evaluation of plant-based foods, especially infant formulations. Previously, the World Health Organization (WHO) adopted an alternate method to estimate protein quality in comparison with the PDCAAS.
is method is used to evaluate amino acid scores for 2-to 5year-old children [22].
Finally, the PDCAAS imparted relatively good protein with improved digestibility. e differences in methods of PDCAAS determination have expounded that reference amino acid score affects the PDCAAS value for a product. e accuracy of PDCAAS is recommended by the WHO to determine the protein quality in numerous commodities [48].

In Vitro Protein Digestibility (IVPD).
IVPD is a key factor in determining the availability of amino acids. erefore, it plays an imperative role in nutritional quality assessment of food proteins. e mean values indicated that the highest value of in vitro protein digestibility was recorded for SPI (87.57 ± 4.41%), followed by FPI (85.41 ± 2.04%) and CPI (82.13 ± 2.86%). However, for soy, the IVPD was observed as 91.35 ± 3.12% while 95.42 ± 2.68% for casein (Table 4). Soy and casein are considered as reference proteins. Increase in IVPD mainly depends on elimination of antinutritional factors as well as denaturation of protein during cooking or its exposure to enzymatic action. e instant findings are in accordance with earlier outcomes that demonstrated 79.50% in vitro protein digestibility for canola flour, 80.60% for sesame protein isolates, and 68.0% for flaxseed protein isolates [49]. Later, it was illustrated that the flaxseed meal and sesame seed meal exhibited 83.90% and 81.40% IVPD, respectively [50]. Similarly, 77.9% IVPD was reported in raw brown sesame seed and 85.7% in roasted sesame seed [51].

Conclusions
e current evaluation of defatted oilseed protein isolates indicated that these contain proteins with a wide range of molecular weight (15-65 kDa) along with a balanced amino  Lys * Aromatic amino acid (phenylalanine + tyrosine). * * Sulfur-containing amino acid (methionine + cysteine). * * * Limiting amino acid. SPI � sesame protein isolates, FPI � flaxseed protein isolates, and CPI � canola protein isolates. e values for Lysine have been presented in bold to indicate that it is the Limiting Amino Acid. acid profile and score. Likewise, the PDCAAS and IVPD indicated sufficient availability of quality proteins for the human body in comparison with the reference pattern. Conclusively, defatted oilseed proteins can play an imperative role in improving the nutritional status as well as health and well-being of individuals, especially in developing and underdeveloped economies of the world. Furthermore, these protein isolates can be potentially utilized as ingredients in various food commodities that will certainly help to uplift the nutritional attributes of the products.

Data Availability
e data used to support the findings of this study are included within the article. Disclosure e current research study presented in this manuscript is a part of the PhD research project/thesis of the main/first author (Muhammad Sibt-e-Abbas); hence, a preprint is available in the HEC repository. (http://prr.hec.gov.pk/ jspui/handle/123456789/8378). e researchers intended to get the result of their research study published in a peer reviewed journal, therefore, decided to submit it here. Proper citations for the preprint have also been made [16].

Conflicts of Interest
All authors declare that they have no conflicts of interest.