PRODUCTION OF PRODIGIOSIN BY Serratia marcescens IBRL USM84 FOR
LIPSTICK FORMULATION
TEH FARIDAH BINTI NAZARI
UNIVERSITI SAINS MALAYSIA
2018
PRODUCTION OF PRODIGIOSIN BY Serratia marcescens IBRL USM84 FOR
LIPSTICK FORMULATION
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
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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
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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
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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 Materials and Methods 97
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
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4.2.2(d) Statistical analysis 100
4.3 Results and Discussion 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
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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 Materials and Methods 131
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 Results and Discussion 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
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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
6.1 Introduction 162
6.2 Materials and Methods 163
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
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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 Results and Discussion 172
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
7.1 Introduction 199
7.2 Materials and Methods 200
7.2.1 Lipstick formulation 200
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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 Results and Discussion 206
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
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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
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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 extract of S. marcescens IBRL USM84
184
Table 6.7 Summary of cytotoxicity levels of extracts obtained from S. marcescens IBRL USM84
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
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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 S. marcescens IBRL USM84
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
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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 S. marcescens IBRL USM84
88
Figure 3.13 Presumptive test for prodigiosin from extracellular extract of S. marcescens IBRL USM84
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
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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, antibacterial activity and growth of S. marcescens 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, antibacterial activity and growth of S. marcescens 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
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antibacterial activity and growth of S. marcescens IBRL USM84
Figure 4.12 Marine broth after cultivated with S. marcescens 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, antibacterial activity and growth of S. marcescens IBRL USM84
121
Figure 4.15 Marine broth after cultivated with S. marcescens 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, antibacterial activity and growth of S. marcescens IBRL USM84
124
Figure 4.17 Effect of percentage of maltose on prodigiosin production, antibacterial activity and growth of S. marcescens 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
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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
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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 extract of S. marcescens IBRL USM84
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 S. marcescens IBRL USM84
191
Figure 6.11 Cytotoxicity result of dichloromethane partition of S. marcescens IBRL USM84 against brine shrimp
193
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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
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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
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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.
.
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PRODUCTION OF PRODIGIOSIN BY Serratia marcescens IBRL USM84 FOR
LIPSTICK FORMULATION
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.
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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.
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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.
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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).
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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
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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).
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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 Antibacterial activity
Glessler et al., (2013)
Fusarium verticillioides
Yellow Naphthoquinone C10H6O2 Antibacterial activity Boonyapranai et al., (2008);
Kurobane et al., (1986)
17 Table 2.2: continued
Fungi Colour Pigment Molecular
formula
Application References
Monascus sp. Yellow
Orange
Red
Monascin Ankaflavin Monascorubrin
Rubropuntatin Monascorubramine
Rubropuntamine
C21H26O5
C23H30O5 C23H26O5
C21H22O5
C23H27O4 C21H23O4
Food colorant Pharmaceuticals Antibacterial activity
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 Table 2.2: continued
Fungi Colour Pigment Molecular
formula
Application References
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 Table 2.2: continued
Fungi Colour Pigment Molecular
formula
Application References
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
