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2014
PHYSICOCHEMICAL PROPERTIES OF LOCAL
FRUITS EXTRACT AS ANTIMICROBIAL AGENTS
AND STAIN REMOVER
NUR AIN BINTI ABD AZIZ
MASTER OF SCIENCE
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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
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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
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SUPERVISOR
IC/PASSPORT NO. NAME OF SUPERVISOR
Date: Date:
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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
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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
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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
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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
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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
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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
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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
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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
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(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
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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
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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
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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
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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 >
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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.
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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.
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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.