001.docx

DOI: 10.3303/CET2189043

Paper Received: 16 June 2021; Revised: 16 September 2021; Accepted: 21 November 2021
Please cite this article as: Soo M.H., Samad N.A., Abang Zaidel D.N., Mohd Jusoh Y.M., Muhamad I.I., Hashim Z., 2021, Extraction of Plant
Based Protein from Moringa oleifera Leaves using Alkaline Extraction and Isoelectric Precipitation Method, Chemical Engineering Transactions,
89, 253-258 DOI:10.3303/CET2189043

CHEMICAL ENGINEERING TRANSACTIONS

VOL. 89, 2021

A publication of

The Italian Association

of Chemical Engineering
Online at www.cetjournal.it

Guest Editors: Jeng Shiun Lim, Nor Alafiza Yunus, Jiří Jaromír Klemeš
Copyright © 2021, AIDIC Servizi S.r.l.
ISBN 978-88-95608-87-7; ISSN 2283-9216

Extraction of Plant Based Protein from Moringa oleifera
Leaves using Alkaline Extraction and Isoelectric Precipitation

Method
Ming Huey Sooa, Nabilah Abdul Samada, Dayang Norulfairuz Abang Zaidelb,*, Yanti
Maslina Mohd Jusoha, Ida Idayu Muhamada, Zanariah Hashima
a Department of Bioprocess and Polymer Engineering, School of Chemical and Energy Engineering, Faculty of Engineering,

Universiti Teknologi Malaysia, Skudai, Malaysia.
b Institute of Bioproduct Development, Faculty of Engineering, Universiti Teknologi Malaysia, Skudai, Malaysia.
dnorulfairuz@utm.my

This study aimed to investigate the effect of solid-to-solvent ratio on the yield and properties of protein
concentrate extracted from dried M. oleifera leaves powder by alkaline extraction and isoelectric precipitation
method. Extraction of protein concentrate was carried out by manipulating the solid-to-solvent ratio (1:10, 1:20
and 1:30) that affects protein yield of MOLPC. The characterization of both chemical and physical functional
properties was performed for dried M. oleifera Leaf Powder (MLP) and M. Oleifera Leaf Protein Concentrate
(MOLPC). Fat absorption capacity (FAC) and water absorption capacity (WAC) of MOLPC were characterized
at the different solid-to-solvent ratio and functional properties of MOLPC such as protein solubility, foaming and
emulsifying capacity were characterized at different pH (2.0 – 11.0). The proximate analysis for the MLP had
shown the moisture content (4.86 ± 1.18 %); ash (9.53 ± 0.07 %); crude protein (17.86 ± 0.23 %); crude fibres
(12.54 ± 0.01 %) and fats (0.50 ± 0.38 %). Based on the findings, MOLPC tends to have higher protein content
(26.58 ± 1.86 to 35.15 ± 3.4 %) compared to MLP. The FAC and WAC increased as the solid-to-solvent ratio
increased (3.21 – 3.82 g/g and 5.19 – 6.41 g/g). For the functional properties of MOLPC; the protein solubility,
foaming and emulsifying properties were all found to be pH-dependent.

1. Introduction
In the prevalent phenomenon of dietary protein deficiency, particularly afflicting the vulnerable pre-school
children, pregnant or nursing mothers and elderly, it necessitates the production of protein concentrates from
viable natural plant-based sources instead of expensive animal source. In the prospect of medicinal value, M.
oleifera has been advocated as a traditional panacea since all the parts of the Moringa tree have long been
used for the treatment of various diseases (Gopalakrishnan et al., 2016). The use of moringa leaves has been
widely spread among medical experts and nutritionists to treat malnutrition and other illnesses due to its high
quality of protein (Thurber and Fahey, 2005). Plant or vegetable proteins from various sources such as soybean
(Preece et al., 2017), sunflower seed (Baurin et al., 2020), and chickpeas (Nguyen et al., 2021) have been
studied for an alternative to animal protein in food application. Protein from M. oleifera can compete favourably
with proteins from animal sources, especially for growth and enzymatic activity of human body (Bhargav et al.,
2015).
It is important to have an appropriate extraction method that promote high yield of plant protein extract with high
functional properties and nutrients for food supplement and nutraceutical production. The most common protein
extraction protocol is by subjecting tissue to the exposure of distilled water or other weak buffers, which then
causing rupture of cells with concomitant release of intracellular proteins as it responds to the hypotonic effect
that gradually emerges (Maehre et al., 2018). In this context, animal cells in the absence of cell walls are suitable
for this protocol, but it is not efficacious for plant cells with presence of cell wall. Due to their hydrophobic groups
and disulfide connections between protein molecules, proteins in plant cells are rarely water soluble. For other
alternatives, aqueous salt or alkaline extraction is one of the most often implemented technique for the isolation

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of plant-based proteins in laboratory scale because high alkalinity assists well in extracting leaf protein by
breaking down the hydrogen bonds, disrupting the leaf tissue and enhancing protein solubility (Rawdkuen,
2020). Because the minimum protein solubility for M. oleifera leaf proteins is attained at the isoelectric point
between pH 3.2 and 4.5, the acid precipitation method is an efficient method for isolating leaf proteins. At this
isoelectric point, white cell sap protein of M. oleifera leaf concentrate is precipitated by acidification and it has
the highest amino acids content and solubility as compared to concentrates extracted by heat coagulation or by
addition of cationic or anionic flocculants (Santamaria-Fernández et al., 2019). Previous study by Ahmed (2016)
has investigated the protein concentrate extraction from M. oleifera using alkaline extraction at fixed parameters.
It has been reported that solid-to-solvent ratio is one of the significant factors that influence protein yield
extracted from plant (Cui et al., 2017). Higher solid-to-solvent ratio coulld produce a higher yield of protein. The
objective of this study was to investigate the effect of different solid-to-solvent ratio on the protein concentrate
extracted from dried M. oleifera leaves powder (MLP) by using alkaline extraction and isoelectric precipitation
method. The dried MLP was characterized for proximate analysis and M. oleifera leaves protein concentrate
(MOLPC) was characterized for its functional properties such as fat and water absorption capacity and protein
solubility, foaming and emulsifying capacity at different pH. The extraction of high yield protein concentrates
from M. oleifera leaves can be incorporated into dietary supplements that has becoming a promising alternative
for the food industry and treatment of malnutrition as well as has a great potential plant that is still underutilized
in its application.

2. Methodology
2.1 Sample collection and pre-treatment of fresh leaves

Samples of fresh M. oleifera leaves (5 kg) were bought from local market in Skudai, Johor, Malaysia and further
processed by tray-drying treatment at 60 ºC for 8 h. The dried leaves were collected, ground into fine MLP and
kept in tightly sealed containers.

2.2 Extraction of M. oleifera leaves protein concentrate

Different parameters of solid-to-solvent ratio (1:10; 1:20; 1:30 w/v of MLP-water) were manipulated to extract
the protein concentrate from MLP (Ahmed, 2016). The suspension was stirred for 1 h using a magnetic stirrer
while adjusting the pH at 8.5 with sodium hydroxide solution (1.0 M). The mixture was then centrifuged at 3,200
rpm, 15 min at room temperature to obtain aliquots of supernatant (S1) and precipitate (P1). At the second stage
of protein coagulation, the S1 was acidified to pH 4.5 by adding HCI (1.0 M), followed by second centrifugation
at 10,000 rpm for 10 min to obtain supernatant (S2) and precipitate (P2). The P2 was rinsed with distilled water
by three times, then subjected to third centrifugation at 10,000 rpm for 10 min to obtain supernatant (S3) and
precipitate (P3). Supernatant (S3) was then discarded, and the slurry was adjusted to neutral pH of 7.0. The
protein cake was freeze-dried to a constant weight until solid matter of MOLPC was achieved.

2.3 Characterization of MLP by proximate composition analysis

2.3.1 Determination of moisture content

Moisture content (MC) was determined following the standard method (AOAC, 1995). The final weight were
being recorded and percentage of moisture (%) for each sample was calculated using Eq(1):

𝑀𝑀𝑀𝑀(%) =
𝑤𝑤1 − 𝑤𝑤2
𝑤𝑤1 − 𝑤𝑤𝑜𝑜

× 100 (1)

where w0 is weight of porcelain crucible, w1 is weight of crucible with fresh sample, w2 is weight of crucible
containing dried sample.

2.3.2 Determination of ash content

Ash content was determined according to standard method (AOAC, 1995) as shown in Eq(2):

𝐴𝐴𝐴𝐴ℎ(%) =
𝑤𝑤2 − 𝑤𝑤𝑜𝑜
𝑤𝑤1 − 𝑤𝑤𝑜𝑜

× 100 (2)

where w0 is weight of porcelain crucible, w1 is weight of crucible with fresh sample, w2 is weight of crucible
containing dried sample.

2.3.3 Determination of crude protein content

The crude protein was conducted following Bradford assay (Kabbashi et al., 2018). A protein standard curve
was performed by dissolving Bovine Serum Albumin (BSA) powder in 10 mL of water at room temperature. A

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calibration standard curve of absorbance against BSA was plotted using Microsoft Excel and an equation of
linear regression was generated. The unknown concentration of M.oleifera samples were determined by referral
to the established protein standard calibration curve.

2.3.4 Determination of crude fibres

Determination of crude fibres was performed according to Offor et al. (2014). The crude fibre was determined
using Eq(3).

Crude fibre (%) =
𝑤𝑤2−𝑤𝑤1

𝑤𝑤
× 100 (3)

where w is weight of the original sample, w1 is weight of the sample after pyrolysis, w2 is weight of the dried
sample.

2.3.5 Determination of crude fat content
Standard protocol of Soxhlet extraction with petroleum ether as solvent was performed to determine the fat
content (Kabbashi et al., 2018). The crude fat content was calculated per 100 g of sample using Eq(4).

Fat Content (%) = 𝑊𝑊2− 𝑊𝑊1
𝑊𝑊

x 100 (4)

where w is weight of original sample, w1 is weight of empty extraction flask, w2 is weight of extraction flask with
fat.

2.4 Characterization of the functional properties of M. oleifera leaves protein concentrate

2.4.1 Fat absorption capacity (FAC) of MOLPC

1.0 g of the MOLPC was mixed thoroughly with 10 mL of corn oil (Ahmed, 2016). The protein-oil mixture was
subjected to centrifugation for 20 min. The supernatant was taken out, and the tube was reweighed. FAC was
then determined by Eq(5):

FAC (%) = 𝑊𝑊2− 𝑊𝑊1
𝑊𝑊

x 100 (5)

where w is weight of dried sample, w1 is weight of tube with sediment, w2 is weight of tube with dried sample.

2.4.2 Water absorption capacity (WAC) of MOLPC

WAC was performed following method by Ahmed (2016) with slight modification. 1.0 g of the MOLPC sample
was weighed into centrifuge tube. Distilled water (10 mL) was added in slowly to the tube and stirred
continuously with glass rod. After 30 min, it was centrifuged for 20 min. Supernatant was taken out and the tube
was reweighed. WAC was calculated using Eq(6) (Ahmed, 2016):

WAC (%) = 𝑊𝑊2− 𝑊𝑊1
𝑊𝑊

x 100 (6)

where w is weight of dried sample, w1 is weight of tube with dried sample, w2 is weight of tube with sediment.

2.4.3 Protein solubility of MOLPC

About 1g of the MOLPC was diffused in 100 mL distilled water and the mixture was adjusted to different pH (2
to 11) with 1.0 N sodium hydroxide and 1.0 N hydrochloric acid. The protein from sample and supernatant were
weighed and the solubility was calculated as shown in Eq(7) (Ahmed, 2016):

Protein solubility (%) =
𝑃𝑃𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 × 50

𝑤𝑤 ×
𝑃𝑃𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠

100

x 100 (7)

where w is weight of original sample, Psupernatant is content of protein in supernatant (mg/mL) and Psample is content
of protein in sample (mg/mL).

2.4.4 Foaming capacity (FC) of MOLPC
0.5 g of the MOLPC was diffused in 50 mL distilled water (Ahmed, 2016). The protein solution was adjusted to
different pH (2 to 11) with 1.0 M sodium hydroxide and 1.0 M of hydrochloric acid. The solution was whipped for
2 min by homogenizer in the graduated tube. FC was calculated as shown in Eq(8):

FC (%) = V2 − V1
V1

x 100 (8)

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where v1 is volume before whipping, v2 is volume after whipping.

2.4.5 Emulsifying capacity (EC) of MOLPC

1.0 g of MOLPC was mixed in 50 mL of 0.1 N NaOH, followed by 50 mL of corn oil (Ahmed, 2016). The dispersion
was magnetically stirred for 1 min at room temperature, then centrifuged at 1,100 rpm for 5 min. The emulsion
was transferred to measuring cylinder. EC was determined as shown in Eq(9):

EC = 𝑉𝑉𝐴𝐴− 𝑉𝑉𝑅𝑅
𝑊𝑊

(9)

where vA is initial volume of oil, vR is volume of oil released, w is weight of original sample.

3. Results and discussion
3.1 Analysis of chemical composition of MLP

The chemical composition of the MLP is presented in Table 1. The MC of dried MLP of less than 5 % was
favourable for maximum nutrient and colour preservation of MLP (Senadeera et al., 2003). Similar range with
previous studies was reported for ash content (8.0 to 9.8 %) (Kabbashi et al., 2018) and crude fibre content (3.4
to 19.4 %) (Yaméogo et al., 2011) in MLP. Crude fat values were lower as compared to previous study (2.3 to
17 %) reported by Yaméogo et al. (2011).

Table 1: Proximate analysis of chemical composition in MLP (dry weight basis)

Parameter(s) Quantity (MLP), %
Moisture content 4.86 ± 1.18
Ash content 9.53 ± 0.07
Crude protein 17.86 ± 0.23
Crude fibre 12.54 ± 0.01
Crude fats 0.50 ± 0.02

3.2 Characterization and functional properties of MOLPC

3.2.1 Effect of solid-to-solvent ratio on protein content, FAC and WAC of MOLPC

Protein content of MOLPC increases with increasing solid-to-solvent ratio (Table 2). Similar trend was reported
by Jain et al. (2019) showing an increase about 48.4 to 67.4 % of protein extractability with increasing solvent-
to-flour ratio from 5:1 to 20:1. Increasing FAC was observed with increasing solid-to-solvent ratio (Table 2) due
to high content of non-polar or hydrophobic amino acids in bulky protein concentrates of plant origin which is
important for them to bind hydrocarbon chains. These findings were coherent within the range of 1.69 g/g to
3.87 g/g as supported by the previous studies (Ahmed, 2016).

Table 2: Protein content, fat absorption capacity and water absorption capacity of MOLPC

Parameters Solid-to-solvent ratio (w/v)
1:10 1:20 1:30

Protein content (%) 26.58 ± 1.86 33.93 ± 0.00 35.15 ± 3.40
FAC (g/g) 3.21 ± 0.24 3.69 ± 0.13 3.82 ± 0.20
WAC (g/g) 5.19 ± 0.35 5.83 ± 0.45 6.41 ± 0.35

WAC increases with increasing solid-to-solvent ratio that aids in reducing moisture loss in MOLPC. WAC
indicates the capacity of hydrophilic peptides in MOLPC binding to water molecules and high hydrogen bonding.
High WAC property can be utilized in MOLPC products which require high water retention with low fat content
to maintain its freshness in viscous form of servings. High WAC may possibly dehydrate other components of
product formulation and moderate WAC in between 3.5 to 5.82 ± 0.47 g/g sample is much encouraged (Azubuike
et al., 2018).

3.2.2 Effect of pH on protein solubility of MOLPC in water

Figure 1 shows the effect of protein solubility at different pH. A U-shaped curve was attained with a minimum
solubility found at pH 3.5-4.0 and similar trends were indicated across three different solid-to-solvent ratios. At
the isoelectric point (pI) which corresponds to minimum solubility at that particular pH, attractive forces are

256

significant and proteins have a net charge of zero, the protein becomes insoluble which resulted from association
of the molecules (Ahmed, 2016). This indicates that interactions with water were minimal for protein molecules
at pH values not far from pI. The net charge becomes negative for pH above pI and protein-water interaction is
greatly enhanced at alkaline pH rather than acidic pH.

Figure 1: Protein Solubility of MOLPC in water against pH

3.2.3 Effect of pH on foaming capacity of MOLPC

Maximum foaming capacity was obtained at alkaline pH of 10-11 (Figure 2a) due to an increase of net charge
of protein molecules which leads to repulsion and weakens the hydrophobic interactions. This enables the
flexibility of protein molecules and allows a faster spreading of protein molecules to the air water interface and
encapsulating air particles (Azubuike et al., 2018). There is an increase in foam formation through dispersions
of gas bubbles with increasing solvent ratio from 1:10, 1:20 to 1:30. Protein isolates' foaming capacity is a key
functional feature that determines their suitability for use in various food systems that require aeration (Shevkani
et al., 2015).

3.2.4 Effect of pH on emulsifying capacity of MOLPC

The maximum emulsifying capacities were observed at pH 11 across three MOLPC-to-solvent ratios (Figure
2b). This could be due to larger contribution of protein in oil–water interfacial reactions by alkali-induced
formation of more soluble protein through unfolding of polypeptide chains.

Figure 2: (a) Foaming capacity (b) Emulsifying capacity (EC) of MOLPC-to-solvent ratios against pH

If emulsifying properties exhibit a gradual decrease, it is due to the increase of protein in the aqueous phase
that inadvertently increases the protein interaction at the protein and oil surface. This result indicates that
emulsifying activity is pH dependent in which alkaline pH can improve the EC more than acidic pH (Okiki and
Balogun, 2015).

4. Conclusion
In this study, protein concentrate was extracted from M. oleifera leaves using alkaline extraction method at pH
8.5, followed by isoelectric precipitation at pH 4.5. The yield of the protein concentrate increases with increasing

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solid-to-solvent ratios. For functional properties, MOLPC has high WAC compared to common leafy vegetable
protein concentrates. The solubility, foaming and emulsifying capacities were found to be dependent on pH. It
was found that at alkaline pHs higher protein yield and improved functional properties of MOLPC was obtained
compared to acidic pHs. Extracted protein from M. oleifera leaves protein concentrate (MOLPC) has great
potential to be an alternative protein supplement in food formulation due to its high protein content than MLP,
high productivity and better functional properties.

Acknowledgments

The author would like to acknowledge the research funding by Ministry of Higher Education Malaysia
(FRGS/1/2018/TK02/UTM/02/7) (4F993) and UTM TDRG (05G90). The author would like to express
appreciation to all laboratory assistants of Biotransformation Lab in SCEE, UTM for technical assistance
throughout this study.

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Extraction of Plant Based Protein from Moringa oleifera Leaves using Alkaline Extraction and Isoelectric Precipitation Method