2014

PHYSICOCHEMICAL PROPERTIES OF LOCAL

FRUITS EXTRACT AS ANTIMICROBIAL AGENTS

AND STAIN REMOVER

NUR AIN BINTI ABD AZIZ

MASTER OF SCIENCE

i

Physicochemical Properties of Local Fruits Extract as

Antimicrobial Agents and Stain Remover

by

Nur Ain Binti Abd Aziz

A thesis submitted in fulfilment of the requirements for the

Master of Science

Faculty of Agro Based Industry

UNIVERSITI MALAYSIA KELANTAN

THESIS DECLARATION

i

I hereby certify that the work embodied in this thesis is the result of the

original research and has not been submitted for a higher degree to any

other University or Institution.

OPEN ACCESS I agree that my thesis is to be made

immediately

available as hardcopy or on-line open

access (full text)

EMBARGOES I agree that my thesis is to be made

available as

hardcopy or on-line (full text) for a

period approved by the Post Graduate

Committee.

Date from until

CONFIDENTIAL (Contains confidential information under the

Official Secret Act 1972)*

RESTRICTED (Contains restricted information as specified

by the

organisation where research was done)*

I acknowledge that Universiti Malaysia Kelantan reserves that right as follows.

1. The thesis is the property of Universiti Malaysia Kelantan

2. The library of Universiti Malaysia Kelantan has the right to make

copies for the purpose of research only

3. The library has the right to make copies of the thesis for academic

exchange

SIGNATURE SIGNATURE OF

i

SUPERVISOR

IC/PASSPORT NO. NAME OF SUPERVISOR

Date: Date:

ii

ACKNOWLEDGEMENT

In the name of Allah, The Most Gracious and The Most Merciful. Peace and

blessing of Allah al Mighty to our beloved, final Prophet Muhammad S.A.W

and his relatives, all his companions and those who have followed.

Alhamdulillah, all praise and thankfulness to Allah S.W.T, The Most

Glorious and Omnipotent, with His willingness has allowed me to complete

this research project. Foremost, I would like to thank to Universiti Malaysia

Kelantan especially Faculty of Agro Based Industry for the research facilities.

My special appreciation to my project supervisor, Assoc. Prof. Dr Seri Intan

Mokhtar for her patience, motivation, enthusiasm, guidance and spending

time in helping me to finish this project. I wish to thanks the lab assistants,

En. Suhaimi and Pn. Sha for their kindness in helping me with my work.

My special thanks goes to my colleagues, Han, Syikin, Lia, Mira and

Anah who has together support me and give beneficial information also to

everyone who has contributed in this project. Finally this research is

dedicated to my beloved family who always give me freedom to explore my

own path, encouragement and support to success. May Allah bless all of them.

Sincerely,

Nur Ain

iii

TABLE OF CONTENTS

PAGE

THESIS DECLARATION i

ACKNOWLEDGEMENT ii

TABLE OF CONTENTS iii

LIST OF TABLES viii

LIST OF FIGURES x

LIST OF ABBREVIATIONS xiii

LIST OF SYMBOLS xiv

ABSTRAK xv

ABSTRACT xvi

CHAPTER 1 INTRODUCTION 1

1.1 General Introduction 1

1.2 Problem Statements 4

1.3 Significance of Study 6

1.4 Research Objectives 6

1.5 Scope and Limitation of Study 7

CHAPTER 2 LITERATURE REVIEW 8

2.1 Fruits 8

2.2 Local Fruits Species 9

2.2.1 Averrhoa bilimbi Linn. 10

2.2.2 Bouea oppositifolia (Roxb.) Meisner 11

2.2.3 Mangifera indica L. 12

2.2.4 Salacca zalacca (Gaertn.) Voss 13

2.2.5 Phyllanthus acidus (L.) Skeels 14

2.2.6 Hibiscus sabdariffa L. 16

2.2.7 Passiflora edulis (F.) Flvicarpa 17

2.2.8 Annona squamosa L. 19

2.2.9 Eleiodoxa conferta (Griff.) Burret 20

iv

2.2.10 Solanum torvum Swartz. 22

2.2.11 Solanum lasiocarpum Syn. 23

2.2.12 Spondias dulcis L. 25

2.2.13 Baccaurea motleyana (Muell. Arg) 27

2.2.14 Garcinia prainiana King 28

2.2.15 Syzygium aqueum (Bum. f.) Alston 30

2.3 Sensory Attributes of Fruit 31

2.3.1 Appearance and Colour of Fruits 31

2.3.2 Flavour 32

2.3.3 Texture 34

2.4 Maturation and Ripening of Fruits 35

2.4.1 Maturity 35

2.4.2 Ripening 37

2.5 Chemical Composition of Fruits 38

2.5.1 Organic Acid 38

2.5.2 Carbohydrates 39

2.5.3 Mineral 41

2.5.4 Vitamin 42

2.6 Microorganisms 44

2.6.1 Bacteria 44

2.6.2 Requirement for Bacterial Growth 46

2.6.2.1 Physical Requirements 46

2.6.2.2 Chemical Requirements 49

2.6.3 Types of Gram Stain Bacteria 51

2.6.3.1 Gram Positive 52

2.6.3.2 Gram Negative 53

2.7 Growth Medium 54

2.7.1 Types of Growth Medium 55

2.7.1.1 Nutrient Medium 55

2.7.1.2 Selective Medium 56

v

2.7.1.3 Enrichment Medium 56

2.7.1.4 Differential Medium 57

2.8 Antimicrobial Properties of Fruits 58

2.8.1 Antimicrobial Compound From Fruits 59

2.8.2 The Value of Fruits Used for Medicine 61

2.9 Laboratory Method of Antimicrobial Susceptibility Testing 63

2.9.1 Disc Diffusion 63

2.9.2 Minimum Inhibitory Concentration (MIC) 64

2.10 Bleaches 66

2.10.1 Chemistry of Bleach 67

2.10.2 Mechanism of Stain Removal 68

2.11 Classification of Stain 69

2.12 Acid of Fruit as Natural Stain Remover 70

CHAPTER 3 RESEARCH METHODOLOGY 73

3.1 Chemical and Reagents 73

3.2 Selected Local Fruits Species 74

3.3 Determination of Ripening Stage 76

3.4 Preparation of Fruit Extracts 81

3.5 Physicochemical Analysis 83

3.5.1 pH Measurement 83

3.5.2 Total Soluble Solids (TSS) 84

3.5.3 Estimation of Titratable Acidity 84

3.6 Quantification of Organic Acid 86

3.6.1 Standard Preparation 86

3.6.2 Preparation of Samples 86

3.6.3 Preparation of Mobile Phase 87

3.7 Antimicrobial Analysis 88

3.7.1 Test Organisms 88

3.7.2 Preparation of Inoculum 89

3.7.3 Preparation of Fruits Extracts 89

vi

3.7.4 Antimicrobial Assay

3.7.4.1 Disc Diffusion 90

3.7.4.2 Antimicrobial Susceptibility Testing 92

3.7.4.3 Minimum Inhibitory Concentration (MIC) 92

3.8 Process of Stain Removal

3.8.1 Materials 93

3.8.2 Commercial Bleach Standards 93

3.8.3 Fruit Extracts Stain Removal Abilities 94

3.9 Data Analysis 94

CHAPTER 4 RESULTS AND DISCUSSIONS 96

4.1 Physicochemical Properties of Local Fruits 96

4.1.1 Physicochemical Changes in Local Fruits during Maturation 99

4.1.1.1 Total Soluble Solids (TSS) 99

4.1.1.2 Titratable Acidity (TA) 101

4.1.1.3 pH 103

4.1.2 Classifications of Local Fruits 105

4.1.2.1 Total Soluble Solids (TSS) 106

4.1.2.2 Titratable Acidity (TA) 108

4.1.2.3 pH 110

4.2 Quantification of Organic Acid 112

4.2.1 Sample Analysis 113

4.3 Evaluation of Local Fruits as Antimicrobial Agent 121

4.4 Qualitative Analysis of Local Fruits against Different Types of Bacteria 128

4.5 Minimum Inhibitory Concentration Analysis 131

4.6 Abilities of Physicochemical Properties in Selected Local Fruits as

Stain Remover

134

4.6.1 Stain Removal Ability Using Commercial Bleaches 135

4.6.2 Natural Bleach Using Selected Local Fruits on Different Types

of Stain

137

vii

CHAPTER 5 CONCLUSION 143

5.1 General Conclusion 143

5.2 Problem Findings 146

5.3 Recommendations of Further Work 147

REFERENCES 148

APPENDIX A 166

LIST OF PROCEEDINGS 174

viii

LIST OF TABLES

NO. PAGE

2.1 Flavour description of some local fruits in Malaysia 33

2.2 Texture of some local fruits in Malaysia 34

2.3 Carbohydrate content of edible portion in fruits 40

2.4 Mineral content in some of tropical fruits in Malaysia 42

2.5 Vitamin content of some tropical fruits 43

2.6 Appropriate temperature (°C) conditions for bacterial species 47

2.7 Types of Gram positive bacteria that commonly infect humans 52

2.8 Types of Gram negative bacteria that commonly infect humans 53

3.1 Botanical name of fifteen local fruits 75

3.2 Changes of fruit parameter at different maturity stages 76

3.3 Peel colour of fifteen local fruits at different maturity stages 77

3.4 Firmness and shape of fifteen local fruits at different maturity stages 78

3.5 Diameter of fifteen local fruits at different maturity stage 81

3.6 Parts of the fruit used for extraction 82

3.7 Concentration range of organic acid standard 86

3.8 Bacterial strain of two Gram positive and three Gram negative 88

3.9 Zone diameter interpretive of ampicillin antibiotic 92

3.10 Stain removal effectiveness scale 94

4.1 Total soluble solids (TSS), titratable acidity (TA) and pH of fifteen local

fruits at different maturity stages

97

4.2 Total soluble solids (TSS) indicator of fifteen local fruits water extracts

at different maturity stages

100

4.3 Titratable acidity (TA) indicator of fifteen local fruits water extracts at

different maturity stages

102

4.4 pH indicator of fifteen local fruits water extracts at different maturity

stages

104

4.5 Refractive index of standard crop juices - °Brix or % sucrose 106

ix

4.6 Correlation relationship established between TSS with different maturity

fruit stages at low refractive juice index (a) and medium refractive juice

index (b)

106

4.7 TA standard range of fruit juice 108

4.8 Correlation relationship established between titratable acidity (TA) with

maturity stages of fruit species at low TA index (a), medium TA index

(b) and high TA index (c)

109

4.9 pH scale of acidic fruit 111

4.10 Correlation relationship established between pH with maturity fruit

stages at strong pH acidity scale (a) and weak pH acidity scale (b)

111

4.11 Regression equation for the calibration curve with limit of detection

and quantification of organic acid standards

113

4.12 Comparison of the organic acids between five local fruits at different

maturity stages

114

4.13 Antimicrobial activity of local fruits as indicated by the diameter of

clearing the inhibition zone at different maturity stage on Gram

positive and Gram negative bacteria after 24 hour incubation at 37°C

122

4.14 Zone diameter interpretive of ampicillin antibiotic 128

4.15 Inhibition interpretation of the susceptibility local fruit extracts at

different maturity stages towards Gram positive bacteria as

summarized from the zone of inhibition results

129

4.16 Inhibition interpretation of the susceptibility local fruit extracts at

different maturity stages towards Gram negative bacteria as

summarized from the zone of inhibition results

129

4.17 Minimum inhibitory concentrations of Averrhoa bilimbi and Eleiodoxa

conferta fruit extracts against E. coli at different maturity stages

132

4.18 Minimum inhibitory concentrations of Averrhoa bilimbi and Eleiodoxa

conferta fruit extracts against S. aureus at different maturity stages

132

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LIST OF FIGURES

NO. PAGE

2.1 Averrhoa bilimbi ( Belimbing Buluh) 10

2.2 Bouea oppositifolia (Kundang) 11

2.3 Mangifera indica (Pauh ringan) 12

2.4 Salacca zalacca (Salak) 13

2.5 Phyllanthus acidus (Cermai) 14

2.6 Hibiscus sabdariffa (Roselle) 16

2.7 Passiflora edulis (Markisa) 17

2.8 Annona squamosa (Nona) 19

2.9 Eleiodoxa conferta (Kelubi) 20

2.10 Solanum torvum (Terung pipit) 22

2.11 Solanum lasiocarpum (Terung asam) 23

2.12 Spondias dulcis (Kedondong) 25

2.13 Baccaurea motleyana (Rambai) 27

2.14 Garcinia prainiana (Chepu) 28

2.15 Syzygium aqueum (Jambu air) 30

2.16 Maturity indices of (A) mango, (B) mangosteen, (C) strawberry and

(D) honeydew based on colour changes

36

2.17 Ripening process of climacteric fruit in papaya 38

2.18 Shape of bacteria 45

2.19 Types of oxygen requirements of bacteria 51

2.20 The position of cell structure on Gram positive and Gram negative

bacteria

54

3.1 Selected local fruits (A) Averrhoa bilimbi, (B) Phyllanthus acidus, (C)

Eleiodoxa conferta, (D) Bouea oppositifolia, (E) Hibiscus sabdariffa,

(F) Baccaurea motleyana, (G) Spondias dulcis, (H) Salacca zalacca,

(I) Solanum torvum, (J) Solanum lasiocarpum, (K) Annona squamosa,

(L) Garcinia prainiana, (M) Passiflora edulis, (N) Syzygium aqueum,

75

xi

(O) Mangifera indica

3.2 Colour changes of fifteen local fruits during maturity stages. (A)

Averrhoa bilimbi, (B) Eleiodoxa conferta, (C) Solanum torvum, (D)

Solanum lasiocarpum, (E) Salacca zalacca, (F) Hibiscus sabdariffa,

(G) Baccaurea motleyana, (H) Mangifera indica, (I) Annona

squamosa, (J) Passiflora edulis, (K) Bouea oppositifolia, (L)

Phyllanthus acidus, (M) Garcinia prainiana, (N) Spondias dulcis,

(O) Syzygium aqueum

80

3.3 Process chart for fruit extraction 82

3.4 Colour changes of extract before and after titration to estimate the

titratable acidity

85

3.5 Disc diffusion assay: 1) bacterial suspension was prepared, 2) bacteria

were inoculated with sterile cotton swab, 3) disc containing extract

was placed on agar surface, 4) diameter of inhibition zone was

measured using Vernier caliper

91

4.1 HPLC chromatogram of organic acid in five local fruits species: (A)

Averrhoa bilimbi, (B) Eleiodoxa conferta, (C) Bouea oppositifolia, (D)

Mangifera indica and (E) Phyllanthus acidus

116

4.2 Concentration of organic acids for selected local fruits at different

maturity stage: (A) young, (B) mature and (C) ripe stage as measured

using HPLC. Graph bar indicates average mean and error bar indicate

standard deviation

119

4.3 Inhibition zone of fruit extract. Red arrow indicates the clearing

diameter on inhibition zone that was measured

123

4.4 Antimicrobial effect of five local fruit extracts at different maturity

stages against Gram positive bacteria of S. aureus and B. cereus.

Graph bar indicates average mean and error bar indicates standard

deviation

125

4.5 Antimicrobial effect of five local fruit extracts at different maturity

stages against Gram negative bacteria of E. coli, P. aeruginosa and

126

xii

Salmonella sp. Graph bar indicates average mean and

error bar indicates standard deviation

4.6 Calibration graph of commercial bleach at different

concentration on lipstick stain

4.7 Effect of natural extracts at different maturity stages on

lipstick stain soaked for 60 minutes in a concentration of 0.5

g/ml of fruit extracts at 90°C. Scale of stain removal on E.

conferta was significantly different with maturity stages (p

< 0.05). Graph bar indicates average mean and error bar

indicates standard deviation

4.8 Effect of natural extracts at different maturity stages on

ink stain soaked for 60 minutes in a concentration of 0.5

g/ml of fruit extracts at 90°C. Scale of stain removal on A.

bilimbi was significantly different with maturity stages (p <

0.05). Graph bar indicates average mean and error bar

indicates standard deviation

4.9 The original colour of stain as control on white cotton

fabric before treatment with fruit extracts

4.10 Fabric and stain colour after tested with fruit extracts. 3.67% stain was

removed by Eleiodoxa conferta (A) and 2.7% stain was removed by

Averrhoa bilimbi extract (B). Fabric colour turns to brown colour (A)

but remain same when tested with Averrhoa bilimbi (B)

135

137

138

141

141

xiii

LIST OF ABBREVIATIONS

HPLC High Performance Liquid

Chromotography TSS Total Soluble Solids

TA Titratable Acidity

DHA Dehydroascorbic acid

TCBS Thiosulphate citrate bile salt

sucrose EMB Eosin methylene blue

MIC Minimum Inhibitory Concentration

RPM Revolutions per minute

xiv

LIST OF SYMBOLS

cm Centimeter

% Percentage

g Gram

m Meter

mm Millimeter

mg Milligram

°C Degree celcius

ml Milimeter

µm Micrometer

nm Nanometer

µl Microliter

µg Microgram

xv

Sifat Fizikokimia Ekstrak Buah-Buahan Tempatan sebagai

Agen Antimikrobial dan Penyingkir Kotoran

ABSTRAK

Tujuan kajian ini adalah untuk mengenal pasti sifat-sifat fizikokimia

lima belas jenis buah-buahan tempatan pada tiga peringkat kematangan

berbeza yang mempengaruhi aktiviti antimikrobial dan kebolehupayaan

sebagai penyingkir kotoran. Lima belas buah-buahan tempatan telah

dianalisis sifat fizikokimia iaitu jumlah pepejal terlarut, keasidan dan pH di

setiap peringkat kematangan buah. Ciri-ciri buah yang mempunyai

keasidan tinggi telah dipilih untuk mengukur kandungan empat jenis asid

iaitu asid oksalik, tartarik, askorbik dan malik dan seterusnya mengukur

aktiviti antimikrobial terhadap dua jenis bakteria Gram positif (S. aureus, B.

cereus) dan tiga jenis bakteria Gram negatif (E. coli, P. aeruginosa,

Salmonella sp.) dengan menggunakan teknik penyerapan cakera dan

pencairan. Kotoran dakwat dan gincu telah diuji ke atas ekstrak buah yang

terpilih bagi menentukan tahap keupayaannya untuk menyingkir kotoran.

Keputusan kajian menunjukkan kandungan asid meningkat dengan

kematangan buah dan sebaliknya untuk kandungan pepejal terlarut dan pH.

Lima buah- buahan tempatan yang mempunyai kandungan asid tinggi iaitu

Averrhoa bilimbi, Eleiodoxa conferta, Phyllantus acidus, Mangifera indica

dan Bouea oppositifolia telah dipilih untuk mengukur dan mengenal pasti

kandungan asid organik. Asid oksalik dan askorbik hadir di setiap peringkat

buah dimana asid askorbik mempunyai kepekatan yang rendah. Asid

tartarik hanya terdapat dalam Bouea oppositifolia dengan kepekatannya

sebanyak 4.44 g/ml di peringkat muda, 5.02 g/ml di peringkat matang dan

5.10 g/ml di peringkat masak. Manakala asid malik hadir dalam setiap buah

pada setiap peringkat kematangan kecuali buah Mangifera indica. Asid

oksalik merupakan asid utama yang hadir dalam buah Averrhoa bilimbi dan

Eleiodoxa conferta. Didapati komposisi fizikokimia buah berhubung kait

dengan aktiviti antimikrobial pada setiap peringkat kematangan. Buah

Eleiodoxa conferta yang mengandungi asid oksalik tinggi mempunyai

ketahanan antimikrobial yang kuat terhadap kesemua jenis bakteria yang

diuji dimana peringkat buah muda mempunyai amtimikrobial yang tinggi.

Antimikrobial aktiviti untuk empat jenis buah yang lain adalah mengikut

turutan berikut Averrhoa bilimbi > Mangifera indica > Bouea oppositifolia >

xvi

Phyllanthus acidus. Kepekatan perencatan minimum Eleiodoxa conferta

melawan bakteria S. aureus mempamerkan perencatan yang minimum

dengan nilai 0.063 g/ml di peringkat muda,

0.125 g/ml di peringkat matang dan masak. Aplikasi buah Eleiodoxa

conferta dan Averrhoa bilimbi yang berasid dan mengandungi kandungan

asid oksalik yang tinggi mempamerkan skala keberkesanan penyingkiran

kotoran yang berbeza Peringkat muda Averrhoa bilimbi lebih berkesan

menanggalkan kotoran gincu pada kain kapas, manakala peringkat muda

Eleiodoxa conferta berkesan menanggalkan kotoran dakwat. Oleh itu, buah-

buahan tempatan yang dipilih sebagai agen antimikrobial dan penyingkir

kotoran dipengaruhi oleh tahap kematangan buah dimana buah pada

peringkat muda menunjukkan potensi lebih baik berbanding peringkat masak.

xvii

Physicochemical Properties of Local Fruits Extract as

Antimicrobial Agents and Stain Remover

ABSTRACT

The aim of this study was to evaluate the physicochemical

properties of fifteen local fruit types and measure the antimicrobial

properties and stain remover abilities at three maturity fruit stages

(young, mature, ripe). Fifteen local fruit types were analysed for their

physicochemical properties included total soluble solids (TSS), titratable

acidity (TA) and pH at different maturity stages. The potential local

fruits, particularly sour taste was selected for quantification of organic

acid (oxalic, tartaric, ascorbic and malic) measured by HPLC and

evaluated for their antimicrobial activities against two species of Gram

positive bacteria (S. aureus, B. cereus) and three species of Gram

negative bacteria (E. coli, P. aeruginosa and Salmonella sp.) using disc

diffusion and broth dilution method. Averrhoa bilimbi and Eleiodoxa

conferta that shows high acid contents were tested on cotton fabric

stained with lipstick and ink to evaluate their abilities as a stain

remover. The results showed that titratable acidity content increased

through maturity stages of fruits and conversely for total soluble solids

content and pH value. Meanwhile, quantification of organic acid from

selected fruits shows that oxalic acid and ascorbic acid were present at

all stages of five local fruit types and ascorbic acid found has low

concentrations. Tartaric acid was only present on Bouea oppositifolia

while malic acid presents in all local fruit types except Mangifera indica.

Oxalic acid was the predominant organic acids in Averrhoa bilimbi

and Eleiodoxa conferta fruit. The highest amount of tartaric acid and

malic acid were detected in Bouea oppositifolia and Phyllanthus acidus.

In the second part, the effect of fruit composition on antimicrobial

activity was investigated which correlated with organic acid content.

Fruit of Eleiodoxa conferta which is rich of oxalic acid have strong

antimicrobial activity against two types of Gram positive bacteria (S.

aureus and B. cereus) and three types Gram negative bacteria (E. coli, P.

xviii

aeruginosa and Salmonella sp.). The minimum inhibitory concentration

(MIC) against S. aureus exhibited the strongest inhibition with a value of

0.063 g/ml at a young stage, 0.125 g/ml at a mature stage and the ripe

stage. Antimicrobial activity for other four local fruit types were in order

of Averrhoa bilimbi > Mangifera indica > Bouea oppositifolia >

Phyllanthus acidus. The application of Eleiodoxa conferta and Averrhoa

bilimbi extracts on stain has shown different effectiveness scale of

stain remover. Young stage of Averrhoa bilimbi was effective to

remove ink stain while the young stage of Eleiodoxa conferta most

effective in removing lipstick stain. Thus, the ability of local fruits as

antimicrobial and stain removal is influenced by the stage of maturity

which shows that the young fruit has the potential than the ripe fruit.