VIETNAM NATIONAL UNIVERSITY

INTERNATIONAL UNIVERSITY
School of Biotechnology
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EXPERIMENTAL DESIGN
PROJECT

Group 1:
Mai Hữu Phước BTBTIU14185
Nguyễn Lê Hà Phương BTBTIU14186
Nguyễn Phạm Quỳnh Nga BTARIU14036
Trần Quang Khải BTARIU14003
Phạm Hồng Phước Tú BTARIU14087
Nguyễn Chí Thông BTARIU13042

I. PROJECT GENERAL INFORMATION

1. NAME OF STUDY:
Screening and optimization of protein isolation from Moringa Oleifera leaves
2. FIELD OF STUDY:
Application of biochemistry techniques to research.
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II. PROJECT CONTENTS

1. AIM

Study the efficiency and optimize the more efficient proteins isolation method
after the comparison of the two techniques: Salting-out and Enzyme-Assisted Aqueous
extraction, on Moringa Oleifera leaves.

2. BACKGROUND

2.1. LITERATURE REVIEW.

Protein and calorie malnutrition is one of the most widespread problems in

developing countries. Since animal proteins are expensive for people in these areas, there
is a constant search for unconventional legumes as new protein sources. In this
connection, Moringa oleifera is considered as a promising plant used in the preparation of
traditional dishes merited the attention.

Moringa oleifera is a highly valued plant, originated and widely used in India,
Pakistan, Africa and Arabia (Somali et al., 1987; Mughal et al.,1999) because of their
impressive range of medicinal uses and high nutritional values. It also refers to
“drumstick tree” or “horseradish tree”. Moringa seeds and leaves are reported to contain
high crude protein content, with 45% (Bridgemohan, Bridgemohan, and Mohamed 2014)
and 28.7% (Teixeira et al. 2014) respectively with a wide range of essential amino acids
and high in vitro protein digestibility (Alain Mune Mune et al. 2016). When compared to
other plants, their protein content is higher than those of several legumes such as cowpea
(22%), Bambara bean (24.78%), Chickpea (23.7%), Horse gram (22.5%) (Mune Mune &
Sogi, 2015; Sreerama, Sashikala, Pratape, & Singh, 2012).

Numerous medicinal properties has been ascribed to various parts of this tree,
including the treatment of inflammation infectious diseases along with cardiovascular,
gastrointestinal and hematological disorders (The Wealth of India, 1962; Singh and
Kumar, 1999; Morimitsu et al., 2000; Siddhuraju and Becker, 2003). Moreover, in some
Asia countries nowadays, including Vietnam, the fruits, seeds, leaves, and flowers are
also eaten as nutritious vegetables. Moringa Oleifera leaf powder has also been
manufactured and produced widely as a “miracle supplement food” to improve infant
malnutrition problem, increasing the production of breast milk for pregnant women and
potentially reducing weight.

2.2. WHY THIS PROJECT NEED TO BE CARRIED OUT

Severe protein deficiencies in various parts of the world have generated

considerable interest in exploring new resources from plant proteins as substitutes for
milk and meat protein. Moringa seeds and leaves residue with such high protein content
could be a good option for human consumption or replacement of soybean meal in
poultry diets. However, the majority of studies conducted on this plant are focused on the
isolation of bioactive compounds, especially with antioxidant and hypotensive activities,
nevertheless, little information about protein extraction is found, and most choose
Moringa seeds as the study subject. Moringa seeds, though with a much higher protein
content, are only produced on an annual basis. On the other hand, Moringa leaves with
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 Thesis proposal
their fast-growing characteristics can be considered as a potential alternative material for
protein resources. By that reason, investigation of protein isolation on the leaves part is
essential and critical.

In addition, to the best of my knowledge, only Salting-out method has been

applied on leaves materials, but no previous studies have been made on the simultaneous
extraction of protein from Moringa leaves through EAE. Hence, the main intension of
this study is to compare the effectiveness between these methods on the recovery process
of protein as well as design an optimum protein extraction procedure for further analysis
or manufacturing purposes.

2.3. THE CONTRIBUTION OF THIS PROJECT INTO SCIENCE

The project outcome can be utilized for further analysis of protein extracted from
Moringa leaves. Furthermore, the isolation procedure can be evaluated to apply in the
manufacture of isolated protein powder product, which can be another supplement beside
soybean plant for patients with protein deficiency or athletes without the ability to
consume protein from animal and dairy sources.

III. CONTENTS
3.1. HYPOTHESIS

EAE method results in a higher yield of protein extracted than Salting-out technique.

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3.2. PROCESS OF PROJECT

3.2. DETAIL CONTENTS:

3.2.1. Sample preparation

Material: Fresh Moringa Oleifera leaves, grinder, sieve, Oven – Dryer, silica crucibles,
desiccator, laboratory kiln and weight.

Methods: The leaves sample is prepared by being dried, ground into powder and preserved
in a glass container with lit for extended self-life. The total ash content and moisture content
are then determined by applying dry ashing and thermogravimetric method.

a. Moisture content determination: This method is to identify the water amount that the
sample contains, by which can help to control moisture levels for material
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preservation during experimental process. The principle of thermogravimetric
techniques is defined as the weight loss of materials mass after being heated.
Moisture is withdrawn from sample by placing them in a dryer at high temperature
o
(105 C) in 5 hours. The crucibles are pre-dried, cooled down and weighted to take the
initial mass before carrying the samples.

b. Total ash content determination: It is designed to quickly identify physiological and

non-physiological ash contained in the sample. The physiological ash derives from
mineral components of the plants; and the other results from foreign matters adhered
to leaves surface by contact with soil and sand. In this process, samples that have
been determined the moisture content will be continued heated until they carbonize.
The samples are then placed into a laboratory kiln for dry ashing process in 6 to 10h,
at 500oC until the ash changes to light grey color. A higher ash content than allowed
indicates inadequate colleting and storing procedure, which will then need to be
adjusted and improved.

Analysis:

a. Moisture content determination: The moisture content of the sample is identified by

calculating the ratio between the weight of the flour after and before drying.

b. Total ash content determination: The light grey ash is then cool down in a desiccator
and weighed until the mass stay constant (the values do not exceed 0.005% between 2
measurements). It is then compared with the initial mass to determine the percentage
of ash content in the sample.

3.2.2. Screening for more effective protein extraction method

Material: Soxhlet extractor apparatus, round-bottom flask, filter paper, magnetic stirrer,
centrifugtor, rotatory evaporator, incubator, leaves flour, enzyme alkaline protease, NaCl,
NaOH, HCl, n-Hexane

Methods:

a. Salting-out method: The ground leaves material is initially defatted by applying

Soxhlet extraction method with n-Hexane. Fats and oils are insoluble in water;
therefore, utilize an organic solvent as n-hexane can solubilize those components and
extract them from flour sample, which is then collected after solvent evaporation. The
leaves powder is contained in a filter paper, which is placed in the Soxhlet extractor.
The solvent is heated by reflux, its vapor rises and is condensed in a condenser,
falling into the Soxhlet extractor below. When the extractor is filled up, the liquid
automatically runs to siphon tube and back to the solvent container, carrying the lipid
component. The process continues for 8 hours to extract all the desired content.

Protein extraction from the defatted material is carried by (Khoa and Nguy 2011)
procedure, utilizing their final optimum values of each variable. The salt solution
used in isolation process is sodium chloride. In the process, 3g of defatted flour
material is dissolved in 100mL of NaCl 0.6M solution and stirred intensively in 180
minutes under the temperature of 30oC. After extraction, NaCl is distilled off by using
rotatory evaporator at 45oC, collecting the remaining residue of the sample.
b. Enzyme-Assisted aqueous extraction: Protein extraction from this method is carried
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by (Latif et al. 2011) procedure, utilizing alkaline protease enzyme. The leaves flour
is mixed with distilled water and boiled, before adjusting pH to the optimal level for
the enzyme using 0.5N NaOH and 0.5N HCl solution. Then, enzyme is added to the
o
mixture and is incubated at 45 C for 120 minutes with continuous stirring. Next,
centrifugation is carried out, leaving the supernatant with three distinct phases (oil
phase, creamy phase and aqueous phase) and the wet meal at the bottom. Creamy
phase is then further treated for more oil recovery, while the wet meal is dried
overnight.

Analysis: Protein content (N X 6.25) of the sample residue after discarding the oil
component estimated using a Kjeldahl apparatus.(AOAC 1990)

3.2.3. Method optimization

Material: Needed materials for method that gives higher protein extraction yield

Methods: Optimization process is carried out using the traditional One-Variable-At-A-Time

method, in which the extraction procedure is operated identically, all variables but one is
held constant at a time to determine its optimum condition for extraction. The optimum
values of this variable will then be utilized to identify the second variable’s optimum. Each
method will have specific variables to consider:

- EAE method: time, temperature, enzyme concentration

- Salting-out method: liquid-solid ratio, time, temperature, concentration of salt

solution

Analysis: statistical analysis

3.3. REFERENCES
1. Alain Mune Mune, Martin, Emilienne Carine Nyobe, Christian Bakwo Bassogog, and
Samuel Ren�� Minka. 2016. “A Comparison on the Nutritional Quality of Proteins
from Moringa Oleifera Leaves and Seeds.” Cogent Food & Agriculture 2 (1). Cogent:4–
11. https://doi.org/10.1080/23311932.2016.1213618.
2. AOAC. 1990. “AOAC Official Methods of Analysis.” Association of
Official Agricultural Chemists. Washington, D.C. 15th (Volume 1):136–38.
3. Bridgemohan, Puran, Ronell Bridgemohan, and Musa Mohamed. 2014. “Chemical
Composition of a High Protein Animal Supplement from Moringa Oleifera” 5 (5):125–
28. https://doi.org/10.14303/ajfst.2014.041.
4. Khoa, C H, and S Nguy. 2011. “NGHIÊN C Ứ U CH Ế T Ạ O VÀ KH Ả O SÁT ĐẶ C
TÍNH C Ủ A DÂY NANO Si.”
5. Latif, Sajid, Farooq Anwar, Abdullah I. Hussain, and Muhammad Shahid. 2011.
“Aqueous Enzymatic Process for Oil and Protein Extraction from Moringa
Oleifera Seed.” European Journal of Lipid Science and Technology 113 (8):1012–
18. https://doi.org/10.1002/ejlt.201000525.
6. Teixeira, Estelamar Maria Borges, Maria Regina Barbieri Carvalho, Valdir Augusto Neves,
Maraíza Apareci Silva, and Lucas Arantes-Pereira. 2014. “Chemical Characteristics and
Fractionation of Proteins from Moringa Oleifera Lam. Leaves.” Food Chemistry 147.
Elsevier Ltd:51–54. https://doi.org/10.1016/j.foodchem.2013.09.135.

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IV. PROGRESS AND EXPECTED RESULTS

Contents Expected results Time

I. SAMPLE PREPARATION
a. Moisture content determination 11%
2 weeks
b. Ash content determination 10.99%
II. SCREENING EXTRACTION
METHODS
a. Salting-out 28.7%
2 months
b. Enzyme-assisted extraction Higher than 28.7%
III. METHOD OPTIMIZATION
Optimization of method that give higher Optimum values of
extraction yield variables are identified 2 months
by statistical analysis

V. SUPERVISOR’S APPROVAL

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