VIETNAM NATIONAL UNIVERSITY
INTERNATIONAL UNIVERSITY

School of Biotechnology
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BSc THESIS PROJECT PROPOSAL
Student’s name: Phan Le Dan Yen
ID: BTBCIU14055
Department: Applied Biochemistry

I. PROJECT GENERAL INFORMATION
1. NAME OF STUDY
STUDY ON EXTRACTION, OPTIMIZATION AND ANALYSIS PECTIN FROM
PASSION FRUIT PEELS FOR COATING ON FRUITS
2. FIELD OF STUDY
Biochemistry
3. SUPPERVISORS
MSc. Le Tran Hong Ngoc (Applied Biochemistry Department- International University)
4. HOST INSTITUTE
IU lab- International University
5. TIME
From 1/3/2019 to 1/7/2019
6. BUDGET ESTIMATION
10 million VND

II.

Thesis
proposal

PROJECT CONTENTS

1. AIM
The goal of this project is that
- Identify the suitable solvent which can be used to extract the highest amount pectin from
Passion fruit peel.
- Optimize some conditions include time, temperature, ratio between solvent and sample effect
on the amount pectin were extracted
- Quantitative and qualitative test of pectin
In addition, pectin extracted can be used to produce coating on strawberries to extend storage
time and quality.
Lastly, reduce the problems of polluted environment cause by waste product

2. BACKGROUND
2.1. LITERATURE REVIEW
a. Passion fruit background
The passion fruit (Passiflora edulis) is a subtropical fruit, wild regions of South America. It is
a vine which can be grown on anything that it can grapple around tendrils. This fruit can
develop quickly and reach 12-20 feet, the lifespans last 5-7 years. It is a pepo, type of berry, in
the maturity stage, fruit are round or oval, can be eaten or juiced, soft to firm, juicy interior
filled with numerous seeds. The juice often added to other fruit juice to enhance aroma.
Passion fruits classified into three types Yellow, Purple, Hybrid passion fruit
(Purple x Yellow) are of commercial importance in India. [1], [2], [3]
Yellow passion fruit: (Passiflora edulis var. flavicarpa)
Fruits of this variety is bigger size than purple type, shape in round, weigh about 60g, with

yellow mottled spots, turn golden yellow at maturity. Juice more acidic than purple type, seed
are brown and the nutrition value lower with purple fruits type.
Purple passion fruit: (Passiflora edulis Sims)
Fruit are 4-5 cm, weigh 35-45g at maturity. The juice content varies from to 31 to 35 percent.
Seed are black. It is known for its quality in flavor and nutrient content.
Hybrid passion fruit (Purple x Yellow)
This type is and hybrid between Purple and Yellow passion fruit, developed by at Central
Horticulture Experimental Station, Indian Institute of Horticulture Research, Chettalli,
Karnataka. Hybrid passion fruits are of commercial importance in India. Fruit are purple,
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weigh 85-110g, fruit quality comparable to that of Purple variety.

Thesis
proposal

b. Pectin background
Pectin is defined as complex mixtures of polysaccharides that make up approximately one
third of the cell-wall dry substance of most types of plants (Van Buren, 1991). The function of
pectin in plants is to contribute structural integrity to the cell walls and adhesion between
cells. Pectin is primarily made up of D-galacturonic acid joined by K-(1-4) glycosidic linkages
(Van Buren, 1991). As a part of the plant structure, it is a complex mixture of blocks of
homogalacturonic acid called ‘smooth regions’ mixed with blocks of homogalaturonic acid
containing many neutral sugars including rhamnose, galactose, arabinose, xylose, and glucose
called ‘hairy regions’ (IPPA, 2001). A percentage of the galacturonic acid residues are
generally esterified with methanol. [4]
In addition, the pectin of a plant can be water-soluble, chelator soluble, or protopectin
(Van Buren, 1991). The methods of extraction will vary based on the actual makeup for
each type plant. Besides, the general makeup of the pectin content varies with ripening of the

plant and it is easily brought into solution depending on the plant type (Van Buren, 1991).
After extraction pectin consists of smooth galacturonic acid regions with a few neutral sugars
still attached (IPPA, 2001). Commercial pectin extraction is mainly from citrus peel and apple
pomace, but several other sources exist such as sugar beets and sunflower heads. [4]
Pectin is the most important gelling agent used for jam jelly and marmalade. However, the
largest use of pectin has changed to be a stabilizer for acidified milk drinks. Pectin has also
been investigated for its usefulness in the pharmaceutical industry. Among other uses it has
been considered in the class of dietary fibers known to have a positive affected on digestive
processes and to help lower cholesterol (Braddock, 1999). It also is utilized to stabilize liquid
pharmaceutical emulsions and suspensions. [4]
There are two types of pectin depending on their Degree of Methylation (DM), pectin is
referred to as high methoxy pectin (DM>50) or low methoxy pectin (DM<50). High methoxy
pectin can form gels in an acidic medium (pH 2.0-3.5) if sucrose is present at a concentration
higher than 55 weight %. Low methoxy pectin can form gels over a larger pH range (2.0–6.0)
in the presence of a divalent ion, such as calcium [5]

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c. Some techniques use to extract pectin

Thesis
proposal

Acid extraction of pectin
Pectin has been extracted using chemical methods to examine structural features, functional
properties of pectin. The extraction conditions vary from facility to facility and are dependent
on the pectin source. Extraction most commonly occurs using a dilute mineral acid, usually
hydrochloric, sulfuric, or nitric acids. [6]
Chemical agents classified into four group include water, buffers, calcium- ion chelator, acid,

base. Acids are strongest agents to extract. It facilitates of insoluble pectin, tightly bound to
the cell matrix.

[6], [7]

Hydrochloric acid shown to be the highest yield of pectin. Presence of high concentration of
hydrogen ions, stimulate the hydrolysis of pectin from proto pectin. However, hydrochloric
acid produced pectin with a smaller DM range in which LM pectin [6], [7]. Nitric acid is also
used commonly to acidify hot water to extract pectin. Pectin yield increased with increasing
extractant strength of nitric acid. Lowest yield was reported for the citric acid. Some studies
had reported that acid type strongly influenced the macromolecular and gelling properties of
isolated pectin. [8]
Microwave extraction:
Microwave Assisted Extraction (ME) involves dielectric heating of plant molecules through
the exposure of microwaves. Dipolar rotation of water is taken place due to the absorbance of
microwave energy, leads to generation of heat inside the plant tissues. Microwave-Assisted
Extraction (MAE) has been recently investigated by many researchers and found that it can
lead to a considerable increase in the yield and quality of extracted pectin. [9]
When increasing the microwave power pectin yield has been increased due to the increase in
microwave irradiation energy, the penetration of solvent into the plant matrix can be enhanced
and can efficiently deliver to plant cells for pectin extraction. [10]
Enzymatic extraction
Plant cell wall is composed of an entangled network of different polysaccharides including
pectin. Cell wall degrading enzymes with minimum pectinolytic activity are used to hydrolyze
plant cell wall components in enzymatic extraction of pectin. Enzymatic extraction of pectin
is environmentally safe and more pectin yield. different enzymes such as polygalacturonase,
hemicellulose, protease and microbial mixed enzymes, cellulose, α-amylase, celluclast,
alcalase and α-amylase,… These enzymes can degrade pectin and modify the properties of
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pectin.

Thesis
proposal

[11]

d. Edible coating
Edible film is a thin layer made from edible material which is used for
food coating or laid

between food components (film) and had function

as a barrier of mass transfer such as humidity, oxygen dan light. The use
of synthetic polymers as packaging material. It is negative effect to the
environment because it is hard to be destructed naturally by biotic agent.
[12]

.

Edible film could be made from 3 different ingredients such as
hydrocolloid, lipid, and combine of both compounds. One kind of
hydrocolloid that can be used as edible film
ingredient is carbohydrate (starch, alginate, pectin, Arabic gum and
modified carbohydrate). Lipid compound that can be used is wax, glycerol
and fatty acid.

[13]


2.2. WHY THIS PROJECT NEED TO BE CARRIED OUT
In Viet Nam, the number of productions from passion fruits is very high. Almost half of these
passion fruits are used to make juice, the remainder as peel considered as wastes. This wasted
peel represents a significant potential for extracting pectin. Therefore, the goal is the
extraction the highest pectin from purple passion peels. The extraction of passion fruits yields
35-38% juice with residues left over 62-65% resulting in a large amount of waste material for
disposal. (USDA National Nutrient Database). It is of interest to explore pectin extraction
from grapefruit peels. The basic pectin extraction procedure for many types of peels as citrus,
guava, ... applied to pectin extraction from several other plant materials will be applied to
passion fruit peel. Besides, edible coating from pectin extracted can storage fruits so the use
preservatives will reduce in fruits preservation.
2.3. THE CONTRIBUTION OF THIS PROJECT INTO SCIENCE
This study will reduce the large amount passion fruit peel to protect the environment. In
addition, it also contributes the best parameters to optimize to extract pectin. Besides, the
coating from pectin can be used to improve the quality and extend storage time for strawberry
after post- harvesting.

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Thesis
proposal

III. CONTENTS
3.1. HYPOTHESIS
The amount of pectin can be affected by time, temperature, concentration, type of acid. In
addition, it can used to perform coating to storage strawberry.

3.2. PROCESS OF PROJECT


HCl/CH3COOH

Milled

P. fruit peels

Solution

P. fruit powders
Heating, Filtrate
Filtrate, dried

Pectin

Qualitative test

Color and solubility in
water and alkali
solution

C2H5OH
Cold at 4°C

Pectin

Quantitative test

Moisture, Equivalent
weight, methoxyl content


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Edible coating solution

Testing performance on
strawberry surface


Thesis
proposal

3.2. DETAIL CONTENTS:
3.2.1. Part I: Extraction and optimize pectin by HCl and CH3COOH and
characterization pectin from passion fruit peel
Material:


Passion fruit peel powder



HCl and CH3COOH solution:



Ethanol solution: 95%, 70%



Acetone solution


Methods:
First, Passion fruit peel was collected by removing the juice. The albedo dried in oven
machine under 55°C until reach constant weight. After albedo dried completely, it was to get
powder. 10 grams powder blend with 0.2N of HCl and CH3COOH solvent. The mixture was
stirred by using magnetic stirrer until forming homologous form. The mixture was heated in
the water bath at the different temperature and time. Next, the hot acid extract will filter with
two layers cheesecloth. Adding ethanol 95% to solution after filtering with the ratio 1:2 (pectin
extract: ethanol) to pectin precipitation. Keeping this mixture at 4°C for overnight. The pectin
precipitation will filter by using filter paper then immersed with ethanol 70 % from 2 to 3
hours and wash with acetone immediately.
Pectin was extracted with different ratio (powder: solvent) and for
different extraction periods. The dried peel to extractant ratios were maintained at 1:10,
1:20, 1:30 and 1:40 (w/v), during extraction. Extraction time varied for 45, 60, 90 minutes to
determine the time of extraction for maximum yield pectin. The temperature of the heating the
mixture at 80, 90,98°C.
Analysis:


Determine the best parameters to extract pectin with the highest yield

3.2.2. Part II: Qualitative and quantitative test
a. Qualitative test
Material:


Dried pectin was extract from the suitable parameter




95% ethanol solution



0.1N NaOH solution
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Thesis
proposal

Methods:
Pectin color:
Observe the color of the pectin after extracting
Solubility in hot and cold water:
First, weighting 0.03g pectin sample and adding 10 ml of 95% ethanol taken in

Erlenmeyer flask, the volume up to 50ml by distilled water. This mixture stirrer and
shake to form suspension. Next, heating it at 85-95°C using magnetic stirrer.
Solubility in hot and cold alkali (NaOH):
Initially, 10ml of 0.1N NaOH taken in Erlenmeyer flask with 0.1g pectin sample and heat
at 85-90°C for 10-15 min by using magnetic stirrer.
b. Quantitative test
Material:


Dried pectin




Sodium chloride NaCl



Ethanol C2H5OH



Phenol red as indicator



Sodium hydroxide NaOH



Hydrochloric acid HCl

The amount of pectin was extract:
Pectin yield can determine by equation:

Moisture content determination:
Methods:
An empty Petri dish was dried in oven then cooled in desiccator. After, weighting from 1 to 2
g of pectin transferred into the Petri dish. The dish contain sample dried in the oven for 1
hours, it cooled in desiccator. Finally, weight residues content.
Equivalent weight determination:
Methods:
Weighting 0.5g pectin into Erlenmeyer flask and adding 5mL ethanol, 1g sodium chloride.
Next, the mixture was added more distilled water to follow up the volume into 100mL, few

drops of phenol red will be added. Ensure that all pectin dissolved, no clumping occurs at the
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Thesis
proposal

side of the flask. Using 0.1N NaOH to titrated with mixture to a pink color at the endpoint.
Methoxyl Content determination:
Adding 25mL of 0.25N NaOH to the neutral solution in the equivalent weight part. Then,
standing the solution for 30 minutes at room temperature. Next, adding 25mL of 0.25N HCl
solution and titrated with 0.1N NaOH until the same endpoint.
Analysis:
The moisture content can determine by using equation:

Equivalent weight can determine by below equation

The methoxyl content identify by formula:

3.2.3. Part III: Perform coating and test the performance of its
Material:


Strawberry fresh



Dried pectin sample




Glycerol solution 1%



CaCl2.2H2O solution

Methods:
Weight 1.5g dried pectin into beaker and mixed with 100mL glycerol solution 1%. The
mixture heated to 70°C keep this temperature for 90 minutes. Next, pouring CaCl2.2H2O
solution slowly into the mixture. Then, keep this mixture at room temperature.
Strawberry were immersed in edible coating for duration 15, 30, 45 seconds with the number
immerse 1, 2, 3 times.
Analysis:
From the coating performance on the surface of the strawberry. There are some criteria to test the
strawberry’s condition include weight, firmness, fungus, …
Weight reduction = initial weight - final weight
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Thesis
proposal

3.3. REFERENCES
[1] USDA National Nutrient Database.
[2] Passion fruit information - />[3] />[4] Pornsak Sriamornsak. "Chemistry of Pectin and Its Pharmaceutical Uses: A Review."
ResearchGate
[5] Kliemann E, Simas KN, Amante ER, Prudencio ES, Teoflo RF, et al. (2009) Original article
optimisation of pectin acid extraction from passion fruit peel (3assiflora edulis flavicarpa) using
response surface methodology. International Journal of Food Science and Technology 2: p476483.

[6] Assoi S, Konan K, Walker LT, Agbo GN, Dodo H, et al. (2014) Functionality and yield of
pectin extracted from Palmyra palm (Borassus aethiopum Mart) fruit. LWT-Food Science and
Technology 58: 214-221.
[7] Maria B, Gama V, Silvab C, Silvab LM, Abudc A (2015) Extraction and characterization of
pectin from citric waste. Chemical Engineering Transactions 44: 259-264.
[8] Yapo BM. (2011) Pectic substances: From simple pectic polysaccharides to complex pectins:
A new hypothetical model. Carbohydrate Polymers 86 :373-385.
[9] Kratchanova M, Pavlova E and Panchev I (2004) The eff‫ٴ‬ect of microwave heating of fresh
orange peels on the fruit tissue and quality of extracted pectin. Carbohydrate Polymers 56: 181185.
[10] Fishman ML, Chau HK, Hoagland PD, Hotchkiss (2006) Microwaveassisted extraction of
lime pectin 20: 1170–1177
[11] Puri M, Sharma D, Barrow CJ (2012) Enzyme-assisted extraction of bioactives from plants.
Trends in Biotechnology 30: 37-44.
[12] Constenla, D., Ponce, A. G., & Lozano, J. E. (2002). Effect of pomace drying on apple
pectin. LWT-Food Science & Technology, 35,
216–221
[13] Koubala, B. B., Mbome, L. I., Kansci, G., Mbiap, T. F., Crepeau, M. J., Thibault, J. F., &
Ralet, M. C. (2008). Physicochemical properties of pectins from ambarella peels (Spondias
cytherea) obtained using different extraction conditions. Food Chemistry, 106, 1202–1207.

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Thesis
proposal

IV. PROGRESS AND EXPECTED RESULTS
Contents



Expected results

Prepare the peel from fruits

Powder passion fruit peel

 Dried and milled into the powder
Part I: Extraction and optimize pectin by HCl
and CH3COOH and characterization pectin
from passion fruit peel

Experiment 1: Effect the ratio sample and solvent Best ratio between sample and
solvent
Best periods
Experiment 2: Effect the time

Time
1/3- 30/4

1/5 – 22/5

Best temperature

Experiment 3: Effect the temperature
Part II: Qualitative and quantitative test
Experiment 1: Qualitative include color and
solubility

The color and solubility reliable,

base on the theory

Experiment 2: Quantitative include yield,
equivalent weight, methoxyl, moisture,

The value of quantitative test in
reliable

26/5- 10/6

Part II: Perform coating and test the
performance of its
Coating can be form

Experiment 1: Perform coating solution from
dried pectin
Experiment 2: Testing the time and
characteristics base on criteria

Coating can be used to extend
time storage and improve quality

Part II: Analysis data

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11/6 -27/6



Thesis
proposal

V. SUPERVISOR’S APPROVAL
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Signature:

Le Tran Hong Ngoc
Date: 23/1/2019

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