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Abdulrahman Ali A, Buba F, Tijani Y, Musa H, Abdullahi Damasak A, M. Adamu M, et al . Isolation and Characterization of Soluble Proteins from Moringa (Moringa Oleifera Lam) Seeds for Possible Use in Food Industries. JNFS 2025; 10 (3) :373-383
URL: http://jnfs.ssu.ac.ir/article-1-1103-en.html
URL: http://jnfs.ssu.ac.ir/article-1-1103-en.html
Ali Abdulrahman Ali * 
, Fatimah Buba , Yahaya Tijani , Hadiza Musa , Ali Abdullahi Damasak , Mohammed M. Adamu , Binta Baba Shehu , Abubakar Gidado
, Fatimah Buba , Yahaya Tijani , Hadiza Musa , Ali Abdullahi Damasak , Mohammed M. Adamu , Binta Baba Shehu , Abubakar Gidado
Department of Biochemistry, University of Maiduguri, Maiduguri, Nigeria
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| Abstract (HTML) (376 Views)
Table 2 shows the protein content of the isolates. The aqueous extract contained 73.0±0.5 g/100 g protein, while the NaOH precipitate fraction and the (NH4)2SO4 fraction had 54.0±0.2 and 45.0±0.3 g/100 g protein.
The results of the in vitro protein digestibility of the aqueous extract, the alkaline precipitated extract, and the ammonium sulphate precipitated extracts are presented in Table 3. The results indicated that a high digestibility value was recorded for the aqueous extract after 6 hours of digestion (82.63±0.27). In contrast, the alkaline precipitated fraction (76.85±0.61) and the ammonium sulphate precipitated fractions (77.91±0.12) had higher values than the aqueous extract after 1 hour of digestion.
The UV absorption spectra of the M. oleifera seed protein isolates are illustrated in Figure 1. The three protein isolates had shown strong absorption at 280 nm which indicates that protein is present in all the protein isolates.
The amino acid composition of the M. oleifera seed protein isolates are shown in Table 4. The essential amino acids are all present in the seed, with histidine having the lowest content and phenylalanine the highest amount 8.93 mg/100g in the (NH4)2SO4 fraction.
Figure 2 describes the solubility of the protein isolates. The solubility of all the fractions was higher at alkaline pH values. The NaOH precipitated fraction and the ammonium sulphate fraction are more soluble than the aqueous
extract.
.PNG)
.PNG)
.PNG)
The water-holding capacity and oil-holding capacity of M. oleifera seed protein isolates are shown in Figure 7. The results showed that (NH4)2SO4 fraction had a higher water-holding capacity (2.8 g/g) than the aqueous extract and the NaOH fractions. The fraction with the most
oil-holding capacity was the aqueous extract
(3.2 g/g).
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Isolation and Characterization of Soluble Proteins from Moringa
(Moringa Oleifera Lam) Seeds for Possible Use in Food Industries
Ali Abdulrahman Ali; MSc*1, Fatimah Buba; PhD1,2, Yahaya Tijani; PhD1, Hadiza Musa; MSc1, Ali Abdullahi Damasak; MSc3, Mohammed Adamu Milala; PhD4, Binta Baba Shehu; PhD1 & Abubakar Gidado; PhD1,2
1 Department of Biochemistry, University of Maiduguri, Maiduguri, Nigeria; 2 Northeast Zonal Biotechnology Center of Excellence, University of Maiduguri, Maiduguri, Nigeria; 3 Department of Nutrition and Dietetics, University of Maiduguri, Maiduguri, Nigeria; 4 Department of Biotechnology, University of Maiduguri, Maiduguri, Nigeria.
(Moringa Oleifera Lam) Seeds for Possible Use in Food Industries
Ali Abdulrahman Ali; MSc*1, Fatimah Buba; PhD1,2, Yahaya Tijani; PhD1, Hadiza Musa; MSc1, Ali Abdullahi Damasak; MSc3, Mohammed Adamu Milala; PhD4, Binta Baba Shehu; PhD1 & Abubakar Gidado; PhD1,2
1 Department of Biochemistry, University of Maiduguri, Maiduguri, Nigeria; 2 Northeast Zonal Biotechnology Center of Excellence, University of Maiduguri, Maiduguri, Nigeria; 3 Department of Nutrition and Dietetics, University of Maiduguri, Maiduguri, Nigeria; 4 Department of Biotechnology, University of Maiduguri, Maiduguri, Nigeria.
| ARTICLE INFO | ABSTRACT | |
| ORIGINAL ARTICLE | Background: Proteins are crucial for the functional properties and sensory characteristics of foods. This study aims to isolate and characterize soluble proteins from Moringa oleifera seeds. Methods: The seeds were defatted using chloroform, methanol, and water (1:2:0.8). Protein isolates were prepared by aqueous extraction, 0.1M NaOH precipitation, and 60% (NH4)2SO4 precipitation. Moreover, proximate composition of the dry seeds, and the protein yield, physicochemical properties, functional properties, and amino acid composition of the isolates were determined using standard methods. Results: 100 g of dry seed contained 29.09±0.40 g proteins. The aqueous extract had the greatest protein content (73.0±0.5 g/100g), followed by NaOH (54.0±0.2 g/100g) and (NH4)2SO4 (45.0±0.3 g/100g). As a result, the aqueous extract showed a significantly (P<0.05) maximum in vitro digestibility (82.63±0.27g) after 6 hours. Strong UV absorption at 280 nm was recorded for all the protein isolates. All essential amino acids were present, and phenylalanine was the chief amino acid (8.93±0.67 mg/100g in (NH4)2SO4 fraction) which was significant at P<0.05. NaOH and (NH4)2SO4 fractions were most soluble at pH 10 which were significant at P<0.05. The NaOH fraction had the peak foaming capacity (28.0±0.4%) at pH 10, and the most stable foam was observed in the NaOH fraction after 10 minutes. Significantly (P<0.05) elevated in the (NH4)2SO4 fraction (32.0±0.6%), followed by aqueous extract (28.0±0.5%) at pH 10 was the emulsifying capacity. The (NH4)2SO4 fraction had significantly (P<0.05) more water-holding capacity (2.80±0.04 g/g); the prime oil-holding capacity was also observed in the aqueous extract (3.20±0.02 g/g). Conclusion: Moringa oleifera seeds possess essential amino acids and functional properties beneficial for food systems, potentially enhancing value or developing new products. | |
| Article history: Received: 7 Aug 2024 Revised: 22 Feb 2025 Accepted: 28 Feb 2025 |
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| *Corresponding author aliabdulrahman@unimaid.edu.ng Department of Biochemistry, University of Maiduguri, Maiduguri, Nigeria. Postal code: 600004 Tel: +234 7030523419 |
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| Keywords Amino acids; Essential amino acids; Phenylalanine; Moringa oleifera; Seeds. |
Introduction
Moringa oleifera (M. oleifera), also called horseradish or drumstick, is a tropical plant native to India and is cultivated all year round in Asia, sub-Saharan Africa, and Latin America (Vijayaraghvan et al., 2011). The seeds, leaves, bark, and roots of this plant possess numerous applications in nutrition, medicine, and commercial purposes (Anwar et al., 2007, Lakshmipriya et al., 2016). Earlier researchers found that M. oleifera seed is not toxic (Bakare, 2016, Yuliastri et al., 2016). The seed of this plant is very common and easy to store, especially in developing countries (Sánchez-Martín et al., 2012). Ndabigengesere et al. argued that the active agents of M. oleifera are found in the seed kernel rather than seed bark (Ndabigengesere et al., 1995). The seed kernel was reported to possess significant quantities of a series of low molecular weight, water-soluble proteins that carry an overall positive charge in solution (Jayalakshmi et al., 2017).
There has been a growing and strong interest in plant proteins; so, the authors turn to alternatives such as plant-based proteins which have reduced carbon footprint, higher sustainability, lower price, and non-allergenicity (Jain et al., 2019). Plant-based proteins are considered vegan food, provide an ample number of amino acids, are directly absorbed by the body, and help in treating various disease ailments. Successful functional plant-based protein product is the soy protein which includes soy protein isolate, soy protein concentrate, and textured soy protein (Singh et al., 2008). Nevertheless, there are people who are allergic to soy protein and need an alternative to soy as well as animal proteins ;also, there is a strong interest in evaluating other plant proteins besides soy (Małecki et al., 2021). M. oleifera seeds represent another source of plant-based proteins with potentially important functional properties that could enhance the structure, texture, and organoleptic properties of food products. This study aims at isolating and characterizing some soluble proteins from M. oleifera seeds.
There has been a growing and strong interest in plant proteins; so, the authors turn to alternatives such as plant-based proteins which have reduced carbon footprint, higher sustainability, lower price, and non-allergenicity (Jain et al., 2019). Plant-based proteins are considered vegan food, provide an ample number of amino acids, are directly absorbed by the body, and help in treating various disease ailments. Successful functional plant-based protein product is the soy protein which includes soy protein isolate, soy protein concentrate, and textured soy protein (Singh et al., 2008). Nevertheless, there are people who are allergic to soy protein and need an alternative to soy as well as animal proteins ;also, there is a strong interest in evaluating other plant proteins besides soy (Małecki et al., 2021). M. oleifera seeds represent another source of plant-based proteins with potentially important functional properties that could enhance the structure, texture, and organoleptic properties of food products. This study aims at isolating and characterizing some soluble proteins from M. oleifera seeds.
Materials and Methods
Sample collection and preparation of M. oleifera seed
Dry M. oleifera seeds were purchased from Gamboru local market, Maiduguri, Nigeria. The seeds were identified by a plant taxonomist at the Department of Biological Science, University of Maiduguri, and a Voucher specimen was deposited at the herberium. The seeds were ground into flour using mortar and pestle and were stored in a clean air-tight container for further analysis.
Proximate composition of M. oleifera seeds
The proximate composition of the seeds of M. oleifera was carried out using the standard procedures of the Association of Official Analytical Chemists (Association of Official Analytical Chemists (AOAC), 2005) on a dry basis.
Extraction and isolation of M. oleifera seed proteins
The ground powdered M. oleifera seeds (110.5 g) were defatted using a mixture of chloroform, methanol, and water in a ratio of 1:2:0.8. This was homogenized in the solvent to extract all lipids, more water was added to the resulting extract, and the mixture was separated into two phases, methanol/water (top phase) and chloroform (bottom phase). The lipids remaining in the chloroform layer were separated from the extract mixture, while the seed proteins in the methanol/water layer were collected and allowed to air dry. The dried seed extract was divided into three portions. The first portion was resolubilized and was considered as the aqueous extract containing seed proteins from M. oleifera seeds. The second portion was prepared by precipitating the proteins with 0.1 M NaOH (1:10, w/v), continuously stirred for 60 min, and subsequently centrifuged at 10000 rpm for 30 minutes. The seed meal was collected, air-dried and then stored in a clean container. The third portion was similarly prepared by the addition of 60% (NH4)2SO4 with continuous stirring for 3 hours and subsequently centrifuged at 10000 rpm for 30 minutes and air-dried and stored in a clean container to obtain the isolate. Protein yield of the isolates were determined as described by Jain et al. using the following formula (Jain et al., 2019):
Protein isolate yield (%) = weig h t of protein in t h e fraction weig h t of defatted moringa seeds X 100
Determination of in vitro protein digestibility
In vitro digestibility was determined according to the method described by (Association of Official Analytical Chemists (AOAC), 2015). One millilitre of 11% Trypsin was introduced into 2 test tubes. Subsequently, 4 ml of phosphate buffer with pH 7.5 was added to each test-tube and 1 ml of 0.1 N HCl was also added and allowed to stand to equilibrate, after which 1 ml of 1% sample was added to all the test tubes (labelled as digestibility at 1 and 6 hrs). The reaction in each test tube was stopped with 5 ml of neutralized formalin at 60 min and 6 hrs. The content of the test tubes were then filtered using filter paper. The filter papers were dried in an oven at 108°C for 3 hr. The nitrogen of the undigested sample was determined by the Kjeldahl method:
Percentage in vitro digestibility = ( CP 1- CP 2)/ CP 1 100
Where CP1 = Total protein of sample;
CP2 = Total protein after digestion with trypsin.
Where CP1 = Total protein of sample;
CP2 = Total protein after digestion with trypsin.
Ultraviolet absorption spectra of protein isolate
After being dissolved in 100 ml of deionized water, the protein was scanned using a UV-visible spectrophotometer in the range of 240 nm to 310 nm (Senthilraja, 2011).
Amino acids analysis
The defatted samples were utilized to estimate amino acids as described by (Association of Official Analytical Chemists (AOAC), 2000). The defatted samples were utilized to estimate amino acids. The sample (30mg) was then hydrolysed with 6N HCI at 110 °C for 24h. Amino acid analysis was performed on reverse phase-high pressure liquid chromatography (HPLC) (Buck scientific BLC 10/11 USA) equipped with UV 338nm detector. A C18, 2.5 x 200mm, 5um column and a mobile phase of 1:2:2 (100mM sodium phosphate, pH 7.2: Acetonitrile: methanol) was used at a flow rate of 0.45 mL/minute and an operating temperature of 40 °C. Mixed standards were analyzed in a similar manner for identification. After that, peak identification was conducted by comparing the retention times of authentic standards and those obtained from the samples, these data were later integrated using peak simple chromatography data system processor; (Buck SCi. chromatopac data processor).
Determination of functional characteristics
Protein solubility: The solubility of the protein was conducted according to the method described by Kumar et al. (Kumar et al., 2014). The sample (100 mg) was dispersed in 5 ml of distilled water. The pH of the mixture was adjusted to 4, 6, 8, 10, and 12 using 0.1 M HCl or 1 M NaOH. Then, the solution was stirred for one hour at room temperature and centrifuged at 4,000 x g for 20 minutes. The percentage solubility of the proteins was calculated as follows:
Solubility (%)= 100
Foaming capacity (FC) and stability (FS): Foaming capacity was measured according to the method described by Lamsal et al. (Lamsal et al., 2007). The sample (500 mg) was added into a beaker containing 50 ml of distilled water, and the pH was adjusted to 4, 6, 8, 10, and 12 with either 0.1 M NaOH or 0.1 M HCl. The solution was whipped using the maximum speed of a homogenizer for 5 min and transferred into a 100 ml graduated cylinder while the volume of foam (V2) was immediately recorded at 0 min. The foam stability was determined by measuring the decrease in the volume of foam as a function of time up to a period of 10, 20, 30, and 40 min.
FC % = 100
Where V2 is the volume after whipping and V1 is the volume before whipping
FS (%)= - 100
Emulsifying capacity (EC) and stability (ES): Emulsifying capacity was determined using the method described by Lamsal et al. (Lamsal et al., 2007). The sample (300 mg) was homogenized for 1 min in 20 ml distilled water, and the pH was adjusted to 3, 4, 6, 8, 10, and 12. The protein solution was mixed with 15 ml of soybean oil followed by homogenising for 1 min. It was then centrifuged at 5,000 rpm for 3 min. EC was calculated using the expression below.
EC % = 100
Emulsifying stability was determined by heating the emulsion at 70 °C for 30 min in a water bath, after which, it was centrifuged at 5,000 rpm for 3 min.
ES % = 100
Water holding capacity (WHC) and oil holding capacity (OHC): WHC and OHC were determined as described by Mariod et al. (Mariod et al., 2010). The sample (0.5 g) was weighed into a 50 ml pre-weighed centrifuge tube and mixed with 10 g of distilled water for WHC determination, while 1.0 gram was mixed with 10 g of soybean oil for OHC determination. Then, the obtained suspensions were vortexed for 2 min and were left to stand for 30 min. The protein-water mixture was centrifuged at 3,000 g for 15 min. The supernatant was removed and the tube was then re-weighed.
WHC or OHC g / g =
Where W1 is the weight of the tube plus the dry sample and W2 is the weight of the tube plus sediment.
Foaming capacity (FC) and stability (FS): Foaming capacity was measured according to the method described by Lamsal et al. (Lamsal et al., 2007). The sample (500 mg) was added into a beaker containing 50 ml of distilled water, and the pH was adjusted to 4, 6, 8, 10, and 12 with either 0.1 M NaOH or 0.1 M HCl. The solution was whipped using the maximum speed of a homogenizer for 5 min and transferred into a 100 ml graduated cylinder while the volume of foam (V2) was immediately recorded at 0 min. The foam stability was determined by measuring the decrease in the volume of foam as a function of time up to a period of 10, 20, 30, and 40 min.
Where V2 is the volume after whipping and V1 is the volume before whipping
Emulsifying capacity (EC) and stability (ES): Emulsifying capacity was determined using the method described by Lamsal et al. (Lamsal et al., 2007). The sample (300 mg) was homogenized for 1 min in 20 ml distilled water, and the pH was adjusted to 3, 4, 6, 8, 10, and 12. The protein solution was mixed with 15 ml of soybean oil followed by homogenising for 1 min. It was then centrifuged at 5,000 rpm for 3 min. EC was calculated using the expression below.
Emulsifying stability was determined by heating the emulsion at 70 °C for 30 min in a water bath, after which, it was centrifuged at 5,000 rpm for 3 min.
Water holding capacity (WHC) and oil holding capacity (OHC): WHC and OHC were determined as described by Mariod et al. (Mariod et al., 2010). The sample (0.5 g) was weighed into a 50 ml pre-weighed centrifuge tube and mixed with 10 g of distilled water for WHC determination, while 1.0 gram was mixed with 10 g of soybean oil for OHC determination. Then, the obtained suspensions were vortexed for 2 min and were left to stand for 30 min. The protein-water mixture was centrifuged at 3,000 g for 15 min. The supernatant was removed and the tube was then re-weighed.
Where W1 is the weight of the tube plus the dry sample and W2 is the weight of the tube plus sediment.
Data analysis
All experiments were performed in triplicate. The results were presented as mean±standard deviation. Moreover, a two-way analysis of variance (ANOVA) with a post hoc test was used to detect statistical differences, particularly in functional properties, and analysis was performed using SPSS software (version 20.0, SPSS Inc., Chicago, IL, USA). The significance level was based on the confidence level of 95% (P<0.05).
Results
Table 1 presents the proximate composition of the dry seeds of M. oleifera. The seed contains high fats (46.41±0.80 g/100 g), followed by proteins (29.09±0.40 g/100 g). The least amount of component regarding the seed was (3.27±0.01 g/100 g) which represents the soluble carbohydrates.
| Table 1. Proximate composition of M. oleifera per 100 g of dry seeds. | |
| Component | Amount |
| Moisture | 6.70±0.05a |
| Ash | 4.45±0.03 |
| Protein | 29.09±0.40 |
| Fat | 46.41±0.80 |
| Fibre | 10.08±0.04 |
| Carbohydrate | 3.27±0.01 |
| a: Mean ± SD. | |
Table 2 shows the protein content of the isolates. The aqueous extract contained 73.0±0.5 g/100 g protein, while the NaOH precipitate fraction and the (NH4)2SO4 fraction had 54.0±0.2 and 45.0±0.3 g/100 g protein.
| Table 2. Protein yield of M. oleifera seed isolates (g/100 g). | |
| Isolate | Amount |
| Aqueous extract | 73.0±0.5a |
| NaOH precipitate fraction | 54.0±0.2 |
| (NH4)2SO4 precipitate fraction | 45.0±0.3 |
| a: Mean ± SD. | |
The results of the in vitro protein digestibility of the aqueous extract, the alkaline precipitated extract, and the ammonium sulphate precipitated extracts are presented in Table 3. The results indicated that a high digestibility value was recorded for the aqueous extract after 6 hours of digestion (82.63±0.27). In contrast, the alkaline precipitated fraction (76.85±0.61) and the ammonium sulphate precipitated fractions (77.91±0.12) had higher values than the aqueous extract after 1 hour of digestion.
The UV absorption spectra of the M. oleifera seed protein isolates are illustrated in Figure 1. The three protein isolates had shown strong absorption at 280 nm which indicates that protein is present in all the protein isolates.
The amino acid composition of the M. oleifera seed protein isolates are shown in Table 4. The essential amino acids are all present in the seed, with histidine having the lowest content and phenylalanine the highest amount 8.93 mg/100g in the (NH4)2SO4 fraction.
Figure 2 describes the solubility of the protein isolates. The solubility of all the fractions was higher at alkaline pH values. The NaOH precipitated fraction and the ammonium sulphate fraction are more soluble than the aqueous
extract.
| Table 3. In vitro digesytibility of M. oleifera seed soluble protein isolates. | ||
| Samples | Amount in grams of protein digestibility at 1 hour | Amount in grams of protein digestibility at 6 hours |
| Aqueous extract | 74.91±0.32a | 82.63±0.27 a |
| NaOH precipitate | 76.85±0.61 a | 68.17±0.42 b |
| (NH4)2SO4 precipitate | 77.91±0.12 a | 68.78±0.25 b |
|
Values with different superscript along a column are significantly different (P<0.05).
.PNG) |
||
| Table 4. Amino acids composition of M. oleifera seed protein isolates (mg/100g). | |||
| Amino acid | Crude extract | NaOH precipitate | (NH4)2SO4 precipitate |
| Tryptophan | 1.54±0.10 | 2.62±0.23a | 4.17±0.35b |
| Histidine | 0.855±0.02 | 1.13±0.01 | 1.32±0.12 |
| Leucine | 1.745±0.01 | 2.25±0.22 | 3.65±0.62a |
| Isoleucine | 2.01±0.12 | 2.84±0.51 | 4.04±0.51a |
| Phenylalanine | 6.25±0.23 | 7.34±0.36 | 8.93±0.67b |
| Valine | 2.75±0.03 | 3.84±0.51a | 3.87±0.44 |
| Lysine | 3.87±0.41 | 4.01±0.23 | 4.26±0.63b |
| Methionine | 2.72±0.22 | 3.78±0.41a | 5.26±0.81b |
| Threonine | 1.535±0.14 | 1.87±0.02 | 3.85±0.46a |
| Asparagine | 3.61±0.02 | 4.23±0.64 | 6.32±0.35a |
| Arginine | 2.80±0.30 | 3.13±0.26 | 3.45±0.23 |
| Alanine | 0.56±0.01 | 0.71±0.02 | 0.98±0.23b |
| Aspartate | 3.23±0.25 | 4.62±0.51a | 5.81±0.63b |
| Glutamate | 1.77±0.11 | 2.71±0.32a | 4.15±0.24b |
| Glycine | 4.57±0.43 | 6.52±0.56a | 7.34±0.45b |
| Tyrosine | 4.43±0.35a | 3.14±0.32 | 4.11±0.75a |
| Cysteine | 3.13±0.12 | 3.41±0.41 | 6.05±0.86a |
| Proline | 1.71±0.50 | 1.83±0.15 | 2.29±0.54 |
| Serine | 2.75±0.32 | 3.12±0.04 | 4.84±0.63a |
| Values are expressed as mean ± SD (n = 3); Values with different superscripts across a row are significantly different (P<0.05).. | |||
The results for the foaming capacity and foaming stability are depicted in Figure 3 and Figure 4 respectively. The fraction for the NaOH precipitate fraction had the highest foaming capacity (28%) at pH 10. Similarly, the highest foaming capacity for the ammonium sulphate precipitated fraction (20%) was observed at pH 10.
Figure 5 presents the emulsifying capacity of M. oleifera seed protein isolates. The highest emulsifying capacity was observed with the (NH4)2SO4 fraction.
Figure 6 shows the emulsifying stability of M. oleifera seed protein isolates. The aqueous extract is the most stable at both pH 10 and pH 12. The (NH4)2SO4 fraction is also significantly (P<0.05) stable at pH 12 and pH 10 with stability of 45% and 40% respectively.
Figure 5 presents the emulsifying capacity of M. oleifera seed protein isolates. The highest emulsifying capacity was observed with the (NH4)2SO4 fraction.
Figure 6 shows the emulsifying stability of M. oleifera seed protein isolates. The aqueous extract is the most stable at both pH 10 and pH 12. The (NH4)2SO4 fraction is also significantly (P<0.05) stable at pH 12 and pH 10 with stability of 45% and 40% respectively.
The water-holding capacity and oil-holding capacity of M. oleifera seed protein isolates are shown in Figure 7. The results showed that (NH4)2SO4 fraction had a higher water-holding capacity (2.8 g/g) than the aqueous extract and the NaOH fractions. The fraction with the most
oil-holding capacity was the aqueous extract
(3.2 g/g).
Discussion
There is an increased awareness of the consumption and use of M. oleifera seeds in food systems in the tropics (Ogunsina et al., 2010). The results of the proximate composition was similar to the findings of Zaku et al. where they reported a protein content of 27.1% per 100 g sample (Zaku et al., 2015). A profile of 30.07% and 33.26% of protein has been reported for mature seeds from M. oleifera cultivated in Pakistan and Brazil, respectively (Manzoor et al., 2007, Oliveira et al., 1999). These variations might have to do with differences in the plant's maturation stage, geographical distribution, post-harvest handling, ambient conditions, and processing methods, all of which can affect the nutritional value of M. oleifera plant parts (Iyapo and Omotosho, 2024).
The observation of the total amino acid content revealed that the essential amino acids tryptophan, leucine, isoleucine, phenylalanine, valine, lysine, methionine, and threonine are available in the range of 3.6 - 8.9 mg/100g of protein (Table 4). It can be vividly noticed that the fraction from the (NH4)2SO4 precipitate contained higher concentrations of the amino acids, compared with aqueous extract and the NaOH precipitate fraction. It was reported that all the parts of this plant contain high percentages of essential amino acids except for methionine, commonly deficient in green leaves (Sánchez-Machado et al., 2010).
While amino acid composition reveals the nutritional quality of protein food, especially the quantity of each amino acid present, protein digestibility shows the utilization potential of such protein (Boye et al., 2012). The decrease in pH during protein digestibility is due to the release of hydrogen ions by the hydrolyzed peptide bonds. A more rapid decrease, which signifies an increased rate of digestion, can be taken as an index of protein digestibility (Malomo and Aluko, 2015). The protein digestibility for the protein isolates shown in Table 2 indicated a high protein digestibility value which might be attributed to the ease of access of the protease to the peptide bonds aided by the lower amount of non-protein materials (Adebowale et al., 2007). In this respect, the (NH4)2SO4 fraction was more digestible, which suggests a conformation with better access to enzymes when compared to the NaOH fraction and the aqueous extract. The values obtained in this study were comparable to 84% and 85% reported for soy protein concentrates and isolates, respectively (Mohamed et al., 2009); however, lower than the 91-95% obtained for mucuna bean protein isolate (Adebowale et al., 2007). Similar values have also been reported such as 68% for flaxseed (Marambe et al., 2013) and 71% for pinto bean (Tan et al., 2014).
Solubility characteristics of proteins are among the most important functional properties since many functional performances of proteins depend upon their capacity to go into solution initially. Once the pH is close to the protein isoelectric point (around pH 4−5), there is a reduction of solubility, as found by Mune-Mune et al., while above/below the protein isoelectric point, the more protein solubility was observed. (Mune Mune et al., 2016). This might be the result of changes in aggregation, solubility, and secondary structure (Amiri et al., 2021). Seena and Sridhar mentioned that at highly acidic and alkaline pH, the protein acquires, respectively, a net positive and negative charge, which promotes the repulsion of molecules, and thus, increases the solubility of the protein (Seena and Sridhar, 2005). This result has been described in many food proteins according to the tendency of protein solubility as a function of pH, including microalgae proteins, mung bean proteins, and sunflower proteins, which have a minimum solubility around an isoelectric point between a pH of 4–5 (Chen et al., 2019, Du et al., 2017, Ulloa et al., 2017). Tang et al. reported the solubility of native water soluble protein from M. oleifera to be 5.56% where ultrasonic treatment increased the solubility significantly to 30.53% (Tang et al., 2021).
Foaming capacity and foaming stability are important functional properties of protein isolates that determine their utilization in different food systems where aeration and overrun are required, for example, whipped toppings, baked foods, and ice-cream mixes (Shevkani et al., 2015). High protein solubility is a prerequisite for achieving better foaming capacity (Deng et al., 2011). Protein molecules should form continuous intermolecular polymers enveloping the air bubble to render good foaming stability since intermolecular cohesiveness and elasticity are important to produce stable foams (Kamara et al., 2009). The higher foam stability observed (Figure 4) after 10 minutes could be attributed to the unfolding of the protein structure which facilitates surface hydrophobic association as well as reduced air leakage leading to the prevention of rupture and coalescence (Shevkani et al., 2015, Wang et al., 2012).
The emulsifying capacity is a measure of the effectiveness of proteinaceous emulsifiers (Pearce and Kinsella, 1978). The emulsifying capacities of the protein are generally dependent on its ability to absorb on the interface whereas emulsion stability is related to the stability properties of this adsorbed layer. The nature of the amino acids in each of the fractions could differ depending on the pH of protein resolubilization which might be the key factor affecting the emulsifying properties of the protein isolates (Figure 6) as well as other functional properties. Protein solubility and hydrophobicity are two major important factors that determine their initial adsorption, and thus, their emulsifying properties (Shevkani et al., 2015).
It was reported that M. oleifera seed protein managed to hold 4.78 ml water/g protein (Paramita et al., 2024). However, the poor water absorption capacity of the aqueous extract may not restrict its application because the salt content in the functional foods formulation would be sufficient to provide the desired solubility. Arrese et al. studied the functional properties of several commercial soy protein isolates and reported that isolates with more denaturation showed a greater water-holding capacity (Arrese et al., 1991). This may be attributed to the unfolding of the polypeptide chain resulting in a matrix that can trap the absorbed water.
The differences in oil absorption capacity might be due to the presence of more non-polar amino acids in the aqueous extract. The presence of several non-polar side chains may bind the hydrocarbon chains of fats, thereby resulting in higher absorption of oil (Sathe et al., 1982). The high oil absorption capacity of M. oleifera seed proteins makes them good ingredients in the cold meat industry, particularly for sausages, where the protein usually bridges the fat and water to obtain good products.
The scope of the study was limited to the isolation and partial characterization of some soluble proteins from the seeds of M. oleifera for use in food industries. This study did not involve any drugs or patient. No laboratory animal was used in the study. This study exclusively examined the properties of protein isolates precipitated with NaOH and ammonium sulfate, without subsequent dialysis to eliminate Na+ and ammonium sulfate residues from the protein isolates. Developing protein supplements or meat alternatives using M. oleifera seed protein isolates, especially the (NH4)2SO4 fraction are recommended for improved digestibility. Incorporating M. oleifera proteins into emulsified products such as dressings or spreads is advised. They should be incorporated into baked goods or whipped toppings due to foaming properties and combined with other proteins to balance amino acids, addressing methionine deficiency.
The observation of the total amino acid content revealed that the essential amino acids tryptophan, leucine, isoleucine, phenylalanine, valine, lysine, methionine, and threonine are available in the range of 3.6 - 8.9 mg/100g of protein (Table 4). It can be vividly noticed that the fraction from the (NH4)2SO4 precipitate contained higher concentrations of the amino acids, compared with aqueous extract and the NaOH precipitate fraction. It was reported that all the parts of this plant contain high percentages of essential amino acids except for methionine, commonly deficient in green leaves (Sánchez-Machado et al., 2010).
While amino acid composition reveals the nutritional quality of protein food, especially the quantity of each amino acid present, protein digestibility shows the utilization potential of such protein (Boye et al., 2012). The decrease in pH during protein digestibility is due to the release of hydrogen ions by the hydrolyzed peptide bonds. A more rapid decrease, which signifies an increased rate of digestion, can be taken as an index of protein digestibility (Malomo and Aluko, 2015). The protein digestibility for the protein isolates shown in Table 2 indicated a high protein digestibility value which might be attributed to the ease of access of the protease to the peptide bonds aided by the lower amount of non-protein materials (Adebowale et al., 2007). In this respect, the (NH4)2SO4 fraction was more digestible, which suggests a conformation with better access to enzymes when compared to the NaOH fraction and the aqueous extract. The values obtained in this study were comparable to 84% and 85% reported for soy protein concentrates and isolates, respectively (Mohamed et al., 2009); however, lower than the 91-95% obtained for mucuna bean protein isolate (Adebowale et al., 2007). Similar values have also been reported such as 68% for flaxseed (Marambe et al., 2013) and 71% for pinto bean (Tan et al., 2014).
Solubility characteristics of proteins are among the most important functional properties since many functional performances of proteins depend upon their capacity to go into solution initially. Once the pH is close to the protein isoelectric point (around pH 4−5), there is a reduction of solubility, as found by Mune-Mune et al., while above/below the protein isoelectric point, the more protein solubility was observed. (Mune Mune et al., 2016). This might be the result of changes in aggregation, solubility, and secondary structure (Amiri et al., 2021). Seena and Sridhar mentioned that at highly acidic and alkaline pH, the protein acquires, respectively, a net positive and negative charge, which promotes the repulsion of molecules, and thus, increases the solubility of the protein (Seena and Sridhar, 2005). This result has been described in many food proteins according to the tendency of protein solubility as a function of pH, including microalgae proteins, mung bean proteins, and sunflower proteins, which have a minimum solubility around an isoelectric point between a pH of 4–5 (Chen et al., 2019, Du et al., 2017, Ulloa et al., 2017). Tang et al. reported the solubility of native water soluble protein from M. oleifera to be 5.56% where ultrasonic treatment increased the solubility significantly to 30.53% (Tang et al., 2021).
Foaming capacity and foaming stability are important functional properties of protein isolates that determine their utilization in different food systems where aeration and overrun are required, for example, whipped toppings, baked foods, and ice-cream mixes (Shevkani et al., 2015). High protein solubility is a prerequisite for achieving better foaming capacity (Deng et al., 2011). Protein molecules should form continuous intermolecular polymers enveloping the air bubble to render good foaming stability since intermolecular cohesiveness and elasticity are important to produce stable foams (Kamara et al., 2009). The higher foam stability observed (Figure 4) after 10 minutes could be attributed to the unfolding of the protein structure which facilitates surface hydrophobic association as well as reduced air leakage leading to the prevention of rupture and coalescence (Shevkani et al., 2015, Wang et al., 2012).
The emulsifying capacity is a measure of the effectiveness of proteinaceous emulsifiers (Pearce and Kinsella, 1978). The emulsifying capacities of the protein are generally dependent on its ability to absorb on the interface whereas emulsion stability is related to the stability properties of this adsorbed layer. The nature of the amino acids in each of the fractions could differ depending on the pH of protein resolubilization which might be the key factor affecting the emulsifying properties of the protein isolates (Figure 6) as well as other functional properties. Protein solubility and hydrophobicity are two major important factors that determine their initial adsorption, and thus, their emulsifying properties (Shevkani et al., 2015).
It was reported that M. oleifera seed protein managed to hold 4.78 ml water/g protein (Paramita et al., 2024). However, the poor water absorption capacity of the aqueous extract may not restrict its application because the salt content in the functional foods formulation would be sufficient to provide the desired solubility. Arrese et al. studied the functional properties of several commercial soy protein isolates and reported that isolates with more denaturation showed a greater water-holding capacity (Arrese et al., 1991). This may be attributed to the unfolding of the polypeptide chain resulting in a matrix that can trap the absorbed water.
The differences in oil absorption capacity might be due to the presence of more non-polar amino acids in the aqueous extract. The presence of several non-polar side chains may bind the hydrocarbon chains of fats, thereby resulting in higher absorption of oil (Sathe et al., 1982). The high oil absorption capacity of M. oleifera seed proteins makes them good ingredients in the cold meat industry, particularly for sausages, where the protein usually bridges the fat and water to obtain good products.
The scope of the study was limited to the isolation and partial characterization of some soluble proteins from the seeds of M. oleifera for use in food industries. This study did not involve any drugs or patient. No laboratory animal was used in the study. This study exclusively examined the properties of protein isolates precipitated with NaOH and ammonium sulfate, without subsequent dialysis to eliminate Na+ and ammonium sulfate residues from the protein isolates. Developing protein supplements or meat alternatives using M. oleifera seed protein isolates, especially the (NH4)2SO4 fraction are recommended for improved digestibility. Incorporating M. oleifera proteins into emulsified products such as dressings or spreads is advised. They should be incorporated into baked goods or whipped toppings due to foaming properties and combined with other proteins to balance amino acids, addressing methionine deficiency.
Conclusion
The high solubility, foaming, and emulsifying properties of M. oleifera seed proteins at alkaline pH levels might play an important role in food systems, thereby improving the quality or texture of foods. Similarly, good in vitro digestibility, further supported by the significant amounts of both dispensable and indispensable amino acids, suggests the possibility of using it as an alternative nutritious plant-based protein.
Acknowledgement
The authors would like to appreciate the support from Petroleum Development Trust Fund (PTDF) Scholarship for Masters for the Local Scholarship Scheme (LSS) Awarded to Abdulrahman AA (PTDF/ED/LSS/MSC/AAA/0537/19 - 19MSC0005) under its Human Capital Development Programmes.
Authors' contributions
Abdulrahman AA, Milala MA, Shehu BB, and Gidado A designed the research; Abdulrahman AA and Damasak AA conducted research; Abdulrahman AA, Buba F, and Tijani Y and Musa H analyzed data and drafted the manuscript; Milala MA, Shehu BB, and Gidado A edited the paper. Abdulrahman AA had the primary responsibility for final content. All authors read and approved the final manuscript.
Conflict of interest
The authors declared no conflict of interest.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
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Association of Official Analytical Chemists (AOAC) 2015. Official methods of analysis. Association of Official Analytical Chemists: Washington D.C.
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Chen Y, et al. 2019. Food hydrocolloids physicochemical and functional properties of proteins extracted from three microalgal species. Food hydrocolloids. 96 (886): 510–517.
Deng Q, et al. 2011. Functional properties of protein isolates, globulin and albumin extracted from Ginkgo biloba seeds. Food chemistry. 124: 1458–1465.
Du M, et al. 2017. Extraction, physicochemical characteristics and functional properties of Mung bean protein. Food hydrocolloids. 76: 131–140.
Iyapo K & Omotosho J 2024. The evaluated proximate profiles of Moringa oleifera plant parts and the utilization of Moringa oleifera seed meal based diets on growth response of the african catfish. FUW Trends in science and technology journal. 8 (3): 323 – 327.
Jain A, Subramanian R, Manohar B & Radha C 2019. Preparation, characterization and functional properties of Moringa oleifera seed protein isolate. Journal of food science and technology. 56 (4): 2093–2104.
Jayalakshmi G, Saritha V & Dwarapureddi B 2017. A review on native plant based coagulants for water purification. International journal of applied environmental sciences. 12 (3): 469–487.
Kamara M, Huiming Z, Kexue Z, Amadou I & Tarawalie F 2009. Comparative study of chemical composition and physicochemical properties of two varieties of defatted foxtail millet flour grown in China. American journal of food technology. 4: 255–267.
Kumar K, Ganesan K, Selvaraj K & Rao P 2014. Studies on the functional properties of protein concentrate of Kappaphycus alvarezii (Doty) Doty - An edible seaweed. Food chemistry. 153: 353–360.
Lakshmipriya G, Doriya K & Kumar D 2016. Moringa oleifera: A review on nutritive importance and its medicinal application. Food science and human wellness. 5 (2): 49–56.
Lamsal B, Koegel R & Gunasekaran S 2007. Some physicochemical and functional properties of alfalfa soluble leaf proteins. LWT- Food science and technology. 40 (9): 1520–1526.
Małecki J, Muszyński S & Sołowiej B 2021. Proteins in food systems-bionanomaterials, conventional and unconventional sources, functional properties, and development opportunities. Polymers. 13 (15): 2506.
Malomo S & Aluko R 2015. Conversion of a low protein hemp seed meal into a functional protein concentrate through enzymatic digestion of fibre coupled with membrane ultrafiltration. Innovative food science and emerging technologies. 31: 151-159.
Manzoor M, Anwar F, IqbalT & Bhanger M 2007. Physico-chemical characterization of Moringa concanensis seeds and seed oil. Journal of the American oil chemists' society. 84 (5): 413–419.
Marambe H, Shand P & Wanasundara J 2013. In vitro digestibility of flaxseed (Linum usitatissimum L.) protein: Effect of seed mucilage, oil and thermal processing. International journal of food science and technology. 48: 628–635.
Mariod A, Farhana S & Ismail M 2010. Preparation and characterisation of protein concentrates from defatted kenaf seed. Food chemistry. 123 (3): 747–752.
Mohamed T, Zhu K, Issoufou A, Fatmata T & Zhou H 2009. Functionality, in vitro digestibility and physicochemical properties of two varieties of defatted foxtail millet protein concentrates. International journal of molecular sciences. 10: 5224–5238.
Mune Mune M, Bakwo Bassogog C, Nyobe E & René Minka S 2016. Physicochemical and functional properties of Moringa oleifera seed and leaf flour. Cogent food and agriculture. 2 (1): 1220352.
Ndabigengesere A, Narasiah K & Talbot B 1995. Active agents and mechanism of coagulation of turbid waters using Moringa oleifera. Water research. 29: 703–710.
Ogunsina B, Radha C & Sign R 2010. Physicochemical and functional properties of full-fat and defatted Moringa oleifera kernel flour. International journal food science and technology. 45: 2433–2439.
Oliveira J, Silveira S, Vasconcelos I, Cavada B & Moreira R 1999. Compositional and nutritional attributes of seeds from the multiple purpose tree Moringa oleifera Lamarck. Journal of the science of food and agriculture. 79 (6): 815–820.
Paramita V, Yuliani H, Rosalin R & Irawati L 2024. Physiochemical and functional properties of Moringa oleifera protein obtained by isoelectric precipitation. Food research. 8 (1): 1 - 8.
Pearce K & Kinsella J 1978. Emulsifying properties of proteins: evaluation of a turbidimetric technique. Journal of agricultural and food chemistry. 26: 716–723.
Sánchez-Machado D, Núñez-Gastélum J, Reyes-Moreno C, Ramírez-Wong B & López-Cervantes J 2010. Nutritional quality of edible parts of Moringa oleifera. Food analytical methods. 3 (3): 175–180.
Sánchez-Martín J, Beltrán-Heredia J & Peres J 2012. Improvement of the flocculation process in water treatment by using Moringa oleifera seeds extract. Brazilian journal of chemical engineering. 29 (03): 495–501.
Sathe S, Deshpande S & Salunkhe D 1982. Functional properties of winged bean [Psophocarpus tetragonolobus (L.) DC] Proteins. Journal of food science. 47: 503–509.
Seena S & Sridhar K 2005. Physiochemical, functional and cooking properties of Canavalia. Food chemistry 32: 406-412.
Senthilraja M 2011. Determination of spectrophotometric absorptivity by analytical ultracentrifugation. Indian journal of pharmaceutical sciences. 73 (4): 436–438.
Shevkani K, Singh N, Kaur A & Rana J 2015. Structural and functional characterization of kidney bean and field pea protein isolates: A comparative study. Food hydrocolloids. 43: 679–689.
Singh P, Kumar R, Sabapathy S & Bawa A 2008. Functional and edible uses of soy protein products. Comprehensive reviews in food science and food safety. 7: 14-28.
Tan E, Ying-Yuan N & Gan C 2014. A comparative study of physicochemical characteristics and functionalities of pinto bean protein isolate (PBPI) against the soybean protein isolate (SPI) after the extraction optimisation. Food chemistry. 152: 447–455.
Tang S, Du O & Fu Z 2021. Ultrasonic treatment on physicochemical properties of water-soluble protein from Moringa oleifera seed. Ultrasonics sonochemistry. 71: 105357.
Ulloa J, et al. 2017. Production, physico-chemical and functional characterization of a protein isolate from jackfruit (Artocarpus heterophyllus) seeds. Journal of food. 15 (4): 947–507.
Vijayaraghvan G, Sivakumar T & VimalKumar A 2011. Application of plant based coagulants for waste water treatment. International journal of advanced engineering research and studies. 1 (1): 88 – 92.
Wang J, et al. 2012. Adsorption and dilatational rheology of heat-treated soy protein at the oil-water interface: relationship to structural properties. Journal of agricultural and food chemistry. 60 (12): 3302–3310.
Yuliastri I, Rohaeti E, Effendi H & Darusman L 2016. The use of Moringa Oleifera Seed powder as coagulant to improve the quality of wastewater and ground water. IOP conference series: Earth and environmental science. 31: 1-10.
Zaku G, Emmanuel S, Tukur A & Kabir A 2015. Moringa oleifera: An underutilized tree in Nigeria with amazing versatility: A review. African journal of food science. 9 (9): 456-461.
Amiri A, et al. 2021. Modification of functional, rheological and structural characteristics of myofibrillar proteins by high-intensity ultrasonic and papain treatment. Innovative food sand emerging technologies. 72: 102748.
Anwar F, Latif S, Ashraf M & Gilani A 2007. Moringa oleifera: a food plant with multiple medicinal uses. Phytotherapy Research. International journal devoted to pharmacological and toxicological evaluation of natural product derivatives. 21 (1): 17−25.
Arrese E, Sorgentini D, Wagner J & Anon M 1991. Electrophoretic, solubility and functional properties of commercial soy protein isolates. Journal of agricultural and food chemistry. 39: 1029–1032.
Association of Official Analytical Chemists (AOAC) 2000. Official Methods of Analytical Chemists. 18th ed. Association of Official Analytical Chemists: Washington D.C.
Association of Official Analytical Chemists (AOAC) 2005. Official methods of analysis of the Association of Official Analytical Chemists. Association of Official Analytical Chemists: Va. USA.
Association of Official Analytical Chemists (AOAC) 2015. Official methods of analysis. Association of Official Analytical Chemists: Washington D.C.
Bakare B 2016. An investigation of moringa oleifera seed extract as a natural coagulant in water treatment. Lecture notes in engineering and computer science. 2226: 566–569.
Boye J, Wijesinha-Bettoni R & Burlingame B 2012. Protein quality evaluation twenty years after the introduction of the protein digestibility corrected amino acid score method. . British journal of nutrition. 108 (S2): S183-211.
Chen Y, et al. 2019. Food hydrocolloids physicochemical and functional properties of proteins extracted from three microalgal species. Food hydrocolloids. 96 (886): 510–517.
Deng Q, et al. 2011. Functional properties of protein isolates, globulin and albumin extracted from Ginkgo biloba seeds. Food chemistry. 124: 1458–1465.
Du M, et al. 2017. Extraction, physicochemical characteristics and functional properties of Mung bean protein. Food hydrocolloids. 76: 131–140.
Iyapo K & Omotosho J 2024. The evaluated proximate profiles of Moringa oleifera plant parts and the utilization of Moringa oleifera seed meal based diets on growth response of the african catfish. FUW Trends in science and technology journal. 8 (3): 323 – 327.
Jain A, Subramanian R, Manohar B & Radha C 2019. Preparation, characterization and functional properties of Moringa oleifera seed protein isolate. Journal of food science and technology. 56 (4): 2093–2104.
Jayalakshmi G, Saritha V & Dwarapureddi B 2017. A review on native plant based coagulants for water purification. International journal of applied environmental sciences. 12 (3): 469–487.
Kamara M, Huiming Z, Kexue Z, Amadou I & Tarawalie F 2009. Comparative study of chemical composition and physicochemical properties of two varieties of defatted foxtail millet flour grown in China. American journal of food technology. 4: 255–267.
Kumar K, Ganesan K, Selvaraj K & Rao P 2014. Studies on the functional properties of protein concentrate of Kappaphycus alvarezii (Doty) Doty - An edible seaweed. Food chemistry. 153: 353–360.
Lakshmipriya G, Doriya K & Kumar D 2016. Moringa oleifera: A review on nutritive importance and its medicinal application. Food science and human wellness. 5 (2): 49–56.
Lamsal B, Koegel R & Gunasekaran S 2007. Some physicochemical and functional properties of alfalfa soluble leaf proteins. LWT- Food science and technology. 40 (9): 1520–1526.
Małecki J, Muszyński S & Sołowiej B 2021. Proteins in food systems-bionanomaterials, conventional and unconventional sources, functional properties, and development opportunities. Polymers. 13 (15): 2506.
Malomo S & Aluko R 2015. Conversion of a low protein hemp seed meal into a functional protein concentrate through enzymatic digestion of fibre coupled with membrane ultrafiltration. Innovative food science and emerging technologies. 31: 151-159.
Manzoor M, Anwar F, IqbalT & Bhanger M 2007. Physico-chemical characterization of Moringa concanensis seeds and seed oil. Journal of the American oil chemists' society. 84 (5): 413–419.
Marambe H, Shand P & Wanasundara J 2013. In vitro digestibility of flaxseed (Linum usitatissimum L.) protein: Effect of seed mucilage, oil and thermal processing. International journal of food science and technology. 48: 628–635.
Mariod A, Farhana S & Ismail M 2010. Preparation and characterisation of protein concentrates from defatted kenaf seed. Food chemistry. 123 (3): 747–752.
Mohamed T, Zhu K, Issoufou A, Fatmata T & Zhou H 2009. Functionality, in vitro digestibility and physicochemical properties of two varieties of defatted foxtail millet protein concentrates. International journal of molecular sciences. 10: 5224–5238.
Mune Mune M, Bakwo Bassogog C, Nyobe E & René Minka S 2016. Physicochemical and functional properties of Moringa oleifera seed and leaf flour. Cogent food and agriculture. 2 (1): 1220352.
Ndabigengesere A, Narasiah K & Talbot B 1995. Active agents and mechanism of coagulation of turbid waters using Moringa oleifera. Water research. 29: 703–710.
Ogunsina B, Radha C & Sign R 2010. Physicochemical and functional properties of full-fat and defatted Moringa oleifera kernel flour. International journal food science and technology. 45: 2433–2439.
Oliveira J, Silveira S, Vasconcelos I, Cavada B & Moreira R 1999. Compositional and nutritional attributes of seeds from the multiple purpose tree Moringa oleifera Lamarck. Journal of the science of food and agriculture. 79 (6): 815–820.
Paramita V, Yuliani H, Rosalin R & Irawati L 2024. Physiochemical and functional properties of Moringa oleifera protein obtained by isoelectric precipitation. Food research. 8 (1): 1 - 8.
Pearce K & Kinsella J 1978. Emulsifying properties of proteins: evaluation of a turbidimetric technique. Journal of agricultural and food chemistry. 26: 716–723.
Sánchez-Machado D, Núñez-Gastélum J, Reyes-Moreno C, Ramírez-Wong B & López-Cervantes J 2010. Nutritional quality of edible parts of Moringa oleifera. Food analytical methods. 3 (3): 175–180.
Sánchez-Martín J, Beltrán-Heredia J & Peres J 2012. Improvement of the flocculation process in water treatment by using Moringa oleifera seeds extract. Brazilian journal of chemical engineering. 29 (03): 495–501.
Sathe S, Deshpande S & Salunkhe D 1982. Functional properties of winged bean [Psophocarpus tetragonolobus (L.) DC] Proteins. Journal of food science. 47: 503–509.
Seena S & Sridhar K 2005. Physiochemical, functional and cooking properties of Canavalia. Food chemistry 32: 406-412.
Senthilraja M 2011. Determination of spectrophotometric absorptivity by analytical ultracentrifugation. Indian journal of pharmaceutical sciences. 73 (4): 436–438.
Shevkani K, Singh N, Kaur A & Rana J 2015. Structural and functional characterization of kidney bean and field pea protein isolates: A comparative study. Food hydrocolloids. 43: 679–689.
Singh P, Kumar R, Sabapathy S & Bawa A 2008. Functional and edible uses of soy protein products. Comprehensive reviews in food science and food safety. 7: 14-28.
Tan E, Ying-Yuan N & Gan C 2014. A comparative study of physicochemical characteristics and functionalities of pinto bean protein isolate (PBPI) against the soybean protein isolate (SPI) after the extraction optimisation. Food chemistry. 152: 447–455.
Tang S, Du O & Fu Z 2021. Ultrasonic treatment on physicochemical properties of water-soluble protein from Moringa oleifera seed. Ultrasonics sonochemistry. 71: 105357.
Ulloa J, et al. 2017. Production, physico-chemical and functional characterization of a protein isolate from jackfruit (Artocarpus heterophyllus) seeds. Journal of food. 15 (4): 947–507.
Vijayaraghvan G, Sivakumar T & VimalKumar A 2011. Application of plant based coagulants for waste water treatment. International journal of advanced engineering research and studies. 1 (1): 88 – 92.
Wang J, et al. 2012. Adsorption and dilatational rheology of heat-treated soy protein at the oil-water interface: relationship to structural properties. Journal of agricultural and food chemistry. 60 (12): 3302–3310.
Yuliastri I, Rohaeti E, Effendi H & Darusman L 2016. The use of Moringa Oleifera Seed powder as coagulant to improve the quality of wastewater and ground water. IOP conference series: Earth and environmental science. 31: 1-10.
Zaku G, Emmanuel S, Tukur A & Kabir A 2015. Moringa oleifera: An underutilized tree in Nigeria with amazing versatility: A review. African journal of food science. 9 (9): 456-461.
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