PHYSICOCHEMICAL PROPERTIES OF LOCAL
FRUITS EXTRACT AS ANTIMICROBIAL AGENTS
AND STAIN REMOVER
NUR AIN BINTI ABD AZIZ
MASTER OF SCIENCE
2014
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.
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I acknowledge that Universiti Malaysia Kelantan reserves that right as follows.
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The thesis is the property of Universiti Malaysia Kelantan
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SIGNATURE
SIGNATURE OF
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SUPERVISOR
IC/PASSPORT NO.
Date:
NAME OF SUPERVISOR
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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
iv
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
v
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
134
Stain Remover
4.6.1 Stain Removal Ability Using Commercial Bleaches
135
4.6.2 Natural Bleach Using Selected Local Fruits on Different Types
137
of Stain
vi
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
vi
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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
97
fruits at different maturity stages
4.2 Total soluble solids (TSS) indicator of fifteen local fruits water extracts
100
at different maturity stages
4.3 Titratable acidity (TA) indicator of fifteen local fruits water extracts at
102
different maturity stages
4.4 pH indicator of fifteen local fruits water extracts at different maturity
104
stages
4.5 Refractive index of standard crop juices - °Brix or % sucrose
vii
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106
4.6 Correlation relationship established between TSS with different maturity
106
fruit stages at low refractive juice index (a) and medium refractive juice
index (b)
4.7 TA standard range of fruit juice
108
4.8 Correlation relationship established between titratable acidity (TA) with
109
maturity stages of fruit species at low TA index (a), medium TA index
(b) and high TA index (c)
4.9 pH scale of acidic fruit
111
4.10 Correlation relationship established between pH with maturity fruit
111
stages at strong pH acidity scale (a) and weak pH acidity scale (b)
4.11 Regression equation for the calibration curve with limit of detection
113
and quantification of organic acid standards
4.12 Comparison of the organic acids between five local fruits at different
114
maturity stages
4.13 Antimicrobial activity of local fruits as indicated by the diameter of
122
clearing the inhibition zone at different maturity stage on Gram
positive and Gram negative bacteria after 24 hour incubation at 37°C
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
129
summarized from the zone of inhibition results
4.16 Inhibition interpretation of the susceptibility local fruit extracts at
129
different maturity stages towards Gram negative bacteria as
summarized from the zone of inhibition results
4.17 Minimum inhibitory concentrations of Averrhoa bilimbi and Eleiodoxa
132
conferta fruit extracts against E. coli at different maturity stages
4.18 Minimum inhibitory concentrations of Averrhoa bilimbi and Eleiodoxa
conferta fruit extracts against S. aureus at different maturity stages
ix
132
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
36
(D) honeydew based on colour changes
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
54
bacteria
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,
x
75
(O) Mangifera indica
3.2 Colour changes of fifteen local fruits during maturity stages. (A)
80
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
3.3 Process chart for fruit extraction
82
3.4 Colour changes of extract before and after titration to estimate the
85
titratable acidity
3.5 Disc diffusion assay: 1) bacterial suspension was prepared, 2) bacteria
91
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
4.1 HPLC chromatogram of organic acid in five local fruits species: (A)
116
Averrhoa bilimbi, (B) Eleiodoxa conferta, (C) Bouea oppositifolia, (D)
Mangifera indica and (E) Phyllanthus acidus
4.2 Concentration of organic acids for selected local fruits at different
119
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
4.3 Inhibition zone of fruit extract. Red arrow indicates the clearing
123
diameter on inhibition zone that was measured
4.4 Antimicrobial effect of five local fruit extracts at different maturity
125
stages against Gram positive bacteria of S. aureus and B. cereus.
Graph bar indicates average mean and error bar indicates standard
deviation
4.5 Antimicrobial effect of five local fruit extracts at different maturity
stages against Gram negative bacteria of E. coli, P. aeruginosa and
xi
126
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
135
137
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
138
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.
141
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)
xi
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141
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
xii
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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
xi
v
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 >
xv
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, buahbuahan 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.
xv
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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.
xv
ii
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.
xv
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