TEH FARIDAH BINTI NAZARI - eprints.usm.my

PRODUCTION OF PRODIGIOSIN BY

Serratia marcescens IBRL USM84 FOR

LIPSTICK FORMULATION

TEH FARIDAH BINTI NAZARI

UNIVERSITI SAINS MALAYSIA

2018




by

TEH FARIDAH BINTI NAZARI

Thesis submitted in fulfilment of the requirements

for the degree of

Doctor of Philosophy

September 2018

ii

ACKNOWLEDGEMENT

Sincerely heart, I would like to express my appreciation to my supervisor,

Professor Dr. Darah Ibrahim for her guidance, advices, patience, and encouragements

given to me during my journey to complete this study. I will never forget her unending

support and every single word from her that had encouraged me so much until I

completed this study. Thank you very much to her.

I also wish to acknowledge the financial support in this study provided by

Ministry of Higher Education (MOHE) for MyBrain15 scholarship programme.

Besides, thousand thanks to all lab members in Industrial Biotechnology

Research Laboratory USM for their endless support in any aspect during completing

this research study. I will remember the many good experience, fun activities and

sweet memories that we have done together.

Finally, special thanks to my husband, Nurfitri Amir Bin Muhammad, my

sons (Luqman Al Hakim and Handzalah), my daughter (Sumayyah) and my family

members for their continuous supports, advices, inspiration, motivations and

unconditionally love.

Teh Faridah Nazari

September, 2018

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENT ii

TABLE OF CONTENTS iii

LIST OF TABLES xiii

LIST OF FIGURES xvi

LIST OF ABBREVIATIONS xxiii

ABSTRAK xxv

ABSTRACT xxviii

CHAPTER 1.0: INTRODUCTION 1

1.1 Problem statement 1

1.2 Rationale of study 2

1.3 Research objectives 5

CHAPTER 2.0: LITERATURE REVIEW 6

2.1 Pigment role in life and classification of pigment 6

2.2 The risk of synthetic pigment usage 8

2.2.1 Human health risks 9

2.2.2 Environmental pollution risks 10

2.3 Pigment distribution 12

2.3.1 Terrestrial environment 13

2.3.1(a) Terrestrial plants 13

2.3.1(b) Terrestrial animal 13

iv

2.3.1(c) Terrestrial microorganism 14

2.3.2 Marine environment 24

2.3.2(a) Marine plants 24

2.3.2(b) Marine animal 24

2.3.2(c) Marine microorganism 26

2.4 Bioactive compounds from marine microorganism 32

2.4.1 Factors influencing the bioactive compounds production 32

2.4.1(a) Marine environmental condition 32

2.4.1(b) Adaptation of marine microorganism 33

2.4.1(c) Predator 36

2.4.1(d) Association 37

2.5 Pigments from marine bacteria 38

2.5.1 Carotenes 38

2.5.2 Prodiginines 39

2.5.3 Melanins 41

2.5.4 Violacein 42

2.6 Market potential of natural pigment 44

2.6.1 Food industry 44

2.6.2 Pharmaceutical industry 45

2.6.3 Textile industry 46

2.6.4 Aquaculture industry 47

2.7 Prodigiosin and its application 48

v

2.7.1 Prodigiosin sources 48

2.7.1(a) Prodigiosin class and structure 49

2.7.1(b) Spectral analysis of prodigiosin pigment 50

2.7.1(c) Various properties and applications of prodigiosin 51

2.7.2 Application of natural pigment in cosmetic industry 52

2.7.2(a) Cosmetic industry 52

2.7.2(b) Natural cosmetic 53

2.7.2(c) Potency of microbial pigment in natural cosmetics 54

2.7.2(d) Disadvantages of synthetic colorants 56

CHAPTER 3.0: ANTIMICROBIAL ACTIVITIY OF THE PRODIGIOSIN

PRODUCED BY A MARINE BACTERIA, Serratia marcescens IBRL USM84 58

3.1 Introduction 58

3.2 Materials and Methods 59

3.2.1 Microorganisms and culture maintenance 59

3.2.1(a) Bacterial strain 59

3.2.1(b) Test microorganism 59

3.2.2 Searching for the existing of antimicrobial activity in the

S. marcescens IBRL USM84 cells using disc diffusion assay 60

3.2.2(a) Cultivation medium 60

3.2.2(b) Extraction of intracellular and extracellular pigments 61

3.2.2(c) Preparation of extract solution 64

vi

3.2.2(d) Preparation of test microorganism for seeded

agar plates 64

3.2.2(e) Preparation of susceptibility test disc 65

3.2.3 Diphenylpicryl-hydrazyl (DPPH) scavenging activity 66

3.2.4 Total phenolic content (TPC) 67

3.2.5 Standard curve of prodigiosin 68

3.2.6 Quantification of prodigiosin 68

3.2.7 Spectral analysis for intracellular and extracellular extracts 69

3.2.8 Presumptive test for prodigiosin from intracellular and

extracellular extracts 69

3.2.9 Macroscopy and microscopy analysis of S. marcescens

IBRL USM84 70

3.2.9(a) Observation of the colony morphology 70

3.2.9(b) Observation using a phase contrast microscope 70

3.2.9(c) Observation of the surface of S. marcescens IBRL

USM84 using Scanning Electron Microscope (SEM) 71

3.2.9(d) Observation of the cross section of S. marcescens IBRL

USM84 with Transmission Electron Microscope (TEM) 71

3.3 Results and Discussion 72

3.3.1 Screening for antimicrobial activity using disc diffusion assay 72

3.3.2 Antioxidant activity of crude 2-propanol extract 78

vii

3.3.3 Quantitification of prodigiosin from intracellular and

extracellular pigment produced by S. marcescens IBRL USM84 81

3.3.4 Characterization of prodigiosin pigment produced by

S. marcescens IBRL USM84 84

3.3.5 Macroscopic and microscopic analysis of S. marcescens

IBRL USM84 90

3.3.5(a) Morphological characteristics 90

3.3.5(b) Microscopic structures of S. marcescens IBRL USM84 92

3.4 Conclusion 95

CHAPTER 4.0: ENHANCEMENT OF PHYSICAL AND CHEMICAL

CULTURE CONDITION FOR PRODUCTION OF PRODIGIOSIN BY

Serratia marcescens IBRL USM84 96

4.1 Introduction 96


4.2.1 Enhancement of cultivation conditions in fermentation process

for cell growth, antibacterial activity and red pigment production 97

4.2.1(a) Physical parameters 97

4.2.1(b) Chemical parameters 98

4.2.2 Determination of cell growth, antibacterial activity and

pigment production 98

4.2.2(a) S. marcescens IBRL USM84 cell growth determination 98

4.2.2(b) Assay for antibacterial activity 98

4.2.2(c) Extraction and analysis of prodigiosin production 99

viii

4.2.2(d) Statistical analysis 100


4.3.1 Enhancement of production by physical and

chemical parameters 100

4.3.1(a) Effect of culture duration 101

4.3.1(b) Effect of light 104

4.3.1(c) Effect of initial pH of medium 106

4.3.1(d) Effect of temperature 108

4.3.1(e) Effect of agitation speed 110

4.3.1(f) Effect of addition of agar into the medium 113

4.3.1(g) Comparison of the growth, antibacterial activity and

prodigiosin production before and after enhancement

for physical parameter 115

4.3.1(h) Effect of carbon sources 117

4.3.1(i) Effect of nitrogen sources 120

4.3.1(j) Effect of inorganic salt 123

4.3.1(k) Effect of percentage of maltose 125

4.3.1(l) Comparison of the growth, antibacterial activity and

prodigiosin production before and after enhancement

for chemical parameter 127

4.4 Conclusion 129

ix

CHAPTER 5.0: BIOASSAY ANALYSIS AND CHARACTERIZATION OF

PRODIGIOSIN PIGMENT IN PARTITIONATED EXTRACT OF

Serratia marcescens IBRL USM84 130

5.1 Introduction 130


5.2.1 Solvent-solvent partitioning 131

5.2.1(a) Spectrophotometric analysis of partitioned extract 132

5.2.1(b) Susceptibility test of partitionated extract 132

5.2.1(c) Antibacterial activity using broth micro dilution assay 134

5.2.1(d) Minimum Bactericidal Concentration (MBC) assay 136

5.2.2 Time kill study 136

5.2.3 Physical characterization of prodigiosin in dichloromethane

partition of S. marcescens IBRL USM84 137

5.2.3(a) Effect of temperature towards stability of prodigiosin 137

5.2.3(b) Effect of pH towards stability of prodigiosin 138

5.2.3(c) Effect of light towards stability of prodigiosin 139

5.2.3(d) Effect of incubation time towards stability

of prodigiosin 140

5.2.4 Statistical analysis 141


5.3.1 Solvent-solvent partitioning process 141

5.3.1(a) Spectrophotometric analysis of partitionated extract 143

5.3.1(b) Antibacterial activity of partitioned extract 144

x

5.3.1(c) Determination of Minimum Inhibitory Concentration

(MIC) and Minimum Bactericidal Concentration

(MBC) of dichloromethane partition extract 147

5.3.2 Time kill study 150

5.3.2(a) Time kill study of MRSA 150

5.3.2(b) Time kill study of A. anitratus 152

5.3.3 Stability of prodigiosin pigment in dichloromethane

partitionated extract of S. marcescens IBRL USM84 154

5.4 Conclusion 160

CHAPTER 6.0: BIOASSAY GUIDED SEPARATION OF PIGMENT

EXTRACTED FROM Serratia marcescens IBRL USM84 162



6.2.1 Thin Layer Chromatography (TLC) 163

6.2.1(a) Bioautography assay using agar overlay method 164

6.2.2 Column Chromatography (CC) technique 165

6.2.2(a) Column packing and development 165

6.2.2(b) Spectrophotometric analysis of fractions 165

6.2.2(c) Thin Layer Chromatography analysis of fraction 166

6.2.2(d) Antimicrobial activity test of fraction 166

6.2.3 Preparative TLC for purification 167

6.2.4 Ultra Performance Liquid Chromatography (UPLC) 168

6.2.5 In vitro toxicity study 169

xi

6.2.5(a) Preparation of artificial seawater (ASW) and

hatching of brine shrimp (Artemia salina) 169

6.2.5(b) Preparation of pigment extract 170

6.2.5(c) Brine shrimp lethality test (BSLT) 171


6.3.1 Thin Layer Chromatography (TLC) 172

6.3.1(a) Bioautography analysis of dichloromethane

partition extract 175

6.3.2 Column Chromatography 176

6.3.2(a) Spectroscopic analysis of fraction 177

6.3.2(b) Thin Layer Chromatography analysis of fraction 178

6.3.2(c) Bioassay analysis of fraction from S. marcescens

IBRL USM84 180

6.3.3 Preparative TLC 185

6.3.4 Ultra performance of Liquid Chromatography (UPLC) 187

6.3.5 In vitro cytotoxicity of extract S. marcescens IBRL USM84 192

6.4 Conclusion 198

CHAPTER 7.0: APPLICATION OF PRODIGIOSIN PIGMENT FROM

Serratia marcescens IBRL USM84 AS COLORING AGENT AND

ANTIMICROBIAL AGENT IN LIPSTICK FORMULATION 199



7.2.1 Lipstick formulation 200

xii

7.2.2 Evaluation of lipstick 200

7.2.2(a) Melting point 202

7.2.2(b) Surface anomalies 202

7.2.3 Antibacterial evaluation test of prodigiosin-formulated lipstick 202

7.2.4 Lipstick formulation for pre-market research 203

7.2.5 Pre-market survey 205

7.2.5(a) Consumer acceptance investigation 205

7.2.5(b) Skin irritation test 205

7.2.5(c) Ranking Test 205


7.3.1 Lipsticks evaluation 206

7.3.2 Antibacterial evaluation test of prodigiosin-formulated lipstick 207

7.3.3 Pre-market research 210

7.4 Conclusion 215

CHAPTER 8.0 GENERAL DISCUSSION 216

CHAPTER 9.0 GENERAL CONCLUSION AND RECOMMENDATIONS

FOR FUTURE STUDY 219

9.1 General conclusion 219

9.2 Recommendation for future study 220

REFERENCES 222

APPENDICES

LIST OF PUBLICATIONS

xiii

LIST OF TABLES

Page

Table 2.1 Quantitative yield of pigments isolated from soil fungi by

submerged fermentation technique

15

Table 2.2 Examples of fungal pigments from soil and their suggested

application

16

Table 2.3 Natural pigments produced by bacteria

22

Table 2.4 Total chlorophyll and carotenoid content of six seaweeds

25

Table 2.5 Bioactive pigments isolated from marine bacteria

30

Table 2.6 Functions and applications of exopolymeric substances

(EPS) produced by marine bacteria

35

Table 3.1 Antimicrobial activity of prodigiosin extract of

S. marcescens IBRL USM84 by disc diffusion assay

73

Table 3.2 Comparison of quantitfication of intracellular and

extracellular pigment from S. marcescens IBRL USM84

82

Table 3.3 Property of intracellular and extracellular extracts of isolate

S. marcescens IBRL USM84

87

Table 4.1 The summary of the culture condition before and after

enhancements

116

Table 4.2 The summary of the medium improvement before and after

enhancements

129

xiv

Table 5.1 Scheme for preparing dilution series of moderate water

soluble extract to be used in MIC assay

135

Table 5.2 Total yield of extract S. marcescens IBRL USM84 from

solvent partitioning

143

Table 5.3 Absorption spectrum of extracts of S. marcescens IBRL

USM84 in different partitionation extracts

143

Table 5.4 Antibacterial activity of different partitionated extract of

isolate S. marcescens IBRL USM84

144

Table 5.5 MIC, MBC and mechanism of antibiosis of dichloromethane

partitionated extract of S. marcescens IBRL USM84 against

test bacteria

149

Table 5.6 Stability of the prodigiosin pigment in dichloromethane

partitionated extract at different characteristics

160

Table 6.1 Scheme for preparing dilution series of moderate water

soluble extract (pink fraction) to be used in MIC assay

167

Table 6.2 Preparation of extract for toxicity test

170

Table 6.3 TLC analysis of the dichloromethane partition extract of

S. marcescens IBRL USM84 in mobile phase of ethanol:

2-propanol (8:2)

174

Table 6.4 The TLC analysis of fractions collected from column

chromatography

179

Table 6.5 Sensitivity test results of fractionated extract in comparison

with inhibition zones of dichloromethane partitionated

181

xv

extract of S. marcescens IBRL USM84

Table 6.6 The MIC and MBC values of Fraction 4 in comparison with

MIC and MBC values of partitionated dichloromethane


184

Table 6.7 Summary of cytotoxicity levels of extracts obtained from


197

Table 7.1 Ingredients with their prescribed quantity in the lipstick

formulation

201

Table 7.2 Lipstick formulation

204

Table 7.3 Evaluation of natural colorant lipsticks

207

Table 7.4 Antibacterial evaluation of lipsticks dyed with antibacterial

prodigiosin pigment against different bacteria

208

Table 7.5 Evaluation of skin irritation test

212

xvi

LIST OF FIGURES

Page

Figure 2.1 Color wheel

7

Figure 2.2 Astaxanthin

39

Figure 2.3 Prodiginines derivatives

40

Figure 2.4 Violacein and deoxyviolacein

44

Figure 2.5 The structure of the archetypal prodiginine, prodigiosin

50

Figure 3.1 Dry pigment paste extracted from pellet cells of


62

Figure 3.2 Flowchart of pigment extraction of intracellular and

extracellular extracts of S. marcescens IBRL USM84

63

Figure 3.3 Disc diffusion assay of crude extract (intacellular) against

different test microorganisms

75

Figure 3.4 Color of extract from intracellular and extracellular

77

Figure 3.5 DPPH free radicals scavenging activity (%) of quercetin and

crude 2-propanol extract (intracellular extract)

80

Figure 3.6 Standard curve of total phenolic content for gallic acid

80

Figure 3.7 Standard prodigiosin calibration curve 82

xvii

Figure 3.8 Pigment extract of S. marcescens IBRL USM84 in a dry

form

84

Figure 3.9 Spectral analysis of intracellular pigment extract of

S. marcescens IBRL USM84 and standard prodigiosin

85

Figure 3.10 Spectral analysis of extracellular pigment extract of

S. marcescens IBRL USM84 and standard prodigiosin

86

Figure 3.11 Spectral analysis of intracellular pigment extract of

S. marcescens IBRL USM84 under alkaline and acidic

condition

87

Figure 3.12 Presumptive test for prodigiosin from intracellular extract of


88

Figure 3.13 Presumptive test for prodigiosin from extracellular extract of


89

Figure 3.14 Colony morphology of isolate S. marcescens IBRL USM84

on Marine agar plates

91

Figure 3.15 Freeze dried biomass cells with pigment of S. marcescens

IBRL USM84

91

Figure 3.16 The observation of S. marcescens IBRL USM84 under the

phase contrast microscope

93

Figure 3.17 SEM micrograph of S. marcescens IBRL USM84

94

Figure 3.18 TEM micrographs of S. marcescens IBRL USM84

94

xviii

Figure 4.1 Effect of culture duration on prodigiosin production,

antibacterial activity and growth of S. marcescens IBRL

USM84

102

Figure 4.2 Pigment extract in 2-propanol obtained at different

cultivation period of S. marcescens IBRL USM84

104

Figure 4.3 Effect of light on prodigiosin production, antibacterial

activity and growth of S. marcescens IBRL USM84

105

Figure 4.4 Effect of initial pH of medium on prodigiosin production,

antibacterial activity and growth of S. marcescens

IBRL USM84

107

Figure 4.5 Effect of temperature on prodigiosin production,


IBRL USM84

109

Figure 4.6 Marine Broth after cultivated with S. marcescens IBRL

USM84 for 48 hours at 120 rpm and at different

incubation temperature

110

Figure 4.7 Effect of agitation speed on prodigiosin production,


IBRL USM84

111

Figure 4.8 Marine broth after cultivated with S. marcescens

IBRL USM84 for 48 hours at 25oC

112

Figure 4.9 Effect of agar on prodigiosin production, antibacterial

activity and growth of S. marcescens IBRL USM84

114

Figure 4.10 Profile of growth, prodigiosin production and antibacterial

activity of S. marcescens IBRL USM84 before and after

physical parameter enhancements

116

Figure 4.11 Effect of carbon sources on prodigiosin production, 118

xix


IBRL USM84


IBRL USM84 for 48 hours at 25oC and 150 rpm

added with different carbon sources

119

Figure 4.13 Pigment extract in 2-propanol obtained after cultivation of

S. marcescens IBRL USM84 of various carbon source

120

Figure 4.14 Effect of nitrogen sources on prodigiosin production,


IBRL USM84

121


IBRL USM84 for 48 hours at 25oC and 150 rpm

added with different nitrogen sources

122

Figure 4.16 Effect of inorganic salt on prodigiosin production,


IBRL USM84

124

Figure 4.17 Effect of percentage of maltose on prodigiosin production,


IBRL USM84

126

Figure 4.18 Marine broth after cultivated with S. marcesens

IBRL USM84 for 48 hours at 25oC, and added

different concentration of maltose

127

Figure 4.19 Profile of growth, prodigiosin production and antibacterial

activity of S. marcescens IBRL USM84 before and after

chemical parameter enhancements

128

Figure 5.1 Flow chart of organic solvent extraction and solvent-solvent

partitioning

133

xx

Figure 5.2 Partitionated extract from crude extract of S. marcescens

IBRL USM84 extracted with different solvents

142

Figure 5.3 Disc diffusion assay of dichloromethane partitionated extract

of isolate S. marcescens IBRL USM84 against MRSA and

A. anitratus

145

Figure 5.4 Disc diffusion assay of various partitionated extract against

test microorganisms

146

Figure 5.5 Time kill study of MRSA exposed to dichloromethane

partition extract of S. marcescens IBRL USM84 at different

concentrations varied from 125 to 500 µg/mL

151

Figure 5.6 Time kill study of A. anitratus exposed to dichloromethane

partitionated extract of S. marcescens IBRL USM84 at

different concentrations varied from 125 to 500 µg/mL

153

Figure 5.7 Stability of prodigiosin pigment at different temperature

155

Figure 5.8 Stability of prodigiosin pigment at different pH

156

Figure 5.9 Disc diffusion assay by the dichloromethane partitionated

extract after treated with different pH for 30 minutes

157

Figure 5.10 Stability of prodigiosin pigment towards light illumination

158

Figure 5.11 Stability of prodigiosin pigment towards incubation time

159

Figure 6.1 Chromatograms of the dichloromethane partition extract of

S. marcescens IBRL USM84 developed using ethanol:

173

xxi

2-propanol (8:2) and graphical illustration

Figure 6.2 The light pink spot with Rf of 0.72 on the TLC plate

exhibited inhibitory activity on MRSA in the

bioautography assay

176

Figure 6.3 The absorbance of different fractions collected from column

chromatography of dichloromethane partition extract of

S. marcscens IBRL USM84

177

Figure 6.4 Chromatograms developed using ethanol: 2-propanol (8:2)

180

Figure 6.5 Inhibition zone of dicholomethane partitionated and Fraction

4 extract of isolate S. marcescens IBRL USM84 against

MRSA and B. subtilis

182

Figure 6.6 Characteristic UV-visible of standard prodigiosin and

preparative TLC purified compound from S. marcescens

IBRL USM84

186

Figure 6.7 UPLC chromatogram of dichloromethane partitionated


188

Figure 6.8 UPLC chromatogram of TLC-preparative purified

compounds of S. marcescens IBRL USM84

189

Figure 6.9 UPLC chromatogram of standard prodigiosin

190

Figure 6.10 Figure 6.10: UPLC chromatogram of standard prodigiosin

and TLC-preparative purified compounds of


191

Figure 6.11 Cytotoxicity result of dichloromethane partition of

S. marcescens IBRL USM84 against brine shrimp

193

xxii

after 6 hours of exposure time (for acute cytotoxicity test)

Figure 6.12 Cytotoxicity result of dichloromethane partition of

S. marcescens IBRL USM84 against brine shrimp after

24 hours of exposure time (for chronic cytotoxicity test)

193

Figure 6.13 Cytotoxicity results of Fraction 4 of S. marcescens

IBRL USM84 against brine shrimp after 6 hours

of exposure time (for acute cytotoxicity test)

194

Figure 6.14 Cytotoxicity results of Fraction 4 of S. marcescens IBRL

USM84 against brine shrimp after 24 hours of exposure

time (for chronic cytotoxicity test)

195

Figure 7.1 Lipstick formulation containing castor oil, shea butter and

bees wax in ratio 5: 1: 1 and 2.5 g of prodigiosin pigment

from S. marcescens IBRL USM84

201

Figure 7.2 The various color of lipstick with different quantity of

pigment

204

Figure 7.3 Consumer acceptance survey

211

Figure 7.4 Skin irritation test on the skin

212

Figure 7.5 Consumer acceptance based on color

213

Figure 7.6 The various concentration of lipstick for pre-market survey,

formulated from natural colorant

214

xxiii

LIST OF ABBREVIATIONS

BUT Butanol

CFU Colony Forming Unit

DCM Dichloromethane

EA Ethyl acetate

EC50 50% effective concentration

FDA Food and Drug Administration

HAI Hospital-acquired infection

HCL Hydrochloric acid

HEX Hexane

HMDS Hexamathyldisilazine

INT p-iodonitrotetrazolium violet salt

LC50 50% lethal concentration

MA Marine Agar

MAP 2-methyl-3-n-amyl-pyrrole

MBC Minimum Bactericidal Concentration

MBC 4-methoxy-2,2‟-bipyrrole-5-carbaldehyde

MHA Mueller Hinton Agar

MHB Mueller Hinton Broth

MIC Minimum Inhibitory Concentration

MRSA Methicillin-resistance Staphylococcus aureus

NA Nutrient Agar

NaOH Sodium Hydroxide

OD Optical density

P.I Polarity index

PDA Potato Dextrose Agar

r/t Retention time

xxiv

Rf Retention factor

SDA Sabouraud Dextrose Agar

SEM Scanning Electron Microscope

TEM Tranmission Electron Microscope

TLC Thin Layer Chromatography

UPLC Ultra Performance Liquid Chromatography

UV-vis Ultra-violet visible

xxv

PENGHASILAN PRODIGIOSIN OLEH

Serratia marcescens IBRL USM84 UNTUK

PEMFORMULASIAN GINCU BIBIR

ABSTRAK

Mikroorganisma menawarkan pelbagai pigmen semula jadi sebagai satu

alternatif selamat kepada pewarna sintetik dalam kebanyakan aplikasi perindustrian

termasuk industri kosmetik, makanan, tekstil, farmaseutikal dan akuakultur.

Penyelidikan ini dijalankan bertujuan untuk mengkaji pigmen merah semulajadi

prodigiosin dengan aktiviti antimikrob oleh bakteria marin. Penemuan pewarna semula

jadi dengan aktiviti antimikrob boleh memberi banyak faedah kepada kebanyakan

industri dan bertindak sebagai agen pewarna semula jadi dengan kesan pengawetan

secara serentak kepada produk perindustrian. Dalam kajian ini, Serratia marcescens

IBRL USM84 telah dipencilkan daripada span laut tempatan Xetospongia testudinaria

dari Pulau Bidong, Terengganu. Analisis tentang kegiatan agen antimikrob daripada

pigmen intrasel dan ekstrasel pencilan IBRL USM84 telah dilakukan dan mendapati

aktiviti antimikrob daripada pigmen intrasel adalah lebih tinggi. Analisis penghasilan

pigmen mendedahkan bahawa pigmen yang diekstrak dari intrasel dan ekstrasel boleh

menghasilkan keamatan warna yang berbeza dalam kuantiti yang berbeza, iaitu

pigmen daripada intrasel menghasilkan 7.02 g/L pigmen berbanding dengan pigmen

ekstrasel sebanyak 0.25 g/L sahaja. Daripada proses peningkatan, S. marcescens IBRL

USM84 telah menghasilkan pigmen prodigiosin yang lebih tinggi dan aktiviti

antibakteria yang lebih baik pada 48 jam tempoh pengkulturan, pH 7 bagi pH awal

medium pengkulturan, dieramkan pada 25oC, dengan 150 psm kelajuan pengocakan,

manakala medium pengkulturan ditambahkan dengan 0.2 % agar-agar dan 1% maltosa

xxvi

dalam kondisi cahaya. Ekstrak diklorometana daripada pigmen intrasel yang

dihasilkan oleh S. marcescens IBRL USM84 menunjukkan kekuatan pigmentasi yang

lebih tinggi dengan aktiviti agen antimikrob yang lebih tinggi berbanding dengan

ekstrak lain. Ekstrak ini mampu merencat pertumbuhan bakteria ujian dalam julat yang

lebih besar dengan nilai kepekatan perencatan minimum yang sama ke arah semua

bakteria ujian iaitu 250 µg/mL. Berdasarkan kajian masa maut, aktiviti antibakteria

ektrak prodigiosin S. marcescens IBRL USM84 adalah bergantung kepada kepekatan.

Keputusan yang diperolehi daripada analisa UV/vis spektoskopi, ujian jangkaan dan

analisa kromatografi menunjukkan bahawa pigmen yang dihasilkan oleh S.

marcescens IBRL USM84 ialah daripada kumpulan prodiginin. Pigmen merah

lembayung ini menunjukkan penyerapan maksimum UV/vis pada 535 nm. Keputusan

daripada analisa penulenan mendedahkan bahawa pigmen prodigiosin yang tulen

menunjukkan aktiviti antibakteria lebih rendah berbanding dengan ekstrak

diklorometana dan ini disebabkan oleh kesan sinergisme antara sebatian yang hadir

dalam ekstrak. Fraksi aktif (Fraksi 4) yang diperolehi daripada kromatografi turus

mempunyai nilai kepekatan perencatan minimum lebih tinggi iaitu 1000 µg/mL

terhadap S. aureus, B. cereus, B. subtilis, MRSA and A. anitratus. Ekstrak prodigiosin

adalah sangat tidak toksik kepada Artemia salina bagi kedua-dua tahap akut dan

kronik. Penghasilan pigmen prodigiosin sebagai satu agen pewarna dalam perumusan

gincu dinilaikan. Aktiviti antibakteria gincu diwarnakan dengan prodigiosin semula

jadi menunjukkan lebih daripada 99.0% daripada penurunan pertumbuhan bakteria

apabila gincu diuji dengan S. aureus, B. cereus, B. subtilis, MRSA and A. anitratus.

Penerimaan pengguna dikaji dengan menggunakan Ranking Test (Skala Likert) dan

mendapati gincu merah semulajadi lebih diminati dalam perbandingan kepada

pewarna sintetik dalam produk kosmetik berdasarkan kecenderungan memilih

xxvii

warnanya. Kesimpulannya, perhatian telah bertambah ke arah penggunaan sumber

warna asli sebagai satu pewarna berpotensi dan agen pengawet semula jadi.

.

xxviii




ABSTRACT

Microorganisms provide plenty of natural pigments as a safe alternative to

the synthetic dyes in many industrial applications including cosmetics, food, textile,

pharmaceutical and aquaculture industries. This research was aimed to study the

natural red pigment prodigiosin with antimicrobial activity of a marine bacterium. The

finding of natural colorant with antimicrobial activity can give benefits to many

industries and acts as a natural coloring agent with preservative value simultaneously

to the industrial products. In this study, Serratia marcescens IBRL USM84 was

isolated from a local marine sponge Xetospongia testudinaria from Pulau Bidong,

Terengganu. The analysis of antimicrobial activity from intracellular and extracellular

pigments was performed and found that the antimicrobial activity from intracellular

pigment was greater. The analysis of pigment production revealed that intracellularly

and extracellularly extracted pigments were able to produce different color intensity at

different quantity, where the pigment from intracellular yielded 7.02 g/L compared to

extracellular pigment that was 0.25 g/L only. From the enhancement process, S.

marcescens IBRL USM84 produced higher prodigiosin pigment and better

antibacterial activity after 48 hours of cultivation period with medium initial pH at 7,

incubated at 25oC and with 150 rpm of agitation speed, the addition with 0.2 % of agar

and 1% of maltose under light condition. The dichloromethane extract from

intracellular pigment produced by S. marcescens IBRL USM84 showed higher

pigmentation strength with greater antimicrobial activity compared to other extracts.

xxix

This extract was able to inhibit broader range of test bacteria with the same MIC value

towards all the tested bacteria which was 250 µg/mL. Based on the time kill assay the

antibacterial activity of prodigiosin extract from S. marcescens IBRL USM84 was

concentration dependent. The results obtained from UV/vis spectrophotometer,

presumptive test and chromatographic analysis indicated that the pigment produced by

S. marcescens IBRL USM84 is of prodiginine group. The pigment was purplish red

and showed a maximum absorption at 535 nm. The results of purification analysis

revealed that the purified prodigiosin pigment exhibited lower antibacterial activity

compared to the dichloromethane extract and this could be due to the synergism effect

between compounds present in the extract. The active fraction (Fraction 4) obtained

from column chromatography had higher MIC value which was 1000 µg/mL against S.

aureus, B. cereus, B. subtilis, MRSA and A. anitratus. The prodigiosin extract was not

toxic towards Artemia salina for both acute and chronic toxicities. The production of

prodigiosin pigment as a colouring agent in lipstick formulation was evaluated. The

antibacterial activity of lipstick dyed with natural prodigiosin showed more than

99.0% of bacterial reduction when the lipstick being treated with S. aureus, B. cereus,

B. subtilis, MRSA and A. anitratus. The consumer acceptance was investigated using

the Ranking Test (Likert Scale) and found that the natural red lipstick was more

preferred in comparison to synthetic colorant in cosmetic product based on its colour

preferences. As conclusion, due to the possible toxicity of the synthetic dyes, an

increasing attention has been directed to natural color resources as a potential colorant

and natural preservative agent.

1

CHAPTER 1.0: INTRODUCTION

1.1 Problem statement

Nowadays, humans are more inclined towards natural resources for survival.

Consciousness of risks and undesirable effects from the use of chemicals, prompted

consumers prefer to choose something more safe and natural. Land and marine

resources provide variety of natural resources that can meet the requirements of

various aspects of life such as food and beverages, medicines, health, economy and

beauty. A wide variety of diseases and medical problems pose a challenging threat to

humans, who since ancient times have searched for natural compounds from plants,

animals, and other sources to treat those problems (Kumar et al., 2015).

Many artificial synthetic colorants, which have broadly been used in food and

beverages, dyestuffs, cosmetics and pharmaceutical manufacturing processes, may

lead to various hazardous effects. To curb the harmful effect of synthetic colorants,

there is worldwide interest in production of pigments from natural sources. Hence, the

deployments of natural pigments in food and beverages, dyestuffs, cosmetics and

pharmaceutical manufacturing processes have been increasing in recent years (Unagul

et al., 2005). Moreover, natural pigments not only provided a good appearance to

increase the marketability of products, but also have biological properties such as

antibiotic, antioxidant, and anticancer activities (Dufosse, 2009).

In the cosmetic industry for instance, the beauty product that has been

produced may not be completely safe for the consumer‟s health. Unsuitable

ingredients and additives formulated in cosmetic products cause side effects to the

skin. Some side effects may be seen after long term usage by appling the cosmetic

products directly on the skin or body part of the user (Vinensia, 2012). In addition, the

2

safety of synthetic colorants has been questioned hence leading to decrease in number

of permitted colorants by Food and Drugs Administration (FDA). The limitation of

permitted colorants conducted by FDA also increased the worldwide interest in uses of

natural colorants in production of consumer goods (Huck & Wilkes, 1996; Azwanida

et. al., 2015).

Besides, the number of drug resistance pathogens has been increasing over

the years. Some of factors that lead to the emergence of new diseases are microbial

adaptation becomes resistant to medicine, misuse of antibiotics, human migration

worldwide, poor sanitation lifestyle, and those who often exposed to disease vectors

and reservoirs (Racaniello, 2004; Wan Norhana & Darah, 2005). All these problems

lead to the increasing number of pathogenic bacteria that are resistant to multiple

antibiotic treatments including methicillin-resistant Staphylococcus aureus (MRSA)

(Sakoultas & Moellering, 2008) and vancomycin-resistant Staphylococcus aureus

(VRSA) (Courvalin, 2008).

Undeniable, the products will have good market value if they are coloured

with natural pigment (Venil et al., 2013). Therefore, the research in natural pigment is

important to develop industrial products with good appearance, attractive colour and

eco-friendly protection.

1.2 Rationale of study

Color plays an important role in our daily life. Color is a basic element for

visual communication. Some examples of colour application in our daily life are the

coloured goods, cosmetic products, food appearances, textiles and decorative items.

3

Color is the main characteristics evaluated and is used as target by the industries to

attract customers.

The production of the synthetic colorants is more economically and

practically advanced with colors covering the whole color spectrum. However,

synthetic colorants are facing some trouble such as dependence on non-renewable oil

resources and sustainability (Venil et al., 2013) of current operation, environmental

toxicity, and human health concerns of some synthetic dyes. Plants also have been

used in production of natural colorants before synthetic dyes were invented, but in

very low yields, low eco-efficiency and less economic (Raisainen et al., 2002). For

example the natural colorants from several plants including Hylocereus polyrhizus

(deep purple), Pandanus amaryllfolius (green), and Clitorea ternatae (blue) have been

used as cosmetic coloring agents (Azwanida et al., 2015).

Among the natural pigments resources, the natural pigments from

microorganisms are preferred for production at commercial scale because of their

stability (Raisainen et al., 2002) and the availability of their cultivation technology

throughout the year (Parekh et al., 2000). Microbial natural pigments can be produced

through fermentation of microbial cells in bioreactor for industrial scale (Dharmaraj et

al., 2009). Technically, it can be valuable sources of natural pigment production as

they are able to produce higher yields of pigment and lower residues. In fact, microbial

fermentations have many advantages such as cheaper production, easier extraction

processes, higher yields, no lack of raw materials, no seasonal variations and more

eco-friendly. Besides, pigmentation is widespread among bacteria in terrestrial and in

marine heterotrophic bacteria that comprises of carotenoids, flexirubin,

xanthomonadine and prodigiosin (Kim et al., 2007; Stafsnes et al., 2010; Soon-Jiun

and Darah, 2011; Yong, 2012).

4

Prodigiosin is an alkaloid group of pigment, synthesized as secondary

metabolite with unique tripyrrole chemical structure (Khanafari et al., 2006). Some

strains of bacteria produce prodigiosin pigment such as Serratia marcescens, Vibrio

psychroerythrus, Streptomyces griseoviridis and Hahella chejuensis where they have

been shown to be associated with extracellular vesicles or present in intracellular

granules (Kobayashi & Ichikawa, 1991; Teh Faridah, 2012). Prodigiosin has been

reported to posses antibacterial (Samrot et al., 2011; Teh Faridah et al., 2009),

antifungal (Croft et al., 2002), cytotoxic (Nakashima et al., 2005), immunological and

antitumor (Perez-Tomas et al., 2003) activities. All these properties are coinciding

with the high demand of effective and non-toxic antibacterial agent in pharmaceutical

industry. The bright red colour of pigment can be used as colouring and preservative

agent in food, cosmetics, textiles and aquaculture products for industrial applications.

Hence, this natural red pigment is a potential product to be commercialized.

In cosmetics industry, colorant play a vital role in the world of beauty as it

determines the aesthetic value of the cosmetic products (Vinensia, 2012). The

development of beauty products with an attractive colour is an important goal in the

cosmetics industry. However, most people are concerned about the long term effect in

the use of synthetic materials, making them more alert and choose products that are

natural and safe. Gradually, cosmetics producers are turning to natural cosmetic

colours, since certain synthetic colour additives have proven to show negative health

issues following their consumption (Bridle & Timberlake, 1997; Ali, 2011). The

application of prodigiosin in cosmetic formulations not only provide an attractive

colour, but can supply natural protection on lips or skins from bacterial infection, since

this pigment demonstrated antibacterial activity.

5

1.3 Research objectives

In this research, the main objective of the study was to enhance on

production of natural pigment form a marine bacterium, S. marcescens IBRL USM84.

Besides, the pigments were evaluated on antimicrobial properties and acted as natural

colorant in lipstick formulations. To complete the research study, several objectives

had been planned and were listed as follows:

i. To determine the antimicrobial activity from the intracellular and

extracellular pigment of S. marcescens IBRL USM84.

ii. To enhance the pigment production, cell growth and antibacterial activity

of S. marcescens IBRL USM84 by physical and chemical parameters in a

shake flask system.

iii. To determine the effectiveness of solvent in partitioning process and

extraction of pigment.

iv. To isolate, purify and characterize the red pigment that is prodigiosin.

v. To evaluate the application of natural red pigment prodigiosin from S.

marcescens IBRL USM84 as a natural colorant and its effectiveness as a

natural antimicrobial agent in cosmetic product, (lipstick formulation).

6

CHAPTER 2.0: LITERATURE REVIEW

2.1 Pigment role in life and classification of pigment

The world would be dull without pigments. As the saying goes “from the

green grass of home to a forest's ruddy autumn hues”, we are surrounded by living

colour. Life presents us with a variety of colours making our life meaningful (Ong &

Tee, 1992; Omayma & Abdel Nasser, 2013). Colors affect us emotionally and

psychologically since colour psychology says that the colour of the product may also

influence the efficacy of therapy (Allam & Kumar, 2011).

Colors also inspire the feelings of joy, sadness and tranquillity. For example,

colors in the red part of the color spectrum are known as “warm colors” and consist of

red, orange and yellow (Figure 2.1). These warm colours induce emotions ranging

from feelings of calm and comfort to feelings of anger and hostility. Colors on the blue

part of the spectrum are known as “cool colors” and consist of blue, purple and green.

These colors are often described as calm, but at different times could be described as

sadness or unresponsive. The studies of psychology effects of colors are also applied

to medications (Allam & Kumar, 2011).

Pigments or colorants can be from either natural or synthetic sources.

Pigments from different sources and origin can be divided into three classes namely

natural, synthetic and inorganic pigments. Natural pigments are extracted from living

organisms such as plant, insects, algae, microorganism, etc. Some color pigments

made by modification of materials from living organisms, where are considered

natural though they are not available in nature. Examples are caramel, vegetable

carbon and Cu-chlorophyllin (vide infra) (Ali, 2011). Natural pigments are safer,

healthier and better than synthetic pigments. Scientists have to intensify research on

natural pigment sources to meet market demand for the natural pigment products.

7

Figure 2.1: Color wheel (Allam & Kumar, 2011)

Synthetic pigments are known as artificial pigments which are manufactured

chemically in laboratories. Synthetic pigments have lower toxicity effect compared to

inorganic pigments. These pigments usually applied for colouring agent in many

industrial applications such as cosmetic materials, textiles, foodstuffs, dyestuffs,

plastics, paints, fish feeds and many more (Ni et al., 2008). Examples include

monoazo pigments, diazo pigments (Mortensen, 2006), Tartrazine, Erythrosine, Sunset

Yellow and Patent Blue V, etc (Allam & Kumar, 2011). Chemical synthesis will cause

inherent waste disposal problems like pollution of heavy metal compounds with high

toxicity (Hamlyn, 1995). In fact, disposable problem could threaten human health,

environmental virginity and ecosystem. Natural pigment is a good alternative to

synthetic pigment for sustainable livelihood.

8

Inorganic pigments are known as mineral pigments are often used to color

foods and drugs. Most of the pigment sources show lack of color intensity and limited

colour spectrum compared to the other two classes of pigments. These pigments are

not recommended due to many that have toxic effects and they are rapidly replaced by

synthetic dyes (Allam & Kumar, 2011). Some examples of inorganic colours are gold,

silver and titanium dioxide (Ali, 2011).

Natural pigments can show better biodegradability, eco-friendly and normally

have a higher compatibility with the environment. People have been using chemicals

or synthetic pigments in food and beverages, cosmetics, textiles and pharmaceutical

products which are having side effects and health complication. Hence, many

researchers searching for alternative pigment from natural sources like microbes and

plants which can synthesize pigments (Kang et al., 1996). These natural pigments are

safe to use and do not have side effects. There is a high demand for microbial

pigments from terrestrial and marine sources due to their innate characters, medicinal

properties (anti-bacterial, anti-fungi, anti-cancer, etc) and simple production (Carels &

Shephard, 1997).

2.2 The risk of synthetic pigment usage

The usage of synthetic chemicals and artificial colors excessively in the last

one and half century via production and application has threatened human health,

environment and eco-system. Most of the countries have banned the use of some

synthetic dyes as food colouring agents and allowed limited number of synthetic

colours under specified maximum limits. For example, Germany and Netherlands have

imposed ban on the use of specific synthetic dyes for textile dyeing in the textile

industry. The 70-odd azo-dyes as colouring agent for textile and other consumer goods

9

are banned in India meanwhile about 118 chemicals have been put up in Red-List

(Kapoor, 2006).

2.2.1 Human health risks

Already centuries, textiles have been dyed with extracts from natural sources

like minerals, plants, and animals. Since 1856, people started to move to synthetic

dyes when scientists discovered how to make them (Zollinger, 1991). The new dyes

successful changed the playing field because the production of synthetic dyes is

cheaper, brighter, color-fast, and easy to apply to fabric. This phenomenon cause

natural dyes to become obsolete for most applications (Lomax & Learner, 2006).

Unfortunately, the chemicals used to produce dyes today are often highly toxic,

carcinogenic, or even explosive alarming the world because it is very dangerous for

consumers. For example, azo dyes are considered deadly poisons and dangerous to

work with, also being highly flammable. In addition, other harmful chemicals used in

the dying process include dioxin cause a carcinogen and possible hormone disrupter,

toxic heavy metals such as chrome, copper, and zinc are known very carcinogens

while formaldehyde a suspected carcinogen (Brit, 2008).

Most of the synthesized synthetic dye manufacturing stage is banned due to

the carcinogenicity of the precursor or product which dangers for dye workers

(Dufosse, 2009; Venil et al., 2013). In the late nineteenth century, there have no

guarantee of safety commensurate for dye workers. In fact, the workers who

manufactured dye and who dyed garments would be facing deadly risks. In Japan, dye

workers were at higher risk of cancers meanwhile in the United States, death among

factory workers caused by cancers, cerebrovascular disease and lung disease are forty

times higher than caused by other diseases (Brit, 2008).

10

Inorganic colors derived from mineral-earth pigment such as lead chromate

and copper sulphate may also cause serious health problems (Hari et. al., 1994).

However, since about thirty years ago, synthetic colours and additives are harshly

criticized and consumers show reluctance toward these products, hence they prefer to

use the natural colorants (Koes et al., 1994). United State in 1960s, the use of such

food additives has been opposed and demonstrated by environmental activists who

campaigned for the natural colorants are the best alternative to synthetic colorants.

They were highlighting the good nutritional characteristics of natural colorants

compared to synthetic colorants as a sales tool. This strategy is not effective at early

stage. Finally, changes in social attitude indicated the health campaign was successful.

As an implication, a worldwide tendency to use natural colorants is generated widely

(Krishnamurthy et al., 2002).

2.2.2 Environmental pollution risks

The textile industry is one among the rapidly developing and growing

industries worldwide. Textile industry utilizes large amounts of synthetic dyes in

manufacturing the textile products. The difficulty is the by-products during

manufacturing process that is often very difficult to treat and dispose consequently

become a serious threat to the environment and has become a very critical problem in

environment conservation. Moreover, they also pollute the ground water resources of

drinking water, fisheries activities and agriculture practices (Krishna et al., 2011).

Most countries require the factories themselves to treat dye waste matter

before it is dumped. Irresponsible actions of some manufacturer by dumping the

chemical wastes into the river without treating them in advance are very disappointing.

In fact, after dying a batch of fabric, the cost is cheaper to dump the used water rather

11

than to clean and reuse the water in the factory. Hence, dye factories all over the world

are dumping millions of tons of dye effluent into rivers. As a result, fields and rivers

near jeans factories in Mexico City are turning dark blue from untreated and

unregulated dye effluent. Factories dying denims for Levi and Gap dump waste-water

contaminated with synthetic indigo straight into the environment without a sense of

responsibility. Local folks and farmers report health problems and became unsure if

the food they are obliged to grow in nearby fields is safe to eat (Brit, 2008).

Undeniable the use of synthetic dyes in textile industry massively has an

undesirable effect on all forms of life. Utilization of various chemical substances such

as sulphur, naphtha, vat dyes, nitrates, acetic acid, soaps, enzymes chromium

compounds and heavy metals like copper, arsenic, lead, cadmium, mercury, nickel,

and cobalt and certain auxiliary chemicals all collectively make the textile effluent

highly toxic. Besides, formaldehyde based dye fixing agents, chlorinated stain

removers and hydrocarbon based softeners are another harmful chemicals present in

the water. These organic materials easily react with many disinfectants especially

chlorine and form by products (DBP‟S) that are often carcinogenic and show allergic

reactions (Kant, 2012).

Water pollution with colloidal matter appears along with colors and oily froth

gives the water a bad appearance and smelly, meanwhile increases the water turbidity.

As a result, it prevents the penetration of sunlight required for the process of

photosynthesis in aquatic ecosystem (Banat et al., 1996). This condition hinders the

oxygen transfer mechanism from the water surface into water. Reduction of dissolved

oxygen in water will threaten aquatic life because the dissolved oxygen is very

essential to survive. In addition, the chemicals can clog the pores of soil and then

12

reduce the plant growth and soil productivity due to soil hardening and penetration of

roots is prevented (Kant, 2012).

2.3 Pigment distribution

Terrestrial and marine environment are natural sources of abundance pigment.

Pigment distribution discovery in plants and microorganisms make researchers race to

find the precious pigment. Almost half of the known drugs that are used to cure illness

originated from natural products produced by terrestrial organisms (Bruckner, 2002).

However, high demand in natural bioactive compounds from land-based makes it

more challenging to find. As an alternative, natural compounds from water-based are

promising sources in many field such as pharmacology, cosmetic and textile for

industrial and commercial applications (Soliev et al., 2011).

In terrestrial environments, the natural pigments are synthesized by higher

plants and microorganisms (fungi, yeasts and bacteria) while natural pigments from

marine environments are produced by lower plants and microorganisms (Ibrahim,

2008). Humans and animals are unable to synthesize pigments de novo (Cvejic et al.,

2007). Hence, they get any essential pigments from their diet either consume directly

from nature sources or formulated diet.

Pigment from plants and animals are not suitable for industrial and

commercial application since the sources become very limited with high potential for

environmental pollution and damage of the biodiversity (Shatila et al., 2013). Besides,

pigments from plant origin have drawbacks such as unstable under light exposure, heat

or adverse pH, insolubility in water, seasonal and limited resources. Meanwhile,

pigment produced by microbial have good stability (Raisainen et al., 2002) and the

13

availability of cultivation technology will increase its applicability for industrial

production (Venil et al., 2013).

2.3.1 Terrestrial environment

2.3.1 (a) Terrestrial plants

Henna is a natural red colour from plant of Middle-East. Natural color from

henna has been used to decorate the hands and feet of bride. In the traditional medical

system, henna has anti-cancer, anti-inflammatory and anti-pyretic properties (Kingston

et al., 2009). Indigofera tinctoria also called true indigo is a species of plant that was

used in eye shadow and body painting. Natural dye from I. tinctoria also used as

coloring agent where it is known as tarum in Indonesia and nila in Malaysia

(Tantituvanont et al., 2008).

Pandanus amaryllfolius (pandan leaves) a well known as a food colorant

among South-East Asian countries and is found across Malaysia, Sri Lanka, India and

Hawaii. The green color from the pandan leaves is due to the synthesis of chlorophyll

which is important for photosynthesis process (Stone, 1978). Butterfly pea (Clitoria

ternatea) is a native plant origin found in equatorial Asia regions which contribute a

deep blue color of the flowers with high amount of anthocyanins (Tantituvanont et al.,

2008). Malaysian people used the flower extract to make “nasi kerabu” which is well

known as a special meal in Kelantan.

2.3.1 (b) Terrestrial animal

Very limited research on natural organic pigments from terrestrial animals

due to it has low potential to use as coloring agent in industrial application. Delgado-

Vargas et al., (2002) reported the pigment from cochineal insect (Dactylopius coccus)

14

has been used as a dye clothes for centuries especially in India, Europe and Persia.

Cleopatra also used the red colour extracted from crushed carmine beetles and ants to

make the red color lips to look beautiful (Garner, 2008; Azwanida et al., 2015).

2.3.1 (c) Terrestrial microorganism

Soil fungi are known as pigment producer that may be actively growing or

closely associated with other organisms. Soil fungi prefer to produce many substances

like bioactive compounds and pigments in acidic condition (Calestino et al., 2014;

Akilandeswari & Pradeep, 2016). Plants, animals, bacteria, fungi, and microalgae have

capability to synthesis pigments. Based on previous study report, fungi have high

potential to produce great amounts of pigments (Mortensen, 2006; Kirti et al,. 2014).

Some of the fungal species produce many pigment colours with high stability (Nagia

& El-Mohamedy 2007). Submerged fermentation is a common technique for

quantitative yield of pigments isolated from few soil fungi as listed in Table 2.1. The

newest alternative to produce pigment in a large scale industry is use of filamentous

fungi cultured on different agro-industrial by-products (Lopes et al., 2013).

Fungi are considered as a potential source of pigments with a broad range of

biological activities (Zhang et al., 2004). The most dominant genera of fungi in the

soil are species of Trichoderma, Penicillium, Paecilomyces, Aspergillus, and

Fusarium (Celestino et al., 2014). Almost all pigment produced by fungi belong to

aromatic polyketide groups which is melanins, quinines (Dufosse et al., 2005; Caro et

al., 2012), falvins, ankaflavin, naphthoquinone, and anthraquinone (Dufosse, 2006).

The fungal pigments would be useful in various industrial applications due to their

simple and fast growth in cheap culture medium which is made from waste product.

The pigments also have different colour shades and being independent of unpredicted

15

weather conditions (Venil & Lakshmanaperumalsamy 2009). Various species of fungi

have been isolated from soil that has the ability to produce natural pigments. Table 2.2

shows some of the fungi isolated from soil and their potential applications.

Table 2.1: Quantitative yield of pigments isolated from soil fungi by submerged

fermentation technique

Fungi pH Colour Yield (g/L) References

Monascus purpureus 5 Red 0.07 Mukherjee &

Singh, (2011)

Paecilomyces sinclairii 6 Red 4.40 Cho et

al.,(2002)

Penicillium

funiculosum IBT3954

8 Red 0.13 Jens et al.,

(2012)

Penicillium sp. 5 Violet 0.20 Ogihara et al.,

(2000)

Penicillium

sclerotiorum

5 Orange 0.31 Lucas et al.,

(2010)

Yeast is known as carotenoids pigment producer and a total of 600-700 g/mL

of carotenoids has been reported to be produced by yeast species such as

Cystofilobasidium capitatum, Rhodosporidium diobovatum, R. sphaerocarpum,

Rhodotorula glutinis, Rh. minuta, and Sporobolomyces roseus (Yurkov et al., 2008).

Apart from pigmented fungus, yeast also have spectrum that is wide, can grow well in

temperature that is high and produce huge amounts of biomass (Kvasnikov et al.,

1978).

16

Table 2.2: Examples of fungal pigments from soil and their suggested application

Fungi Colour Pigment Molecular

formula

Application References

Aspergillus

niger

Black Aspergilin C24H35NO4 Antimicrobial activity Ray & Eakin, (1975)

Aspergillus

niger

Brown - - Textile dyeing

Antibacterial activity

Atalla et al., (2011);

Aishwarya, (2014)

Aspergillus

sclerotiorum

Yellow Neoaspergilic acid - Antibacterial activity Micetich & Macdonald,

(1965);

Teixeria et al., (2012)

Aspergillus

versicolor

Yellow Asperversin C47H58O10 Antifungal activity Miao et al., (2012)

Fusarium

oxysporum

Pink/

violet

Anthraquinone C14H8O2 Textile dyeing


Glessler et al., (2013)

Fusarium

verticillioides

Yellow Naphthoquinone C10H6O2 Antibacterial activity Boonyapranai et al., (2008);

Kurobane et al., (1986)

17

Table 2.2: continued


formula


Monascus sp. Yellow

Orange

Red

Monascin

Ankaflavin

Monascorubrin

Rubropuntatin

Monascorubramine

Rubropuntamine

C21H26O5

C23H30O5

C23H26O5

C21H22O5

C23H27O4

C21H23O4

Food colorant

Pharmaceuticals


Anticancer activity

Antioxidant

Juzlova &Martinkova, (1996);

Mostafa &Abbady, (2014);

Babitha et al., (2008);

Moharram et al., (2012);

Babula et al.,(2009);

Yang et al., (2014)

Penicillium

herquei

Yellow Atronenetin - Food additive

Antioxidant

Takahashi & Carvalho, (2010)

Penicillium

oxalicum

Red Anthraquinone C14H8O2 Anticancer effectin food and

pharmaceuticals

Textile dyeing

Dufosse, (2006);

Atalla et al.,(2011)

18



formula


Penicillium

purpurogenum

Orange

to

Yellow

Yellow

to

Orange

Red

Purpurogenone

Mitorubrin

Mitorubrinol

C14H12O5

C21H18O7

C21H18O8

Dyeing of cotton fabrics

Food, pharmaceuticals,

and cosmetics

Buchi et al., (1965);

Martinkova et al., (1995);

Mapari et al.,(2005);

Takahashi & Carvalho, (2010);

Teixeria et al., (2012);

Santos-Ebinuma et al., (2013)

Penicillium

sclerotiorum

Yellow

to

Orange

Pencolide

C9H9NO4

Antibacterial

activity

Brikinshaw et al., (1963);

Sclerotiorin

C21H23ClO5

Antibacterial

activity

Chidananda & Sattur, (2007);

19



formula


Isochromophilone

VI

Antibacterial

activity

Lucas et al., (2007);

- Antifungal

activity

Lucas et al., (2010)

Trichoderma

viride

Yellow

Green

Brown

Viridin

-

C20H16O6

-

Textile dyeing

Antifungal activity

Food industry

Chitale et al.,(2012);

Neethu et al., (2012);

Gupta et al.,(2013)

Trichoderma

virens

Yellow Viridol

Virone

C20H18O6

C22H24O4

Textile dyeing

Antifungal

activity

Mukherjee & Kenerley,

(2010);

Sharma et al., (2012);

Kamal et al., (2015)

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There are various Rhodotorula type species that are being isolated from plant

leaves, flowers, fruits, slime fluxes (or exudates) of deciduous trees, soil, refinery

waste water, air and yoghurt (Phaff et al., 1972; Sakaki et al., 2000; Aksu & Eren,

2007). Carotenoid production by Rhodotorula sp. is usually influenced by the

species, media content and environmental circumstances. Carotenoid total production

category produced by this species can be classified as low (less than 100 μg/g),

medium (101 to 500 μg/g) and high (more than 500 μg/g) as reported by earlier

researcher (Costa et al., 1987; Squina et al., 2002; Aksu & Eren, 2007; Maldonade et

al., 2008; Luna-Flores et al., 2010; Saenge et al., 2011).

Melanin is a special dark coloured pigment that could be generated by

halophilic black yeast, Hortaea werneckii. Melanins can be used widely in various

fields like agriculture, cosmetic, and pharmaceutical industries. Survey results also

showed melanin of Hortaea werneckii has inhibition activity on pathogenic bacteria

namely Salmonella typhi and Vibrio parahaemolyticus. This could be due to the

melanin of Hortaea werneckii isolated from solar salterns is new discovery of natural

product that pose an antimicrobial agent (Kalaiselvam et al., 2013).

Besides, bacteria are also great contributor to natural pigment production. The

application of pigment produced by bacteria as natural colorants has been investigated

by many scientists (Joshi et al., 2003; Venil & Lakshmanaperumalsamy, 2009). The

hazardous effects from synthetic color usage (Venil et al., 2013) caused an increasing

demand for natural colors from industries. Bacteria provided large scope for

commercial production of biological pigments such as carotenoids, anthraquinone,

cholorophyll, melanin, flavins, quinones, and violacein (Keneni & Gupta 2011).

Duc et al., (2006) reported some pigmented spore-forming bacterial isolates

were found from human faeces from subjects in Vietnam. Two different pigments

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found in vegetative cells are yellow pigment and orange pigments found in spores

were identified as carotenoids pigment. All these colonies were identified with close

relatives of Bacillus cibi, Bacillus jeotgali and Bacillus indicus. (Duc et al., 2006).

This finding is very closely related with fermented seafood condiment known as uoc

Mam or Vietnamese fish sauce in Vietnamese diet. A novel strain has identified as

Bacillus vietnamensis sp. nov. in Nuoc Mam (Noguchi et al., 2004).

From 216 bacterial species that were isolated from five different samples

types namely vermicompost soil, cattleshed soil, garden soil, rhizosphere banana and

rhizosphere papaya, only 13 isolates produced diverse colours of pigment. Five

selected bacteria with different pigment color which is orange, red, bluish, lemon

yellow and golden yellow were selected and identified as Micrococcus

nishinomiyaensis, Serratia marcescens, Psedomonas aeruginosa, Micrococccus

luteus, and Staphylococcus aureus (Rajguru et al., 2016).

Some of the natural pigments produced by bacteria as listed in Table 2.3

(Malik et al., 2012). Both red and yellow pigments become the main focus in

development of pigment production study. For example, monascus produced by

Monascus sp. (Yongsmithet al., 1998), carotenoid from Phaffia rhodozyma (Vazquez

et al., 1997), Micrococcus roseus (Chattopadhyayet al., 1997), Brevibacterium linens

(Guyomarc‟h et al., 2000) and Bradyrhizobium sp. (Lorquin et al., 1997) and

xanthomonadin from Xanthomonas campestrispv (Poplawskyet al., 2000). However,

only a few bacterial isolates are able to produce blue pigment.

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Table 2.3: Natural pigments produced by bacteria (Malik et al., 2012)

Bacteria Pigments/ Molecule Colour Applications

Agrobacterium aurantiacum,

Paracoccus carotinifaciens,

Xanthophyllomyces dendrorhous.

Astaxanthin Pink-red Feed supplement

Rhodococcus maris Beta-carotene Bluish-red Used to treat various disorders such as erythropoietic

protoporphyria, reduces the risk of breast cancer

Bradyrhizobium sp., Haloferax

alexandrines

Canthaxanthin Dark-red Colorant in food, beverage and pharmaceutical

preparations

Corynebacterium insidiosum Indigoidine Blue Protection from oxidative stress

Rugamonas rubra,

Streptoverticillium rubrireticuli,

Vibrio gaogenes,

Alteromonas rubra,

Serratia marcescens,

Serratia rubidaea

Prodigiosin Red Anticancer,immunosuppressant, antifungal, algicidal;

dyeing (textile, candles, paper, ink)

Pseudomonas aeruginosa Pyocyanin Blue-green Oxidative metabolism, reducing local inflammation

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Bacteria Pigments/ Molecule Colour Applications

Chromobacterium violaceum,

Janthinobacterium lividum

Violacein Purple Pharmaceutical (antioxidant, immunomodulatory,

antitumoral, antiparasitic activities); dyeing (textiles),

cosmetics (lotion)

Flavobacterium sp., Paracoccus

zeaxanthinifaciens,

Staphylococcus aureus

Zeaxanthin Yellow Used to treat different disorders, mainly with affecting

the eyes

Xanthomonas oryzae Xanthomonadin Yellow Chemotaxonomic and diagnostic markers

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2.3.2 Marine environment

2.3.2 (a) Marine plants

Natural pigment from marine algae sources are widely used in food, cosmetic

and pharmacology (Pangestuti and Kim, 2011). Seaweeds are one of the marine plants

and it is definitely algae because they lack vascular tissue. Macroalgae species

classified into three different groups based on their photosynthetic pigment and

chemical composition namely browns pigment is Phaeophyta, red pigment from

Rhodophyta and green pigment is Chlorophyta (Gupta and Abu-Ghannam, 2011).

Chinnadurai et al., (2013) reported six seaweeds namely Cheatomorpha

antennina, Enteromorpha intestinalis,Grateloupia lithophila, Hypnea valentiae,

Padina gymnospora, Ulva fasciata collected from Pondicherry coast were identified

and analysed for the estimation of photosynthetic pigment content. The maximum total

chlorophyll content of 0.68±0.01 mgg-1

produced by seaweed Cheatomorpha

antennina and the maximum carotenoid content 0.63±0.02 mgg-1

produced by Padina

gymnospora. The rich pigment content can be formulated in supplementary diet. The

estimation of total chlorophyll and carotenoid content of six seaweeds collected from

Pondicherry coast as listed in Table 2.4 (Chinnadurai et al., 2013).

2.3.2 (b) Marine animal

The wonderful colours ranging between yellow, green, blue, brown, orange,

red, purple and black also can be seen in marine animals. The various colourations in

sea fan and sea feathers (Gorgonacea), blue coral (Coenothecalia), and organ pipe

coral (Stolonifera) is due to rich of carotenoids or carotene proteins in marine

invertebrates skeleton (Goodwin, 1968; Bandaranayake, 2006).

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Table 2.4: Total chlorophyll and carotenoid content of six seaweeds (Chinnadurai

et al., 2013).

Seaweeds Total chlorophyll (a&b)

(mgg-1

)

Carotenoids (mgg-1

)

Cheatomorpha antennina 0.68±0.01 0.26±0.03

Enteromorpha intestinalis 0.54±0.02 0.38±0.01

Ulva fasciata 0.60±0.02 0.37±0.01

Padina gymnospora 0.66±0.01 0.63±0.02

Grateloupia lithophila 0.51±0.03 0.61±0.03

Hypnea valentiae 0.63±0.03 0.49±0.02

Sea coral have no ability in photosynthesis process like plant. Elde et al.,

(2012) reported three different deep-water coral species contained carotenoids

pigments which are astaxanthin and canthaxanthin-like carotenoid. There are two

morphologies basic color for Lophelia pertusa namely orange and white (Waller &

Tyler, 2005). Paragorgia arborea shows color pigment is white and range color

pigment from deep red to white-pink. Besides, Primnoa resedaeformis shows common

color pigment is orange-yellow (Mortensen & Buhl-Mortensen, 2005). Basically, the

variation of pigment color among these species depends on probability of carotenoids

bound to specific proteins (Elde et al., 2012).

Sponges have existed since more than 800 million years (Radjasa et al., 2007)

and are known to produce bioactive compounds including pigments (Donia &

Hamann, 2003; Hamid et al., 2013). These secondary metabolites play important role

for protecting them from predators or other competitors (Pawlik et al., 2002). Almost

all sponges appear in bright and conspicuous colors whether cryptic, hiding in

secluded caves or exposed. But the research about the bioactive compounds from

pigmented sponges (Porifera) and other marine invertebrates include anemones, corals,

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