proposal for thesis

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 t

VIETNAM NATIONAL UNIVERSITY INTERNATIONAL UNIVERSITY

School of Biotechnology

-

* -https://books.google.com.vn/books?

id=xteiARU46SQC&pg=PA314&lpg=PA314&dq=equation+pectin+precipitation&source=bl&ots=HBIweQU KqE&sig=ACfU3U0dYkE3nWoei_0B5XR9EM4uiCEhdw&hl=vi&sa=X&ved=2ahUKEwj0np3yva3gAhWYf d4KHQIlDVAQ6AEwBHoECAYQAQ#v=onepage&q=equation%20pectin%20precipitation&f=false

BSc THESIS PROJECT PROPOSAL

Student’s name: Phan Le Dan Yen ID: BTBCIU14055

Department: Applied Biochemistry

I PROJ ECT GENERAL INFORMATI ON

1 N AM E OF S TU D Y

STUDY ON EXTRACTION, OPTIMIZATION AND ANALYSIS PECTIN FROM PASSION FRUIT PEELS FOR COATING ON FRUITS

2 F I E LD OF S TU D Y

Biochemistry

3 S U PP ER V I S OR S

MSc Le Tran Hong Ngoc (Applied Biochemistry Department- International University)

4 H OS T INS T I T UTE

IU lab- International University

5 TI M E

From 1/3/2019 to 1/7/2019

6 BU D G ET E S TI M ATI O N

10 million VND

Thesis proposal

II O PR J E C T C O N TE N T S

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 BA C KG ROU N D

2.1 LI T ER A TU R E REVIE W

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,

Thesis proposal

weigh 85-110g, fruit quality comparable to that of Purple variety

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]

Thesis proposal

c Some techniques use to extract pectin

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

Thesis proposal

pectin [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 P ROJECT NE E D TO BE C A R R I E D 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 CONTRIBUTI O N OF THIS P ROJECT INTO S CIENCE

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

Thesis proposal

III C O N TE N TS

3.1 HY P O T H ES IS

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 P ROCE S S OF P ROJECT

P fruit peels

Mille d

P fruit powders

HCl/CH 3 COOH

Heating, Filtrate

Solution

C

2 H

5 OH

Pectin

Cold at 4°C Filtrate, dried

Pectin

Qualitative test Quantitative test Edible coating solution

Testing performance on strawberry surface

Moisture, Equivalent weight, methoxyl content

Color and solubility in

water and alkali

solution

Thesis proposal

3.2 DE T AIL CONTENT S :

3.2.1 P a r t I: Extraction and optimize pectin by HCl and CH 3 COOH and

characterization pectin from passion fruit peel

M a ter ia l :

 Passion fruit peel powder

 HCl and CH3COOH solution:

 Ethanol solution: 95%, 70%

 Acetone solution

Me

tho d s:

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

Ana l ysi s :

 Determine the best parameters to extract pectin with the highest yield

3.2.2 P a r t II: Qualitative and quantitative test

a Qualitative test

M

a ter ia l :

 Dried pectin was extract from the suitable parameter

 95% ethanol solution

Thesis proposal

Me

tho d s:

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

M

a ter ia l :

 Dried pectin

 Sodium chloride NaCl

 Ethanol C2H5OH

 Phenol red as indicator

 Sodium hydroxide NaOH

 Hydrochloric acid HCl

Pectin yield can determine by equation:

Moisture content determination:

Me

tho d s:

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:

Me

tho d s:

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

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 P a r t III: Perform coating and test the performance of its

M

a ter ia l :

 Strawberry fresh

 Dried pectin sample

 Glycerol solution 1%

 CaCl2.2H2O solution

Me

tho d s:

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, …

Thesis proposal

3.3 RE F EREN C ES

[1] USDA National Nutrient Database

[2] Passion fruit information - http://www.passionfruit.org.nz/

[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: p476-483

[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: 181-185

[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