PRODUCTION of PRODIGIOSIN by Serratia Marcescens IBRL USM84 for LIPSTICK FORMULATION

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

TEH FARIDAH BINTI NAZARI

Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

September 2018

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

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

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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 iv

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

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

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

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

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

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 15 submerged fermentation technique

Table 2.2 Examples of fungal pigments from soil and their suggested 16 application

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 35 (EPS) produced by marine bacteria

Table 3.1 Antimicrobial activity of prodigiosin extract of 73 S. marcescens IBRL USM84 by disc diffusion assay

Table 3.2 Comparison of quantitfication of intracellular and 82 extracellular pigment from S. marcescens IBRL USM84

Table 3.3 Property of intracellular and extracellular extracts of isolate 87 S. marcescens IBRL USM84

Table 4.1 The summary of the culture condition before and after 116 enhancements

Table 4.2 The summary of the medium improvement before and after 129 enhancements

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Table 5.1 Scheme for preparing dilution series of moderate water 135 soluble extract to be used in MIC assay

Table 5.2 Total yield of extract S. marcescens IBRL USM84 from 143 solvent partitioning

Table 5.3 Absorption spectrum of extracts of S. marcescens IBRL 143 USM84 in different partitionation extracts

Table 5.4 Antibacterial activity of different partitionated extract of 144 isolate S. marcescens IBRL USM84

Table 5.5 MIC, MBC and mechanism of antibiosis of dichloromethane 149 partitionated extract of S. marcescens IBRL USM84 against test bacteria

Table 5.6 Stability of the prodigiosin pigment in dichloromethane 160 partitionated extract at different characteristics

Table 6.1 Scheme for preparing dilution series of moderate water 167 soluble extract (pink fraction) to be used in MIC assay

Table 6.2 Preparation of extract for toxicity test 170

Table 6.3 TLC analysis of the dichloromethane partition extract of 174 S. marcescens IBRL USM84 in mobile phase of ethanol: 2-propanol (8:2)

Table 6.4 The TLC analysis of fractions collected from column 179 chromatography

Table 6.5 Sensitivity test results of fractionated extract in comparison 181 with inhibition zones of dichloromethane partitionated

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extract of S. marcescens IBRL USM84

Table 6.6 The MIC and MBC values of Fraction 4 in comparison with 184 MIC and MBC values of partitionated dichloromethane extract of S. marcescens IBRL USM84

Table 6.7 Summary of cytotoxicity levels of extracts obtained from 197 S. marcescens IBRL USM84

Table 7.1 Ingredients with their prescribed quantity in the lipstick 201 formulation

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 208 prodigiosin pigment against different bacteria

Table 7.5 Evaluation of skin irritation test 212

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 S. marcescens IBRL USM84

Figure 3.2 Flowchart of pigment extraction of intracellular and 63 extracellular extracts of S. marcescens IBRL USM84

Figure 3.3 Disc diffusion assay of crude extract (intacellular) against 75 different test microorganisms

Figure 3.4 Color of extract from intracellular and extracellular 77

Figure 3.5 DPPH free radicals scavenging activity (%) of quercetin and 80 crude 2-propanol extract (intracellular extract)

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 84 form

Figure 3.9 Spectral analysis of intracellular pigment extract of 85 S. marcescens IBRL USM84 and standard prodigiosin

Figure 3.10 Spectral analysis of extracellular pigment extract of 86 S. marcescens IBRL USM84 and standard prodigiosin

Figure 3.11 Spectral analysis of intracellular pigment extract of 87 S. marcescens IBRL USM84 under alkaline and acidic condition

Figure 3.12 Presumptive test for prodigiosin from intracellular extract of 88 S. marcescens IBRL USM84

Figure 3.13 Presumptive test for prodigiosin from extracellular extract of 89 S. marcescens IBRL USM84

Figure 3.14 Colony morphology of isolate S. marcescens IBRL USM84 91 on Marine agar plates

Figure 3.15 Freeze dried biomass cells with pigment of S. marcescens 91 IBRL USM84

Figure 3.16 The observation of S. marcescens IBRL USM84 under the 93 phase contrast microscope

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, 102 antibacterial activity and growth of S. marcescens IBRL USM84

Figure 4.2 Pigment extract in 2-propanol obtained at different 104 cultivation period of S. marcescens IBRL USM84

Figure 4.3 Effect of light on prodigiosin production, antibacterial 105 activity and growth of S. marcescens IBRL USM84

Figure 4.4 Effect of initial pH of medium on prodigiosin production, 107 antibacterial activity and growth of S. marcescens IBRL USM84

Figure 4.5 Effect of temperature on prodigiosin production, 109 antibacterial activity and growth of S. marcescens IBRL USM84

Figure 4.6 Marine Broth after cultivated with S. marcescens IBRL 110 USM84 for 48 hours at 120 rpm and at different incubation temperature Figure 4.7 Effect of agitation speed on prodigiosin production, 111 antibacterial activity and growth of S. marcescens IBRL USM84

Figure 4.8 Marine broth after cultivated with S. marcescens 112 IBRL USM84 for 48 hours at 25oC

Figure 4.9 Effect of agar on prodigiosin production, antibacterial 114 activity and growth of S. marcescens IBRL USM84

Figure 4.10 Profile of growth, prodigiosin production and antibacterial 116 activity of S. marcescens IBRL USM84 before and after physical parameter enhancements

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 119 IBRL USM84 for 48 hours at 25oC and 150 rpm added with different carbon sources

Figure 4.13 Pigment extract in 2-propanol obtained after cultivation of 120 S. marcescens IBRL USM84 of various carbon source

Figure 4.14 Effect of nitrogen sources on prodigiosin production, 121 antibacterial activity and growth of S. marcescens IBRL USM84

Figure 4.15 Marine broth after cultivated with S. marcescens 122 IBRL USM84 for 48 hours at 25oC and 150 rpm added with different nitrogen sources

Figure 4.16 Effect of inorganic salt on prodigiosin production, 124 antibacterial activity and growth of S. marcescens IBRL USM84

Figure 4.17 Effect of percentage of maltose on prodigiosin production, 126 antibacterial activity and growth of S. marcescens IBRL USM84

Figure 4.18 Marine broth after cultivated with S. marcesens 127 IBRL USM84 for 48 hours at 25oC, and added different concentration of maltose

Figure 4.19 Profile of growth, prodigiosin production and antibacterial 128 activity of S. marcescens IBRL USM84 before and after chemical parameter enhancements

Figure 5.1 Flow chart of organic solvent extraction and solvent-solvent 133 partitioning

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Figure 5.2 Partitionated extract from crude extract of S. marcescens 142 IBRL USM84 extracted with different solvents

Figure 5.3 Disc diffusion assay of dichloromethane partitionated extract 145 of isolate S. marcescens IBRL USM84 against MRSA and A. anitratus

Figure 5.4 Disc diffusion assay of various partitionated extract against 146 test microorganisms

Figure 5.5 Time kill study of MRSA exposed to dichloromethane 151 partition extract of S. marcescens IBRL USM84 at different concentrations varied from 125 to 500 µg/mL

Figure 5.6 Time kill study of A. anitratus exposed to dichloromethane 153 partitionated extract of S. marcescens IBRL USM84 at different concentrations varied from 125 to 500 µg/mL

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 157 extract after treated with different pH for 30 minutes

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 173 S. marcescens IBRL USM84 developed using ethanol:

2-propanol (8:2) and graphical illustration

Figure 6.2 The light pink spot with Rf of 0.72 on the TLC plate 176 exhibited inhibitory activity on MRSA in the bioautography assay

Figure 6.3 The absorbance of different fractions collected from column 177 chromatography of dichloromethane partition extract of S. marcscens IBRL USM84

Figure 6.4 Chromatograms developed using ethanol: 2-propanol (8:2) 180

Figure 6.5 Inhibition zone of dicholomethane partitionated and Fraction 182 4 extract of isolate S. marcescens IBRL USM84 against MRSA and B. subtilis

Figure 6.6 Characteristic UV-visible of standard prodigiosin and 186 preparative TLC purified compound from S. marcescens IBRL USM84

Figure 6.7 UPLC chromatogram of dichloromethane partitionated 188 extract of S. marcescens IBRL USM84

Figure 6.8 UPLC chromatogram of TLC-preparative purified 189 compounds of S. marcescens IBRL USM84

Figure 6.9 UPLC chromatogram of standard prodigiosin 190

Figure 6.10 Figure 6.10: UPLC chromatogram of standard prodigiosin 191 and TLC-preparative purified compounds of S. marcescens IBRL USM84

Figure 6.11 Cytotoxicity result of dichloromethane partition of 193 S. marcescens IBRL USM84 against brine shrimp

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

Figure 6.12 Cytotoxicity result of dichloromethane partition of 193 S. marcescens IBRL USM84 against brine shrimp after 24 hours of exposure time (for chronic cytotoxicity test)

Figure 6.13 Cytotoxicity results of Fraction 4 of S. marcescens 194 IBRL USM84 against brine shrimp after 6 hours of exposure time (for acute cytotoxicity test)

Figure 6.14 Cytotoxicity results of Fraction 4 of S. marcescens IBRL 195 USM84 against brine shrimp after 24 hours of exposure time (for chronic cytotoxicity test)

Figure 7.1 Lipstick formulation containing castor oil, shea butter and 201 bees wax in ratio 5: 1: 1 and 2.5 g of prodigiosin pigment from S. marcescens IBRL USM84

Figure 7.2 The various color of lipstick with different quantity of 204 pigment

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, 214 formulated from natural colorant

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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

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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

xxv

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

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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.

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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

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.

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).

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.

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).

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.

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.

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

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).

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

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

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

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)

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

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 8 Red 0.13 Jens et al., funiculosum IBT3954 (2012)

Penicillium sp. 5 Violet 0.20 Ogihara et al., (2000)

Penicillium 5 Orange 0.31 Lucas et al., sclerotiorum (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).

Table 2.2: Examples of fungal pigments from soil and their suggested application Fungi Colour Pigment Molecular Application References formula

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

Aspergillus Brown - - Textile dyeing Atalla et al., (2011); niger Aishwarya, (2014) Antibacterial activity

Aspergillus Yellow Neoaspergilic acid - Antibacterial activity Micetich & Macdonald, sclerotiorum (1965); Teixeria et al., (2012)

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

Fusarium Pink/ Anthraquinone C14H8O2 Textile dyeing Glessler et al., (2013) oxysporum violet Antibacterial activity

Fusarium Yellow Naphthoquinone C10H6O2 Antibacterial activity Boonyapranai et al., (2008); verticillioides Kurobane et al., (1986)

Table 2.2: continued

Fungi Colour Pigment Molecular Application References formula

Monascus sp. Yellow Monascin C21H26O5 Food colorant Juzlova &Martinkova, (1996);

Ankaflavin C23H30O5 Pharmaceuticals Mostafa &Abbady, (2014);

Orange Monascorubrin C23H26O5 Antibacterial activity Babitha et al., (2008);

Rubropuntatin C21H22O5 Anticancer activity Moharram et al., (2012);

Red Monascorubramine C23H27O4 Antioxidant Babula et al.,(2009);

Rubropuntamine C21H23O4 Yang et al., (2014)

Penicillium Yellow Atronenetin - Food additive Takahashi & Carvalho, (2010) herquei Antioxidant

Penicillium Red Anthraquinone C14H8O2 Anticancer effectin food and Dufosse, (2006); oxalicum pharmaceuticals Atalla et al.,(2011) Textile dyeing

Table 2.2: continued

Fungi Colour Pigment Molecular Application References formula

Penicillium Orange Purpurogenone C14H12O5 Dyeing of cotton fabrics Buchi et al., (1965); purpurogenum to Yellow Martinkova et al., (1995);

Mapari et al.,(2005); Takahashi & Carvalho, (2010); Yellow Teixeria et al., (2012); to Mitorubrin C H O Food, pharmaceuticals, Orange 21 18 7 Santos-Ebinuma et al., (2013) and cosmetics Red

Mitorubrinol C21H18O8

Penicillium Yellow Pencolide C9H9NO4 Antibacterial Brikinshaw et al., (1963); sclerotiorum to activity Orange

Sclerotiorin C21H23ClO5 Antibacterial Chidananda & Sattur, (2007); activity

Table 2.2: continued

Fungi Colour Pigment Molecular Application References formula

Isochromophilone Antibacterial Lucas et al., (2007); VI activity

- Antifungal Lucas et al., (2010) activity

Trichoderma Yellow Viridin C20H16O6 Textile dyeing Chitale et al.,(2012); viride Green Neethu et al., (2012); Antifungal activity Gupta et al.,(2013) Food industry Brown - -

Trichoderma Yellow Viridol C20H18O6 Textile dyeing Mukherjee & Kenerley, virens (2010); Virone C22H24O4 Antifungal Sharma et al., (2012); activity Kamal et al., (2015)

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

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.

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

Bacteria Pigments/ Molecule Colour Applications

Agrobacterium aurantiacum, Astaxanthin Pink-red Feed supplement Paracoccus carotinifaciens, Xanthophyllomyces dendrorhous.

Rhodococcus maris Beta-carotene Bluish-red Used to treat various disorders such as erythropoietic protoporphyria, reduces the risk of breast cancer

Bradyrhizobium sp., Haloferax Canthaxanthin Dark-red Colorant in food, beverage and pharmaceutical alexandrines preparations

Corynebacterium insidiosum Indigoidine Blue Protection from oxidative stress

Rugamonas rubra, Prodigiosin Red Anticancer,immunosuppressant, antifungal, algicidal; Streptoverticillium rubrireticuli, dyeing (textile, candles, paper, ink) Vibrio gaogenes, Alteromonas rubra, Serratia marcescens, Serratia rubidaea

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

Table 2.3: continued

Bacteria Pigments/ Molecule Colour Applications

Chromobacterium violaceum, Violacein Purple Pharmaceutical (antioxidant, immunomodulatory, Janthinobacterium lividum antitumoral, antiparasitic activities); dyeing (textiles), cosmetics (lotion)

Flavobacterium sp., Paracoccus Zeaxanthin Yellow Used to treat different disorders, mainly with affecting zeaxanthinifaciens, the eyes Staphylococcus aureus

Xanthomonas oryzae Xanthomonadin Yellow Chemotaxonomic and diagnostic markers

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).

Table 2.4: Total chlorophyll and carotenoid content of six seaweeds (Chinnadurai et al., 2013). Seaweeds Total chlorophyll (a&b) Carotenoids (mgg-1) (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,

jellyfish, cnidarians and ascidians is still in early phase. Pigment colours from the sponges generated from pigment granules located in the amebocytes or from the symbiosis interaction. Meanwhile, cnidarians and ascidians may secrete their pigment through the membrane cell or localise in spicules or skeleton (Bandaranayake, 2006).

2.3.2 (c) Marine microorganism

Marine fungi are recognized sources for novel bioactive pigments from secondary metabolites (Bugni & Ireland, 2004) with high potential to produce different metabolites compared to terrestrial organisms (Spery et al., 1998).

Penicillium sp is one of the well known fungal strains has capability to produce natural pigment. According to Ogihara and Oishi, (2002), earlier studies showed the pigment produced by marine Penicillium has similarity in chemical structure with pigment from monascus namely monascorubrine and monascuscorubramine. Mohan and Vijay-

Raj, (2009) also reported radical scavenging activity and pigment production from a

Penicillium NIOM-2 isolated from marine sediment in India Ocean. Besides,

Chintapenta et al., (2014) stated, the pigment production from a mangrove Penicillium is red in color.

About three halophilic fungal strains were isolated from the saltern in eastern coast of the Adriatic Sea which is Hortaea weneckii, Phaeotheca triangularis, and

Trimmatostroma salinum. These isolates can produce melanin in high salinity environment (Tina et al., 2006). Cirrenalia pygmea Kohlmeyer is a hyphomycetous fungus which grows on stilt roots of the mangrove tree Rhizophora and has dark brown mycelium and conidia. This obligate marine fungus can produce melanin pigment in its hyphae (Ravishankaret al., 1995).

The word "yeast" originates from the Old Dutch word gist and the German word gischt, which is related to fermentation process (Kurtzman &Fell, 1989). The obligate marine yeast could be found from the marine environment, while the facultative marine yeast could be found from terrestrial habitats (Anusha et al., 2014).

Torula sp. (red yeast) and Mycoderma sp. (white yeast) are the first marine yeasts isolated by Bernhard Fischer in 1894 from the Atlantic Ocean. Following the first yeast discovery by Fisher's study, a lot of marine yeast species have been isolated from various marine sources including seawater, seaweeds, seabirds, marine fish and mammals (Kutty & Philip, 2008; Zaky et al., 2014). However, previous study described that marine yeasts have capability to produce various bioactive substances, such as amino acids, glucans, glutathione, toxins, enzymes, phytase and vitamins with high potential application in the food, pharmaceutical, cosmetic and chemical industries (Chi et al., 2009; Zaky et al., 2014).

Ushakumari & Ramanujan, (2013) reported astaxanthin present in marine yeast which was isolated from the marine sediments collected from Cochin, Kerala during the month of August 2012. Astaxanthin is a pigment from the carotenoid group, responsible for the orange-red colour of some living organisms (Higuera-Ciapara et al., 2006). It is classified as a xanthophylls and also known as “yellow leaves”. Mostly pigments from carotenoid group are colorful and oil-soluble pigment (Lorenz &

Cysewski, 2000; Moren et al., 2002). They had isolated marine yeast strain from the

Pacific Ocean and identified as Rhodotorula glutinisYS-185. It is also found to be capable of producing astaxanthin (He et al., 2011; Zaky et al., 2014). Most researchers are more attracted to marine bacteria compared to terrestrial bacteria. This is because they can potentially produce compounds with unique biological properties (Fenical,

1993). Various marine bacteria such as Streptomyces, Pseudomonas,

Pseudoalteromonas, Bacillus, Vibrio and Cytophaga isolated from seawater, sediments, algae, and marine invertebrates are proven to produce bioactive compounds. They are having capability to synthesize bioactive pigments such as indole derivatives (quinines and violacein), alkaloids (prodiginines and tambjamines), polyenes, macrolides, peptides, and terpenoids (Soliev et al., 2011). ZoBell and

Feltham, (1934) reported that about thousands of colonies growing on media agar formulated with sea water or marine mud and the results showed 69.4% were chromogenic. From this percentage 31.3% were yellow, 15.2% were orange, 9.9% were brown and 5.4% were red or pink. For further investigation by ZoBell and

Upham, (1944) reported that, a total 60 new marine species were identified which 19 yellow, 5 brown, 5 pink or salmon coloured, 4 orange and 1 red.

There are different type and potential of pigments such as the red pigment shows the presence of active compound like prodigiosin and its derivatives, whereas yellow pigment with phenazine and its derivatives. The different potential of pigmentation from marine bacteria is supported by the study of Du et al., (2006) from

China. Some bioactive pigments from marine bacteria are summarized as listed in

Table 2.5 (Soliev et al., 2011). The discovery between autumn 2004 and summer 2005 reported more than 1,500 pigmented heterotrophic bacteria strains were isolated from the coastal surface water in Mid-Norway. The isolates were identified and able to produce pigments with various color pigment like golden, yellow, red, pink and orange. The LC-MS analyses showed that most of the pigments probably from carotenoids group namely zeaxanthin, nostoxanthin and sarcinaxanthin, whereas some with novel glycosylation patterns (Stafsnes et al., 2010). Moreover in pigment production of marine bacteria is Serratia sp. BTWJ8 was recognized to synthesize a

prodigiosn-like pigment (Krishna et al., 2008) and marine Pseudomonas sp. capable of melanin production (Tarangini & Mishra, 2013).

Table 2.5: Bioactive pigments isolated from marine bacteria (Soliev et al., 2011).

Pigment Activity Bacterial strains References

Undecylprodigiosin Anticancer Streptomyces ruber Gerber, (1975)

Heptyl prodigiosin Antiplasmodial α-Proteobacteria Lazaro et al., (2002)

Prodigiosin Antibacterial; Anticancer; Algicidal Pseudoalteromonas rubra Gerber & Gauthier, (1979) Hahella chejuensis Kim et al., (2007)

Astaxanthin (carotene) Antioxidation Agrobacterium aurantiacum Misawa et al., 1995

Violacein Antibiotic; Antiprotozoan; Pseudoalteromonas tunicata Matz et al.,(2004) Anticancer Pseudoalteromonas sp. 520P1 Yada et al., (2008)

Phenazine derivatives Cytotoxic Bacillus sp. Li et al., (2007)

Pyocyanin and pyorubrin Antibacterial Pseudomonas aeruginosa Saha et al., (2008)

Melanins Protection from UV irradiation Alteromonas nigrifaciens Ivanova et al., (1996)

Table 2.5: continued

Pigment Activity Bacterial strains References

Scytonemin Protection from UV irradiation, Anti-inflammatory, Cyanobacteria Stevenson et al., (2002) Antiproliferative

Tryptanthrin Antibiotic Cytophaga/ Flexibacteria Wagner-Dobler et al., AM13,1strain (2002)

Phenazine-1-carboxylic acid Antibiotic Pseudomonas aeruginosa Nansathit et al., (2009)

5,10-dihydrophencomycin Antibiotic Streptomyces sp. Pusecker et al., (1997) methyl ester

Fridamycin D, Himalomycin A, Antibacterial Streptomyces sp. B6921 Maskey et al., (2003) Himalomycin B

Chinikomycin A and Anticancer Streptomyces sp. M045 Li et al., (2005) Chinikomycin B, Manumycin A

Tambjamines (BE-18591, Antibiotic, Anticancer Pseudoalteromonas tunicate Franks et al., (2005); pyrrole and their synthetic analogs) Pinkerton et al., (2010)

2.4 Bioactive compounds from marine microorganism

2.4.1 Factors influencing the bioactive compounds production

2.4.1 (a) Marine environmental condition

Almost all bioactive compounds yielded by marine and terrestrial microorganism are different due to respective environmental circumstance influence.

Most bacteria isolated from marine sources are Gram-negative rods. Gram-negative marine bacteria have capability to survive and grow in the marine environment with insufficient nutrient, high salinity and high stress. That is why their outer membrane structure is evolutionarily adapted to aquatic environmental factors. As such, marine habitat can be divided by living at low pressure (psychrophiles), high salinity

(halophiles) and under high pressure (barophile) (Soliev et al., 2011; Pimpliskar &

Jadhav, 2014). The extreme conditions like low temperature characterized by below

4oC, high pressures considered as higher than 100 × 105 Pa (Delong & Yayanos,

1987), while extreme halophiles has been achieved at 10% (w/v) NaCl (Bowers et al.,

2009). The environmental conditions that are extreme causing marine microorganism produce various bioactive compounds that make them sustain live in the marine environment.

However, adaptations to the sea conditions will influence the production of secondary metabolite. Jensen & Fenical, (1996) found four actinomycetes strains that produced novel metabolites which were isolated from marine sources and then were transferred to laboratory for further research. In this study, the seawater was replaced by synthetic medium which is complex fermentation medium in deionized water. The result shows only one of the four was able to produce the metabolite without seawater addition in the medium (Jensen & Fenical, 1996). Another example is the production of the metabolite by a marine actinomycete, Chainia purpurogena was dependent on

the addition of an extract of the seaweed Laminaria. This proves that the production of secondary metabolites by marine bacteria could depend on the existence of nutrients

(Okazaki et al., 1975). Hence, the understanding about the mechanisms used by bacteria in various marine microhabitats is very important. It makes it easier to decide the proper selection pressure to maintain a high production level of the interesting compounds for industrial purpose (de Carvalho & Fernandes, 2010).

2.4.1 (b) Adaptation of marine microorganism

Marine environments diversity has been forcing bacteria to new strategies adaptation and driving to synthesize the new metabolites known as secondary metabolites (Jensen & Fenical, 1996; Valentine, 2007). One of the adaptations at the cellular membrane is production of specialized lipids. The maintenance of the integrity of the cellular membrane is vital to bacterial cells because it can affect the maintenance of the energy status of the cells which is related to signal transduction and other energy-transduction processes and for keeping turgor pressure (Sikkema et al., 1995). Besides, the cells have to change the degree of saturation of the fatty acids of the membrane phospholipids for keeping the membrane fluidity via a mechanism named “homeoviscous adaptation” (Sinensky, 1974). Moreover, the strains were grown under high pressure conditions mostly have membranes with a high degree of saturation, commonly will be better adapted to compete for available nutrients under high salinity conditions (de Carvalho & Fernandes, 2010).

The production of exopolymers also includes as one of the bacteria adaptations. Bacterial exopolysaccharides also known as EPS (exopolymeric substances) have several functions including protection from dehydration, cryoprotection, stabilization of enzymes through buffering pH and salinity

fluctuations, surface adherent, nutrient storage and elimination of toxic compounds

(Flemming & Wingender, 2001). Novel EPS have been isolated from marine bacteria which are collected from the deep-sea area near hydrothermal vent. This area considered as high pressure and temperature with high levels of sulphur and heavy metals. Debnath et al., (2007) described that, EPS has ability in tissue regeneration that useful for the treatment of cardiovascular and oncological diseases. Some of the

EPS properties that have been exploited are listed in Table 2.6 (de Carvalho &

Fernandes, 2010).

In addition, the permeability of bacteria cell membrane can be adapted to extreme environmental conditions by the production of hopanoids. Hopanoids is pentacyclic triterpenoid compounds that play a role similar to sterols in eukaryotes

(Kannenberg & Poralla, 1999; Schouten et al., 2000). This compound has been suggested to maintain the membrane fluidity and to reduce the diffusion of ions through the cell membrane and also increases in response to environmental stress, such as to temperature increases and to the presence of alcohols (Joyeux et al., 2004;

Schmidt, 2005). One of the unique characteristic attributes of marine bacteria is that a large part of them are pigmented (Zobell, 1946). The special characteristic feature is some pigments produced by microorganisms have been strong enough to inhibit the growth of other bacteria was reported by earlier study (Balraj et al., 2014).

Furthermore, several bacteria are capable in molecules production that prevents the attachment, growth or survival of challenging organisms in competitive environments.

The broad application of bioactive compounds produced by marine bacteria as an adaptive response to demanding conditions makes them suitable to be commercialised

(de Carvalho & Fernandes, 2010).

Table 2.6: Functions and applications of exopolymeric substances (EPS) produced by marine bacteria (de Carvalho & Fernandes, 2010).

Examples of functions of EPS in bacterial cells

Type of EPS Function Bacterium Reference

Glycolipid Biosurfactant Halomonadaceae Yakimov et sp. strain MM1 al.,(1996) Polysaccharide Benefit during Pseudoalteromonas Debnath et al., competition for tunicata (2007) space and nutrients on surfaces Polysaccharide Allow survival in Bacillus sp. Pfiffner et oil wells al.,(1986) Polysaccharide and Helps microbial Nocardia amarae Decho & Herndl, proteins interactions (1995)

Examples of applications of EPS from marine bacteria

Bacterium Applications Reference

Alteromonas infernus Bone-healing material Colliec-Jouault et al., (2004) Bacillus circulans Biosurfactant; antimicrobial Das et al., (2008) action Vibrio and Alteromonas Tissue regeneration; Debnath et al., (2007) antithrombotic effects P. tunicata Antifouling activity Egan et al.,(2002) Flavobacterium uliginosa Antitumor activity Sangnoi et al., (2009) Bacillus sp. Pseudoplastic behavior Pfiffner et al.,(1986)

2.4.1 (c) Predator

Predator is one of the core factors to induce the production of secondary metabolites by marine bacteria. Bacteria also become one of the food sources to other marine life. This natural phenomenon has forced the bacterial communities to develop inedible morphologies such as cell filaments and aggregates which are leading to changes in the structural and taxonomic composition (Hahn & Hofle, 2001; Ronn et al., 2002).

Undeniably, the long exposure of bacteria community to the predator can enhance the capabilities of bacteria to produce the secondary metabolites. The production and accumulation of bioactive compounds in bacteria may play a vital role in protection against protozoan predators (Matz et al., 2004). Most of the bioactive compounds have biological properties like antibiotic and cytotoxic activity (Fenical,

1993). Earlier report by Hay & Fenical, (1988) stated in plant-herbivore interactions, the production of bioactive compounds is generally considered as a chemical defence strategy against predator grazing. Besides, one of the effective defence mechanisms of sea hare is producing a purple ink and opaline when attacked by predators (Derby,

2007; Love-Chezem et al., 2013; Berriman et al., 2015).

Apart from that, several previous studies described about the correlation between the production of bioactive compounds and the presence of pigments in bacteria isolates (Holmstrom et al., 1996; Egan et al., 2002). According to Matz et al.,

(2004), they found a total of pigmented bacteria have increased when they enhanced the protozoan grazing pressure. This discovery suggested that pigment production by heterotrophic bacteria act as a protective mechanism against protozoan grazing (Singh,

1942) and against solar radiation (Margalith, 1992). Additionally, Singh, (1945) also

reported that bacterial colonies with red, green, or violet pigmentation were inedible by soil amoebae or even prevented amoebae growth.

2.4.1 (d) Association

Competitive advantages among marine organisms are encouraging the secretion of secondary metabolites in competitive surroundings. Undeniably, the interaction among microorganisms with marine plants or marine animals indicates that most bioactive compounds are synthesized by associated microorganisms (Osinga et al., 2001). Besides, the microbe community have to compete for limited space and nutrient. This pressure becomes a predisposing factor for microbes to yield several bioactive compounds to be applied in industrial application (Armstrong et al., 2001).

Previous studies also found the secondary metabolites produced by platonic bacteria which are growing on the surface of sponges with space and nutrients are limited

(Burgess et al., 1999; Slattery et al., 2001; Lu et al., 2009). Moreover, the biological active compounds identified in sponges have high similarities to compounds isolated from their associated microorganisms (Thiel &Imhoff, 2003; Radjasa et al., 2007).

According to Bruhn et al., (2007), Roseobacter clade is the most dominant in association among bacterial communities with marine algae. The Roseobacter clade also known as aggressive colonizers are able to colonize rapidly and form biofilm that authorized these bacteria to colonize alga. They also reported about nine of 14 members of the Roseobacter clade. Half of the isolates were isolated from cultures of the dinoflagellate Pfiesteria piscicida, which have capability in production of antibacterial compounds (Bruhn et al., 2005). However, the production of secondary metabolites from this strain is influenced by the growth conditions (Bruhn et al.,

2006). The optimum conditions for growth is when bacteria were cultured in liquid

medium under static conditions without agitation. Under static conditions, the bacterial cells easier attached to one another, support the rosettes and biofilm formation and enhance the production of antibacterial compound (Bruhn et al., 2007).

2.5 Pigments from marine bacteria

2.5.1 Carotenes

Carotenes are pigments comprised of 40 carbon atoms per molecule and naturally found in plants. Carotenoids are considered to be the major group of pigment. In prokaryote, they can be appearing either yellow, orange or red due to a wide diversity of isoprenoid compounds (phytoene, phytofluene, lycopene, and β- carotene). The pigment distribution is widespread among bacteria (prokaryotes) to higher plants (eukaryotes) (Kim et al., 2007; Soliev et al., 2011).

Carotenoids play a vital role as protective agent of photoxidative damage by quenching singlet oxygen and harmful radicals that are produced when cells are illuminated (Demmig-Adams & Adams, 2002). Besides, β-carotene is also needed in the synthesis of vitamin A (nutrition) and retinal (vision) for mammals (Giovannucci,

2002; Mares-Perlman et al., 2002; Duc et al., 2006). Commercially, carotenoids play an important role as colouring agent with antioxidant properties which are widely used as food colorants, animal feed supplements and nutraceuticals for cosmetic and pharmaceutical need. In early stage, the main sources of carotenoid production are the extraction from plant tissues or chemical synthesis. But, in meeting the worldwide market demanding, microbial production has large potential based on the efficiency of production and the variety of carotenoid structures (de Haan et al., 1991; Buzzini &

Martin, 1999; Frengova et al., 2003; Stafnes et al., 2010). For example, the carotenoids production of microbial sources include the unicellular algae Dunaliella

salina, Spirulina (Borowitzka, 1999), Haematococcus (Lorenz & Cysewski, 2000;

Guerin et al., 2003) as well as the filamentous fungus Blakeslea trispora (Quiles-

Rosillo et al., 2005) are used commercially.

Astaxanthin is one of the natural pigments from carotenoid group (Figure

2.2). This pigment have commercial value and broadly used as a food supplement for humans and as food additives in aquaculture industry. Misawa et al., (1995) reported that the marine bacterium Agrobacterium aurantiacum can produce astaxanthin. Lee et al., (2004) also reported another marine bacterium have capability in astaxanthin production was isolated and identified as Paracoccus haeundaensis.

Figure 2.2: Astaxanthin (Soliev et al., 2012)

2.5.2 Prodiginines

Prodigiosin is the first pigment identified by Gerber, (1969) and recognized as one of the prodiginine derivatives. This pigment produced by Bacillus prodigiosus bacterium and today known as Serratia marcescens (Gerber, 1975). Prodiginines have a diversity of biological properties such as antibacterial, antifungal, antimalarial, antibiotic, immunosuppressive, and anticancer activities (Montaner & Perez-Tomas,

2003; Williamson et al., 2007). Figure 2.3 shows the chemical structure of prodiginines derivatives, Prodigiosin (2-methyl-3-pentyl-prodiginine) (Figure 2.3 A),

Heptyl prodigiosin (2-methyl-3-heptyl-prodiginine) (Figure 2.3 B),

Undecylprodigiosin (Figure 2.3 C) and Cycloprodigiosin (Figure 2.3 D).

A B

C D

Figure 2.3: Prodiginines derivatives, (A) Prodigiosin (2-methyl-3-pentyl- prodiginine), (B) Heptyl prodigiosin (2-methyl-3-heptyl-prodiginine), (C) Undecylprodigiosin and (D) Cycloprodigiosin

Apart from Serratia, several species of marine bacteria from some genera including Streptomyces (Gerber, 1975), Pseudomonas (Gandhi et al., 1976),

Actinomadura (Gerber, 1975) and Pseudoalteromonas (Sawabe et al., 1998) have been identified and reported to produce prodigiosin and related compounds. For example Pseudoalteromonas denitrificans (Gauthier et al., 1995) and

Pseudoalteromonas rubra can produce cycloprodigiosin (Gauthier, 1976) while α-

Proteobacteria was reported to produce heptyl prodigiosin (Lazaro et al., 2002).

Earlier report also stated the cycloprodigiosin has medical properties like

immunosuppressive, antimalarial, and apoptosis-inducing activities (Kawauchi et al.,

1997; Yamamoto et al., 1999; Kim et al., 1999). Other bacteria reported to produce red pigments isolated from the coasts of Korea, Taiwan, and Japan include Hahella

(Lee et al., 2001), Vibrio (Shieh et al., 2003), and Zooshikella (Yi et al., 2003).

The research team from Japan have investigated the Pacific Ocean in the

South and the Sea of Japan in the North and West to isolate the marine microorganisms and their respective metabolites. They found the prodigiosin- producing bacterium Pseudoalteromonas rubra is Gram-negative with rod-shaped morphology. According to physicochemical analysis, the pigment synthesized by this strain consists of seven structurally similar prodiginine compounds. Only four chemical structures of these were successfully determined. These bioactive compounds were further identified as prodigiosin and its analogues 2-methyl-3-butyl- prodiginine, 2-methyl-3-pentyl-prodiginine (prodigiosin), 2-methyl-3-hexyl- prodiginine, and 2-methyl-3-heptyl-prodiginine (Yada et al., 2003; Soliev et al.,

2011). The bioactive compounds from prodiginine derivatives have already entered clinical tests as potential drugs against different stages of several tumours (Williamson et al., 2007).

2.5.3 Melanins

Based on chemical structure and pigment color, melanins comprise of three classes namely eumelanins, pheomelanins and allomelanins. Eumelanins can produce black to brown pigment, pheomelanins are brown, red or yellow in colour whereas allomelanins is one of the nitrogen free heterogeneous groups of polymers which is synthesized by catechol precursors (Harki et al., 1997; Gomez-Marin & Sanchez,

2010; Tarangini & Mishra, 2013). Usually, the eumelanins and pheomelanins are

present in animal species, otherwise allomelanins occur in microorganisms and plants

(Coyne & Al-Harthi, 1992).

Naturally, melanins play a vital role in against UV and visible light

(photoprotectants), charge transport mediators, scavenge the free-radical, antioxidants, balance the metal ion and so on (Geng et al., 2010). These bioactive pigments also have broad applications in agriculture, medicine, cosmetic and pharmaceutical industries. Furthermore, melanins are hydrophobic, negatively charged, high molecular weight compounds and also insoluble in common organic solvents, aqueous acids and water (Sajjan, 2010; Gomez-Marin & Sanchez, 2010).

Several of marine bacterial strains described to produce melanin or melanin- like pigments include Vibrio cholerae, Shewanella colwelliana, and Alteromonas nigrifaciens (Kotob et al., 1995; Ivanova et al., 1996). Other bacteria reported to produce melanin are Aeromonas salmonicida, Azotobacter, Mycobacterium,

Micrococcus, Bacillus, Legionella, Streptomyces, Rhizobium, Proteus, Azospirillum,

Pseudomonas aeruginosa, Hypomonas sp, Burkholderia cepacia, E. coli, Bordetella pertusis, Campylobacter jejuni, Yersinia pestis and etc (Coyne & Al-Harthi, 1992;

Geng et al., 2010).

2.5.4 Violacein

The blue-black pigment violacein (3-[1,2-dihydro-5-(5-hydroxy-1H-indol-3- yl)-2-oxo-3H-pyrrol-3-ylidene]-1,3-dihydro-2H-indol-2-one) is an indole derivative pigment described already since 1882 (DeMoss & Evans, 1960; Hakvag et al., 2009).

Mostly, this bioactive pigment isolated from bacteria of the genus Chromobacterium from soil and water of tropical and subtropical areas (Rettori & Duran, 1998). For example, Chromobacterium marinum was isolated from sea water and this strain

produced a blue pigment was then identified as violacein via physicochemical characteristics (Hamilton & Austin, 1967). Besides, Pseudoalteromonas luteoviolacea was isolated from the surface of the marine sponge (Acanthella cavernosa), has been reported to produce violacein, which is the major pigment found in the genera

Chromobacterium and Janthinobacterium (Dessaux et al., 2004; Matz et al., 2004).

Violacein shows a variety of biological properties such as antibacterial, antiviral, anticancer, antioxidant, antiulcerogenic, and antileishmanial activities (Duran

& Menck, 2001; Konzen et al., 2006; Duran et al., 2007). Additionally, this pigment showed a strong anti-bacterial activity includes inhibition of Staphylococcus aureus,

Neisseria meningitidis, Streptococcus spp., Bacillus spp., Mycobacterium and

Pseudomonas (Hakvag et al., 2009). Earlier report described the production of violacein by Chromobacterium violaceum was influenced by physical parameters such as incubation temperature, agitation speed and pH (Riveros et al., 1989). Whereas, the formation of violacein carbon skeleton involves the two molecules of L-tryptophan which is lack of molecular oxygen will affect pigment production (DeMoss & Evans,

1960; Momen & Hoshino, 2000; Hakvag et al., 2009). Yada et al., (2003) stated two groups of novel violacein and deoxyviolacein produced by marine bacteria were isolated from the Pacific Ocean and characterized in detail (Yada et al., 2008). The chemical structure of the violacein and deoxyviolacein were showed in Figure 2.4.

2.6 Market potential of natural pigment

2.6.1 Food industry

Nowadays, food industry prefers to more use of natural color rather than synthetic color. Several fermentation-derived pigments were used in the food industry namely β-Carotene produced by fungus Blakeslea trispora in Europe while pigments produced by Monascus in Asia (Lampila et al., 1985; Wissgott & Bortlik, 1996;

Downham & Collins, 2000). Besides possessing beautiful colors, natural pigments also have medical benefits like antibiotic, antioxidant and anticancer properties

(Dufosse, 2009) make people crazy toward the use of natural products compare to synthetic color with hazardous effects (Lampila et al., 1985).

Figure 2.4: Violacein and deoxyviolacein

Β-Carotene also known as pro-vitamin A is a yellowish carotenoid pigments.

It has antioxidant activities and has potential against certain diseases (Kumar et al.,

2015). Several microbes have been reported to produce β-Carotene including

Blakeslea trispora, Mucor circinelloides and Phycomyces blakesleeanus (Navarro et al., 2001; Dufosse, 2006). Riboflavin is yellow food coloring widely used in cereal- based products. However, slightly bitter tasting and a little odour has limited usage in

food production. Fermentation of Ashbya gossypi is preferred in the riboflavin production because of higher yield and greater genetic stability has been reported

(Stahmann et al., 2000; Kumar et al., 2015).

The natural pigments from Monascus sp. already known in industrial purpose and consist of three types of pigment colour namely red, orange, and yellowish colorants (Kumar et al., 2015). One of the common name of this fungal product are

Red Yeast Rice (RYR) containing monacolins that has been used to control cholesterol level which reduces the LDL-cholesterol and increase the HDL-cholesterol

(Vidyalakshmi et al., 2009). Futhermore, Monascus can produce angkak which convert the starchy substrates into several metabolites such as alcohols, antibiotic agents, antihypertensive, enzymes, fatty acids, flavour compounds, flocculants, ketones, organic acids, pigments and vitamins. Monascus pigment has been used as a coloring agent in food give a special flavour added value in food products (Kumar et al., 2015).

2.6.2 Pharmaceutical industry

Colorants play a vital role in pharmaceutical field. Pharmacist also regarded colorants as a cosmetic in the pharmaceutical preparation. Among the colorants factors involved in the pharmaceutical preparation are for acceptability increase of drugs, identification, standard preparations, and stability purpose. Medicine products with attractive colors assist in improving acceptability especially for children which always avoid injections, make it easier to treat them at home with syrups, tablets, or capsules

(Allam & Kumar, 2011).

Many research projects are going on for treating various diseases such as cancer, leukaemia, and diabetes mellitus by using bioactive pigments from pigmented microbes. These pigments already have their own natural biological properties like antibiotics, anticancer, antiploriferative and immunosuppressive activities (Kumar et al., 2015). Anthocyanins is one of the bioactive pigments that have pharmaceutical values including decrease the risk of cancer (Lazze et al., 2004), reduce inflammatory

(Yuodim et al., 2002) and modulate immune response (Wang & Mazza, 2002).

Natural red pigments produced by genus Streptomyces or Serratia have been found to have antibiotic and antimalarial activities (Lazaro et al., 2002). In 2007,

Pandey and his team also reported the immunosuppressant with anticancer activity of prodigiosin. Furthermore, the active compounds in prodigiosin have ability to prevent and treat diabetes mellitus (Hwanmook et al., 2006).

Violacein is a violet pigment produced by the bacteria Chromobacterium violaceum and some other species was reported to have antitumour, antiparasit, antiprotozoan (Matz et al., 2004), anticancer (Ferreira et al., 2004; Kodach et al.,

2006), antibacterial (Nakamura et al., 2003) and antioxidant activities (Konzen et al.,

2006). Previous report described the antimycobacterial activity of two pigments namely violacein (a purple violet pigment) and flexirubin (a yellow-orange pigment) isolated from Janthinobacterium sp. Ant5-2 (J-PVP) and Flavobacterium sp. Ant342

(F-YOP) respectively. These bioactive pigments might be valuable compounds for chemotherapy of tuberculosis (Richard, 2003).

2.6.3 Textile industry

Undeniably, approximately 1.3 million tons of dyes, pigments and dye precursors have been manufactured synthetically with high cost reaching U$23 billion 46

in textile industry. However, the use of chemicals and synthetic colorants in fabric materials can cause skin allergy, generate hazardous wastes during its synthesis, creating worker safety concerns and not environment friendly (Venil et al., 2013;

Kumar et al., 2015). The limitations of synthetic colorants usage make industries are shifting towards the use of natural pigment which more economic and environment friendly to replace the synthetic dyes. Some microorganisms have high potential to produce natural pigments in high yields including species of Monascus (Hajjaj et al.,

2000), Serratia (Williams et al., 1971) and Streptomyces (Oshima et al., 1981; Ryu et al., 1989) have been reported.

According to Kang et al., (1996), the use of natural pigments in textile colouration shows anti-UV and anti-microbial properties. Alihosseini et al., (2008) described the bright red pigment prodigiosin from Vibrio spp. can be used to dye many types of fabrics such as wool, nylon, acrylics and silk. Besides, Ahmad et al., (2012) also reported the red pigment prodigiosin and violet pigment violacein produced by

Serratia marcescens and Chromobacterium violaceum, respectively, have different dyeing efficiency, depending on the types of fabric. The results obtained suggested that prodigiosin has good performances in dyeing acrylic while violacein was observed could be used to dye pure rayon, jacquard rayon and silk satin.

2.6.4 Aquaculture industry

Microbial colours have been used in the fish industry, for example to enhance the pink colour of farmed salmon (Venil & Lakshmanaperumaisamy, 2009). Studies have shown that color is very important for making purchasing decisions about salmon. Almost all consumers perceive that redder salmon is equated to these

characteristics including fresher, better flavor, higher quality and higher price

(Anderson, 2000).

The presence of chromatophores like melanophores, xanthophores, erythrophores, iridophores, leucophores and cyanophores are giving the colour effect on the fish skin. These chromatophores containing pigments such as melanins, carotenoids, pteridines and purines that give color to the skin and tissues of animals and plants (Chatzifotis et al., 2005). Carotenoids give the skin the yellow and red colours and also give the orange and green colours to the egg, skin and flesh of many fish (Fuji, 1969). These natural pigments are synthesized from geranyl diphosphate by all photosynthetic organisms (Giuliano et al., 2000).

As already known, salmonids cannot endogenously synthesize astaxanthin.

Therefore, it must be supplemented in the fish feed formulation. Besides having good effect in coloration, astaxanthin also has biological functions related to growth, reproduction and tissue health in salmonids and shrimp (Bell et al., 2000).

2.7 Prodigiosin and its application

2.7.1 Prodigiosin sources

The natural red pigment prodiginines are bioactive secondary metabolites produced by both Gram-negative and Gram-positive bacteria (Darshan & Manonmani,

2015). Prodigiosin is one of the famous members from this group pigment. Prodigiosin was first isolated and characterized from S. marcescens (Gerber, 1975) and this strain known as the main producer of prodigiosin pigment. A total of ten species of Serratia have been identified, but only three species are capable of producing prodigiosin

which is S. plymuthica, S. rubidaea and some biogroups of S. marcescens (Grimont et al., 1977; Wai & Chen, 2005). This antibiotic red pigment also synthesized by some other microbial strains namely Vibrio psychroerythrus, Streptomyces griseoviridis,

Hahella chejuensis (Hubbard and Rimington, 1950). Gram positive actinomycetes including Streptoverticillium rubrireticuli and Streptomyces longisporus ruber are able to produce prodigiosin or its derivatives (Khanafari et al., 2006). Harris et al., (2004) also reported the actinomycete Streptomyces coelicolor A3(2) has been shown to synthesize a closely prodigiosin related lineartripyrrole, undecylprodigiosin, and a cyclic derivative, butylmeta-cycloheptylprodiginine in a 2 : 1 ratio.

2.7.1 (a) Prodigiosin class and structure

Prodiginines members are identified and characterized by a common pyrrolyl dipyrromethene skeleton that consists of a common 4-methoxy, 2–2 bi pyrrole ring system. This group pigment has been divided into two classes which is linear and cyclic derivatives. Prodigiosin and undecylprodigiosin are in class of linear derivatives while streptorubin B, cycloprodigiosin, and cyclononylprodigiosin are in class of cyclic derivatives (Williamson et al., 2006; Mo et al., 2008).

The chemical structure of archetypal prodiginine (prodigiosin) was described since 1960s by partial and total chemical synthesis revealing a pyrrolyl dipyrromethene core skeleton (Wasserman et al., 1960; Rapoport & Holden, 1962).

Prodigiosin exists in solution as a mixture that consist of two interconverting rotamers namely cis (Beta, β) and trans (Alpha, α). The balance between these forms was influenced by pH of the solution (Furstner et al., 2001). Figure 2.5 shows the chemical structure of archetypal prodiginine, prodigiosin.

Figure 2.5: The structure of the archetypal prodiginine, prodigiosin (Furstner et al., 2001).

2.7.1 (b) Spectral analysis of prodigiosin pigment

According to Hubbard & Rimington, (1950), prodigiosin have different spectral curves at acid, alkaline and neutral pH. The existence of prodigiosin has been shown to be influenced by the presence of hydrogen ion in the solution. In acidic condition, the red pigment indicates a sharp spectral peak at 535 mμ. Meanwhile, in alkaline condition, the pigment turned to orange-yellow color and shows a broader spectral curve centred at 470 mμ (Williams et al., 1956). Prodigiosin pigment exhibits maximum UV/Vis spectrum in range 530 to 540 nm of wavelength. This pigment has

an ability to absorb the green color and perceive the orange to red color (Feng et al.,

1982; Alihosseini et al., 2008; Yang et al., 2013).

In general, prodigiosin has molecular weight of 300.00 to 330.00 Dalton.

Various types of prodigiosin and its source can leverage on prodigiosin molecular weight. For example, Hubbard and Ramington since 1950 have reported the molecular weight of prodigiosin obtained from Bacillus prodigiosus (Serratia marcescens) is

323.4 Dalton. Followed by Casullo de Araujo et al., (2010) found prodigiosin was isolated from S. marcescens UCP 1549 has molecular weight of 324.00 Dalton.

Furthermore, two types of prodigiosin have been isolated from Zooshikella rubidus

S1-1 namely prodigiosin and cyclo prodigiosin with 324.00 Dalton and 322.00 Dalton of molecular weight respectively (Lee et al., 2011). Naik et al., (2012) also reported that prodigiosin obtained from Serratia marcescens CF-53 achieved the molecular weight at 325.20 Dalton. However, the molecular weight of prodigiosin produced by

Serratia sp. (Nadaf et al., 2016) and Serratia marcescens UCP/WFCC1549 (Lins et al., 2014) indicated the same molecular weight is 323.00 Dalton.

2.7.1 (c) Various properties and applications of prodigiosin

Prodigiosin has diverse properties and broad area of application, mainly in pharmaceutical and textile industries (Kumar et al., 2015). Several characteristics that make prodigiosin pigment appropriate for medical applications such as antimicrobial, antitumor and antibiotic (Vijayalakshmi & Jagathy, 2016). Prodigiosin and its derivatives are induces of apoptasis in various cancer cell lines without toxicity effects towards normal cell lines (Darshan & Manonmani, 2015). Besides, prodigiosin has been reported to have antimalarial (Kim et al., 1999), algicidal (Park et al., 2012),

insecticidal (Wang et al., 2012) and immunosupressor (Montaner et al., 2000) properties.

Apart from medical utility, prodigiosin with strong dyeing activity is very useful in many industries involved in colour applications. Ahmad et al., (2012) described the potential of prodigiosin as a colouring agent in candles, paper, soap and pencil case pouch and also observed the potential of prodigiosin as ink in ball point pen and high lighter pen. Furthermore, Gulani et al., (2012) stated that prodigiosin being resistant to acid, alkali and detergents may find broad applications in textile industry. Ryazantseva & Andreyeva, (2014) also reported the use of prodigiosin as a colorant for polyolefines is more economical and environmental friendly.

2.7.2 Application of natural pigment in cosmetic industry

2.7.2 (a) Cosmetic industry

Recently, the natural cosmetic has expanded to be a great trend compared to chemical-based product and aiming for future developing tendency of cosmetic industry should be more safe and environmentally-friendly. Old Chinese said that everyone loves beauty and people have more choices in choosing cosmetic whether from natural or synthetic resources (Chen, 2009).

According to the drugs and cosmetic Act 1940 defines a cosmetic as any article intended to be rubbed, poured, sprinkled or sprayed on or introduced into or applied on human body skin or any part for several purposes such as for cleansing, beautifying, promoting attractiveness or altering the appearance without affecting the skin (Elsner & Maibach, 2005). However, many customers assumed that natural is good and synthetic is bad. That is why more environmentally-friendly companies are

emerging to do everything possible to fulfil the demand of customers (Johri &

Sahasakmontri, 1998; Elsner & Maibach, 2005).

The application of natural cosmetics in the beauty industry may still at early stage. Because of the great customer demand, they already offer a variety of beauty products and suitable for all skin types such as foundation, eye shadow, and lipstick which are appropriate irrespective of the skin tone. People with skin problem like oily or sensitive skin can also use the natural cosmetics and never have to worry about degrading their skin condition (Winter, 2009). The benefits of natural cosmetics are safer to use, cheaper cost, no side effects, environmental friendly and so on (Joshpi &

Pawar, 2015). Therefore, further research in natural cosmetics is needed which can make human closer to nature indirectly. Women have better choices in beauty products while saving the environment from the synthetic threat.

2.7.2 (b) Natural cosmetic

Natural pigments have been used for cosmetic since ancient times. The annatto (Bixa orellana) plant produced a beautiful orange red carotenoid pigment is commonly known as the lipstick plant. The ancient women used the pigment as a lipstick on their lips (Tantituvanont et al., 2008). The ancient Mesopotamian women like to decorate their lips and eyes area by using crushed semi-precious jewels.

Besides, the ancient Egyptian women applied a purplish-red dye that obtained from seaweed to colour their lips. Furthermore, Queen Elizabeth I also introduced and popularized the black colour lips that become fad once upon a time (Garner, 2008).

Azwanida et al., (2015) had evaluated the colour stability of Hylocereus polyrhizus, Clitorea ternatae and Pandanus amaryllfolius as cosmetic colorants. From

the survey results obtained, consumer preferred the natural colorant from H. polyrhizus extract compared to synthetic colorant. Consumer refused to use the synthetic colorant on their lips because of the occurrences of chapped and dry lips appeared on lipstick consumer lips nowadays. According to colour stability and consumer acceptance, H. Polyrhizus was the best pigment to be formulated in a new lipstick formulation.

2.7.2 (c) Potency of microbial pigment in natural cosmetics

Microbial pigments in natural cosmetics considered as a new research and very limited information are available about this finding. Previous studies focus more on plants-based as main resources for natural cosmetics. Undeniable, in natural cosmetics, plants can be better resources compared to synthetic resources. However, microbial pigments have a great future ahead as compared to the plant resources. The natural pigments from plants have some unfavorable conditions such as very sensitive to light, heat or adverse pH, low water solubility and are often not accessible throughout the year. Meanwhile, microbial pigments promising the great stability ease and fast growth in the cheap culture medium, free from seasonal influences and capable in producing the different shades of color (Parekh et al., 2000).

Suryawanshi et al., (2015) had reported that prodigiosin and violacein have great potential as UV-protectants. These pigments were added to commercial sunscreens as additives which are intended to prevent UV induced damage to human skin. As a result, both pigments are able to increase the SPF of commercial sunscreens that have high value as ingredients for a new range of sunscreens utilizing substances of biological resources. Several features of sunscreens include chemically inert, non-

irritating, non-toxic, non-allergic, non-carcinogenic and photostable (Schalka & Reis,

2011; Latha et al., 2013).

In addition, prodigiosin and violacein also have antimicrobial activity against pathogenic bacteria found on human skin. Prodigiosin is known to posses bactericidal activity towards Gram-positive and the Gram-negative bacterial pathogens. This substance may cause a breakdown of the intracellular pH gradient and inhibit proton pumps as mode of action against test bacteria (Sato et al., 1998; Matsuya et al., 2000).

Thus, the human skin could be protected naturally from pathogenic bacteria when applying the sunscreen formulations that contained photoprotectant agents on the skin.

Torulene and torularhodin are other examples of microbial pigments produced by several red yeasts, such as Rhodotorula glutinis and Sporobolomyces ruberrimus (Tinoi et al., 2005). Torularhodin exhibits a considerable antioxidant activity (Breierova et al., 2008) that increases the stabilization of membranes under stress conditions (Gorbushina et al., 2008). These carotenoids member are valuable because they are precursors of vitamin A and hormones and they have antiaging and antioxidant properties (Buzzini et al., 2005; Breierova et al., 2008). The potential of prodigiosin as antioxidant agent has been reported in many research studies (Gulani et al., 2012; Suryawanshi et al., 2015; Vijayalakshmi & Jagathy, 2016).

Likewise, the variety of colors synthesized by several microbial species caused the microbial colors having good opportunity in natural cosmetics. For example the predominant color in prodigiosin is red, carotenoid (yellow, orange and red), violacein (purple), phenazines (yellow to purple), chlorophylls (green), allomelanins (yellow to brown), anthraquinones (red to purples) etc (Delgado-Vargas et al., 2000).

Additionally, cosmeceuticals is combination between cosmetic- pharmaceutical products intended to improve the health and beauty of the skin. The concepts providing a specific result ranging from acne-control and anti-wrinkle effects, including sun protection (Gediya et al., 2011). As such, prodigiosin pigment exhibits a great potential in cosmeceuticals field due to special characteristic present in this pigment like photoprotectant, antimicrobial and antioxidant properties. This proven that the prodigiosin has multipurpose function and broad applications from beauty to health properties.

2.7.2 (d) Disadvantages of synthetic colorants

Almost all people believe that natural cosmetics would cause less side effects compared to chemical-based cosmetics. According to the simple survey about cosmetic users and side effects to the skin conducted by Chen, (2009), the results obtained 87.1% of the people use cosmetics, 94% of users believe that the chemical- based cosmetic would cause side effects while only 6% of users believe that natural cosmetics may cause side effects.

In chemical-based cosmetic formulations, BHA (Butylated Hydroxyanisole) and BHT (Butylated Hydroxytoluene) are known as synthetic antioxidants and are widely used as preservatives in lipsticks and moisturizers (Suzuki, 2010). These substances may cause allergic reactions in the skin. The international Agency for

Research on Cancer evaluated BHA as a possible human carcinogen (IARC, 1978). In contrast, in natural cosmetic formulations contain natural antioxidants like vitamin C

(Kadam et al., 2013). Moreover, coal tar-derived colors are also used widely in

cosmetic products (chemical-based). Winter, (2009) reported that the coal tar with high toxic can cause cancer on human.

CHAPTER 3.0: ANTIMICROBIAL ACTIVITIY OF THE PRODIGIOSIN

PRODUCED BY A MARINE BACTERIA, Serratia marcescens IBRL USM84

3.1 Introduction

Almost all marine microorganisms have their own natural pigment. Bacteria is one of the major groups that produce natural pigment for survival. Various types of pigments are found in marine heterotrophic bacteria such as carotenoid, flexirubin, xanthomonadine, and prodigiosin (Reichenbach et al., 1980; Stafsnes et al., 2010).

The rapid emergence of new infectious diseases and antibiotic-resistant bacteria such as Methicillin Resistant Staphylococcus aureus (MRSA) are getting worse because the drugs created are mostly analogues of the existing compounds (Berdy, 2005). All these cause problems in medical treatment which encourage seeking for new metabolites that are vigorous even against multi-resistant pathogens (Bull, 2004).

Most of the marine pigments are found to posses antimicrobial activity such as carotenoids (Lee et al., 2004) prodigiosin (Nadaf et al., 2016), violacein (Yada et al., 2008) and melanins (Geng et al., 2010). Natural pigments have big potential in the giant industries such as cosmetic, food, textile, and pharmaceutical can act as colorant as well as preservatives simultaneously. In this chapter, the crude extract of intracellular and extracellular pigment were extracted from the natural pigment produced by a local marine isolate, Serratia marcescens IBRL USM84 and tested against five species of fungi, five species of yeasts and nine species of bacteria in which four of them were Gram positive and five Gram negative bacteria. The test microorganisms that were inhibited by the crude extract were then used in antibacterial activity of partitionated extract and fraction analysis while the test microorganisms with poor or partial antimicrobial activity were discarded.

This chapter dealt with the production of natural red pigment with prodigiosin activity produced by the marine bacterial isolate, S. marcescens IBRL USM84 in a submerged system. The location of pigments in the cell was determined and the antimicrobial activity of the pigment against pathogenic microorganisms was performed. The prodigiosin activity was also determined.

3.2 Materials and Methods

3.2.1 Microorganisms and culture maintenance

3.2.1 (a) Bacterial strain

The strain, Serratia marcescens IBRL USM84 was provided by the Industrial

Biotechnology Research Laboratory, School of Biological Sciences, Universiti Sains

Malaysia, Penang, Malaysia. It was isolated from the surface of a marine sponge

Xetospongia testudinaria originated from Pulau Bidong, Terengganu. The subculturing was done monthly to ensure the viability of the bacterial strain. This bacterial culture was maintained on marine agar slant (Difco, United Kingdom) at

25oC for 24 hours aerobically before storing it at 4oC until further use (Darah et al.,

2014). The culture was kept as 20% glycerol stock at -20oC for long term storage.

3.2.1 (b) Test microorganism

The test microorganisms used for the bioassay test was provided by the

Industrial Biotechnology Research Laboratory, School of Biological Sciences,

Universiti Sains Malaysia, Penang, Malaysia. A total of four Gram positive bacteria were tested, Staphylococcus aureus, Bacillus cereus (ATCC 27853), Bacillus subtilis, and Methicillin Resistant Staphylococcus aureus (MRSA) whereas the Gram negative

bacteria used were Escherichia coli, Yersinia enterocolitica, Acinetobacter anitratus,

Klebsiella pneumoniae and Proteus mirabilis. Besides, five yeast cultures (Candida albicans, Candida tropicalis, Candida oleophila, Candida utilis and Cryptococcus neoformans) and five fungal cultures (Aspergillus flavus, Aspergillus fumigatus,

Aspergillus niger, Fusarium solani and Trichophyton rubrum) were also used. The test bacteria were maintained on nutrient agar (NA) (Merk, Germany) slant at 37oC for 24 hours aerobically (Darah et al., 2014). The test yeasts were maintained on Sabouraud

Dextrose agar (SDA) (Himedia) slant at 30oC for 24 hours aerobically while the test fungi were maintained on SDA (Himedia) slant at 30oC for 5 days aerobically. Then, all these test microorganisms were stored at 4oC until further use. The subculturing was done monthly to ensure the viability of the cultures (Teh Faridah, 2012). The cultures were kept as 20% glycerol stock at -20oC.

3.2.2 Searching for antimicrobial activity in the S. marcescens IBRL USM84 cells using disc diffusion assay

3.2.2 (a) Cultivation medium

A loop full of the 48 hours old colonies of the IBRL USM84 isolate was inoculated into 250 mL Erlenmeyer flask containing 50.0 mL of marine broth medium

(Difco, United Kingdom) and incubated at 26oC with 120 rpm of agitation speed for

17 hours. The experiment containing (v/v: 1 x 109 cells/mL) 17 hours old bacterial inoculum as a preculture (Darah et al., 2014). Inoculum 2%, was inoculated into flask containing 0.3 % of agar in 100.0 mL of marine semi-solid medium (Difco, United

Kingdom), initial pH at 7.5 and incubated at 25oC with 120 rpm of agitation speed for

72 hours cultivation period.

3.2.2 (b) Extraction of intracellular and extracellular pigments

At the end of cultivation period, the cells culture was centrifuged (4000 rpm,

45 minutes at 4oC) to separate the pellet and supernatant. The extraction of pigments from pellets (intracellular pigments) was done according to Slater et al., (2003) with slight modifications. The pellet was extracted by adding the acidified 2-propanol

(Qrec) (4% of 1M HCL in 1L 2-propanol) until 30 mL. The mixture was then vortexed and centrifuged at 4000 rpm for 30 minutes to separate the pellet from 2-propanolic supernatant. The supernatant was collected in the fresh vial. The pellet was re- extracted by adding the acidified 2-propanol until 20 mL and subjected to vigorous vortexing, followed by centrifugation at the same condition stated above. The supernatant was then transferred into the same vial. The intracellular pigment extract was concentrated in a rotary evaporator (Heidolph, Laborota 4000), poured into a glass

Petri dish and dried under fume hood until the pigment turned into a paste form. The pigment was present with salt granules. Methanol (Qrec) was then added to the paste in order to dissolve the pigment. The pigment was then placed in a new glass Petri dish and dried in the fume hood for further analysis. Figure 3.1 shows the dry pigment paste with the salt granules (Figure 3.1A) and after dissolving the paste by using methanol (Figure 3.1B). The dry paste was weighed until a constant weight (expressed in gram per litre) was obtained.

The pigment was extracted from the supernatant (extracellular pigments) by using an equal volume of ethyl acetate at a ratio of 1:1 in a separatory funnel

(Jafarzade et al., 2013a). The mixture was vigorously shaken and was placed on a tripod to allow the separation of phases where the upper layer (organic phase) was collected, while, the bottom layer (aqueous phase) was discarded. The organic phase

(ethyl acetate containing pigment) was then concentrated using a rotary evaporator,

placed in a glass Petri dish and was dried in a fume hood for further analysis. The dry paste was weighed until a constant weight (expressed in gram per litre) was obtained.

Figure 3.2 summarizes the extraction procedure for pellets and supernatants.

A B Pigment

Salt granules

Figure 3.1: Dry pigment paste extracted from pellet cells of S. marcescens IBRL USM84 (A) Pigment present with salt granules; (B) Pigment after dissolved with methanol

Culture broth (48 hours old culture) Centrifuged (4000 rpm, 4oC, 45 minutes)

Pellet (intracellular) Supernatant (extracellular) Acidified 2-propanol added Ethyl acetate vortexed, centrifuged (30 minutes) added

Pellet Supernatant

-Acidified 2-propanol added, vortexed, centrifuged (30 minutes) *This step repeated Upper layer Bottom layer until the pellets became colorless

White Pellet Supernatant

Rotary Rotary evaporator evaporator Discard Discard

Figure 3.2: Flowchart of pigment extraction of intracellular and extracellular extracts of S, marcescens IBRL USM84

3.2.2 (c) Preparation of extract solution

A total 50 mg of the dried pigments (intracellular and extracellular) in the glass Petri dish (Section 3.2.2b) were scraped and transferred into a 2.0 mL Eppendorf tube, respectively. Both pigments were re-dissolved in 1000 µl of methanol (Qrec) and thoroughly vortexed to ensure all the paste was completely dissolved.

3.2.2 (d) Preparation of test microorganism for seeded agar plates

All test microorganisms (Section 3.2.1 b) were used in the screening test for antimicrobial activity. Test bacterial inocula were prepared by inoculating nine pure colonies from 24 hours old bacterial culture on NA into a Universal bottle containing

5.0 ml of 0.85 % saline, NaCl. The suspension was mixed by vortexing and its turbidity was visually adjusted by adding a sterile saline or more colonies. The turbidity of each suspension was compared with 0.5 Mcfarland standards to obtain a cell density of approximately 1.0 x 108 cells per ml. A sterile cotton swab was dipped into the adjusted bacterial suspension and was gently pressed on the inside wall of the

Universal bottle to force down the excess inocula from the swab. The swab was then streaked on the surface of Mueller-Hinton agar (MHA) (Himedia) thrice. The agar plate was rotated approximately 360o to ensure the inoculum was evenly dispersed

(CLSI, 2006).

Test yeast inocula were prepared by inoculating five pure colonies from 24 hours old of yeast culture on SDA into a Universal bottle containing 5.0 mL of 0.85 % saline, NaCl. The suspension was mixed by vortexing and its turbidity was visually adjusted by adding a sterile saline or more colonies. The turbidity was compared with

0.5 Mcfarland standards to yield a yeast density of approximately 1.0 x 106 cells per

ml. A sterile cotton swab was dipped into the adjusted yeast suspension and was gently pressed on the inside wall of the Universal bottle to force down the excess inoculum from the swab. The test yeast inocula were then evenly dispersed on SDA using swab streaking technique.

The fungal spore suspension was prepared by using the five days old cultures or good conidial growth was present on SDA. Five millilitre of sterile saline was gently poured into the fungal culture and was gently scraped using an inoculating loop to spread the spores. The spore suspension was then transferred to another sterile

Universal bottle. The spore suspension density was measured by microscopic enumeration method using a Haemocytometer (Neubauer, German) (Aberkane et al.,

2002). The dilution was performed until the spore concentration of approximately 1.0 x 106 spores per ml was obtained. A sterile cotton swab was dipped into the adjusted spores and was then seeded on SDA using swab streaking technique

3.2.2 (e) Preparation of susceptibility test disc

About 20 µl of the intracellular or extracellular extracts (50.0 mg/mL) were loaded into the sterile antibiotic disc (Whatman AA disc, 6mm) on a sterile glass Petri dish. The discs were allowed to dry for a few minutes to ensure complete evaporation of solvent from the disc. The final concentration of the extract was 1.0 mg/disc. Then, the extract impregnated discs were firmly placed on MHA and SDA plates by using a sterile forcep. Each disc was gently pressed to ensure the disc closely contacted the agar. Each test was prepared in triplicate. Chloramphenicol was used as a positive control for antibacterial evaluation test, while, Ketoconazole was used as a positive control for antiyeast and antifungal tests. About 20 µl of the Chloramphenicol and

Ketoconazole (1.5 mg/mL) were prepared with 100% (v/v) ethanol and were loaded into the sterile disc placed on a sterile glass Petri dish. The final concentration of the control was 30 µg/disc. A disc loaded with methanol was used as a negative control.

The discs were allowed to dry for a few minutes to ensure complete evaporation of solvent from the disc before placing them on the MHA and SDA plates. After 10 minutes, the plates were inverted and incubated. The MHA plates seeded with bacteria were incubated at 37oC for 24 hours, while the SDA plates seeded with fungi and yeast were incubated at 30oC for 24 hours (yeasts) and 72 hours (fungi). Each plate was examined for the formation of inhibition zone (measured in millimetre, mm) around the discs.

3.2.3 Diphenylpicryl-hydrazyl (DPPH) scavenging activity

About 2.0 mg/mL of crude 2-propanol extract and quercetin were dissolved in

100% DMSO as the stock solution, followed by a serial of two-fold dilution to prepare

10 sample solutions with 10 different initial concentrations, ranging from 3.91 - 2000

µg/mL.

Diphenylpicryl-hydrazyl (DPPH) was prepared by dissolving 0.16 mM of commercial DPPH (Sigma-Aldrich) in absolute ethanol. The DPPH solution was prepared and kept in the dark until use (Choi et al., 2002).

The test was conducted with a microtitre plate in a dark condition. Each well consisted of 150 µL DPPH solution and 50 µL of extract sample with designated concentration. Blank DPPH served as control. The microtitre plate was covered and wrapped with aluminium foil and incubated at 40oC for 30 minutes. The absorbance of the content in the wells was measured using spectrophotometer at 517 nm.

The method was repeated by substituting the extract with quercetin solutions.

DPPH scavenging activity was then plotted by using quercetin as the standard. 50 % of the effective concentration (EC50) of the extract and quercetin were determined

(Prior et al., 2005). Where Ac is the absorbance of the control and As is the absorbance of the sample (Kulisic et al., 2004).

A - A Scavenging activity (%) = c s X 100

3.2.4 Total phenolic content (TPC)

TPC assay was done using Folin-Ciocalteu reagent according to the method described by Oliveira et al., (2009) with slight modification. Gallic acid was prepared in 2.0 mg/mL in 100% DMSO and diluted into 10 different concentrations through twofold dilution while the extract (2.0 mg/mL) was dissolved in 100% DMSO. Then,

10% of Folin-Ciocalteu solution (10ml) was prepared in distilled water, thoroughly mixed and was kept in the dark until use.

About 50 µL of gallic acid, 25 µL of Folin-Ciocalteu solution, 25 µL of 2% sodium bicarbonate (Na2CO3) and distilled water (100 µL) were added into the well as a standard. The same ingredients were added into the other wells using different concentrations of gallic acid.

For the test sample, the same ingredients were added in the well with an exception of replacing the gallic acid with the extract (2.0 mg/mL). The plate was incubated at room temperature for 30 minutes. The absorbance of the contents was measured at 760 nm.

3.2.5 Standard curve of prodigiosin

Prodigiosin from Serratia marcescens (Sigma, Aldrich) was used as an authentic sample. Serial dilution was performed for standard prodigiosin to obtain final concentrations of 50.00 µg/mL, 25.00 µg/mL, 12.50 µg/mL, 6.25 µg/mL, 3.12

µg/mL, 1.56 µg/mL, 0.78 µg/mL and 0.39 µg/mL. The absorbance of each dilution was recorded at 535 nm of wavelength using a spectrophotometer. A standard prodigiosin graph (Absorbance versus concentration) was plotted.

3.2.6 Quantification of prodigiosin

About 50.0 mg of the dry paste pigment from intracellular and extracellular extracts were dissolved in 2.0 mL of acidified 2-propanol. The concentration of prodigiosin was determined by measuring the absorbance at 535 nm using a spectrophotometer. The absorbance at 535 nm multiplied by the dilution factor (if any) to express the concentration of prodigiosin. The OD535 was converted to mass concentration in µg/mL via the calibration curve using purified prodigiosin (Sigma,

Aldrich) as standard (Kim et al., 2007; Wang et al., 2012). The quantitification of prodigiosin in the next chapter had focused on the pigment extracted from the intracellular only. The extracted and collected supernatant containing prodigiosin from the pellet cells was determined spectrophotometrically at 535 nm before concentrating

or drying it into the paste form. The expression of prodigiosin mass concentration in

µg/ml was used the same method as mentioned in Section 3.2.5.

3.2.7 Spectral analysis for intracellular and extracellular extracts

Maximum absorbance of the dried crude extract (intracellular and extracellular) extract from Serratia marcescens IBRL USM84 was determined by using UV spectrophotometer (Genesys; Thermoscientific) according to Williams et al., (1955) with slight modifications. This test was carried out by scraping the dried pigment from intracellular and extracellular extracts into test tube and suspending them with 10.0 ml of absolute 2-propanol, respectively. The mixture was then vortexed until it mixed properly and divided into two portions for acidic and alkaline condition. Acidic 2- propanol was used as blank for acidic condition, while alkaline 2- propanol for alkaline condition. The absorbance spectrum was measured within the wavelength region of 400 to 600 nm and the maximum absorbance (λmax) of the pigment was determined, and compared with the authentic sample of prodigiosin

(Sigma, Aldrich). The graph was plotted as absorbance (A) versus wavelength (nm).

3.2.8 Presumptive test for prodigiosin from intracellular and extracellular extracts

The presumptive color test for prodigiosin was employed according to Ding

& Williams, (1982) with slight modifications. This test was carried out by scraping the dried pigment extracted from Serratia marcescens IBRL USM84 into test tubes. The dried extract was suspended in 15.0 ml of 95% methanol and was vigorously vortexed for proper mixing. The clear solution was then divided into three portions, of which

the first portion was acidified with three drops of 1.0 M HCl solution, the second portion was alkalinized with three drops of 1.0 M NaOH solution and the third portion was kept as a control. A red or pink color in the acidified solution and a yellow or tan color in the alkaline solution indicated a positive presumptive test for prodigiosin.

3.2.9 Macroscopy and microscopy analysis of S. marcescens IBRL USM84

3.2.9 (a) Observation of the colony morphology

The colony of S. marcescens IBRL USM84 on MA plate incubated at 25oC was examined after 24 hours of culturing. The streak plate method was used to obtain a single colony. The following characteristics of the colonies were observed and the colour appearance (pink, red, blood red or brick red), size (pinpoint to big), form or shape (round to irregular), elevation (raised, convex or flat), margin (entire, serrate, filamentous or undulate), surface appearance (smooth, rough or glistening) and also opacity (transparent or opaque) were described.

3.2.9 (b) Observation using a phase contrast microscope

The Phase Contrast Microscope was used to visualise S. marcescens IBRL

USM84 cells in unstained state at 40x and 100x magnifications to determine the motility and the morphological shape of the cells. The strain was grown on MA, diluted and emulsified by placing a small piece of cultures using the tip of sterile inoculation loop into a drop of water on a clean glass slide. The culture mixture was spread using the inoculation loop for better observation. The slide was then examined using a Phase Contras Microscope (Olympus BX50).

3.2.9 (c) Observation of the surface of S. marcescens IBRL USM84 using

Scanning Electron Microscope (SEM)

The Scanning Electron Microscope (SEM) was used to observe the outer structure of S. marcescens IBRL USM84. The 48 hours old bacterial culture from 100 mL of marine broth was transferred into Eppendorf tubes and centrifuged at 4000 rpm for 15 minutes. The supernatant was discarded and the pellet was mixed with

McDowell-Trump fixation to fix and preserve the cells. The cells were washed with

0.1 M phosphate buffer, stained with 1% Osmium tetroxide, rinsed with sterile distilled water and dehydrated with 50%, 75%, 95% and 100% alcohol. The cells were centrifuged at 4000 rpm for 15 minutes for each solution. The cells were then immersed in Hexamethyldisilazane (HMDS) for 10 minutes and the specimen was then kept in a dessicator at room temperature (28oC). Before examination under a scanning electron microscope, the specimen was coated with 100 Å of gold with

Polaron SC515 SEM sputter coater and observed under SEM (Leica, Cambridge S-

360).

3.2.9 (d) Observation of the cross section of S. marcescens IBRL USM84 with

Transmission Electron Microscope (TEM)

The Transmission Electron Microscope (TEM) was used to observe the inner structure of S. marcescens IBRL USM84. The samples were centrifuged to obtain the pellet. The formation of the antibiotic-producing pigments within the cells was determined. The samples were fixed and preserved using McDowell-Trump fixation.

The cells were washed with 0.1 M phosphate buffer, stained with 1% Osmium tetroxide and dehydrated with 50%, 75%, 95% and 100% alcohol. The cells were centrifuged at 4000 rpm for 15 minutes for each alcohol solution. The cells were

dehydrated with 100% acetone as a final step. Specimens were infiltrated in the mixture of Acetone: Spurr‟s resin (1:1) for 24 hours in a rotator and the resin were left to solidify and cut. The prepared sample were observed and analyzed under TEM

Libra 120 model.

3.3 Results and Discussion

3.3.1 Screening for antimicrobial activity using disc diffusion assay

The screening for antimicrobial activity on S. marcescens IBRL USM84 was performed using disc diffusion assay against laboratory strains of Gram positive bacteria, Gram negative bacteria, yeasts and fungi. The results were taken by measuring the diameter of clear inhibition zones formed around the disc as it proved the presence of antimicrobial activity of the extract towards the test microorganisms.

The results revealed that all Gram positive bacteria (Staphylococcus aureus, Bacillus cereus, Bacillus subtilis and methichillin-resistant Staphylococcus aureus) and Gram negative bacteria Acinetobactor anitratus were susceptible to the intracellular prodigiosin extract. The other Gram negative bacteria (Escherichia coli, Salmonella paratyphi, Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis), all yeasts

(Candida albicans, Candida tropicalis, Candida oleophila, Candida utilis,

Cryptococcus neoformans) and fungi (Aspergillus flavus, Aspergillus fumigatus,

Aspergillus niger, Fusarium solani, Trichophyton rubrum) were resistant to the intracellular extract.

Antimicrobial activities of prodigiosin are listed in Table 3.1.The diameter of inhibition zones formed around the extract disc were in the range of 13.0 mm to 26.0 mm (Figure 3.3). Jafarzade et al., (2013b) found the inhibition zones of crude extract

produced by some marine pigmented bacteria were within the same range in this study i.e. from 8.0 to 30.0 mm. Undeniable, the fact that the Gram positive bacteria were more susceptible towards the extract compared to Gram negative bacteria. The similar patterns of results were reported previously by Olowosulu et al., (2005) and Alvarez-

Suarez et al., (2010). This could be due to the layer differences in the cell wall of both bacteria where Gram positive bacteria possess a porous peptidoglycan layer in the cell wall which is more permeable to most antimicrobial agents. Meanwhile, apart from peptidoglycan layer, the cell wall of Gram negative bacteria possesses an outer membrane comprising lipopolysaccharide (LPS), proteins and phospholipids. This membrane acts as a permeation barrier to prevent the entrance of active compounds into the cells (Pankey & Sabath, 2004; Tian et al., 2009). Moreover, the LPS acts as a toxin to protect the Gram negative bacteria against predator and can also cause the cell wall impermeable to lipophilic solutes (Barnett, 1992; Pandey et al., 2004).

Table 3.1: Antimicrobial activity of prodigiosin extract of S. marcescens IBRL USM84 by disc diffusion assay

Test Microorganism IN EX CH/ KZ M

Gram positive bacteria: Staphylococcus aureus ++ - ++ - Bacillus cereus ++ - ++ - Bacillus subtilis ++ - ++ - MRSA ++ - ++ -

Table 3.1: continued

Gram negative bacteria: Escherichia coli - - ++ - Yersinia enterocolitica - - ++ - Acinetobactor anitratus ++ - ++ - Klebsiella pneumoniae - - ++ - Proteus mirabilis - - ++ -

Yeast: Candida albican - - ++ - Candida tropicalis - - ++ - Candida oleophila - - ++ - Candida utilis - - ++ - Crytococcus neuformans - - ++ -

Fungi: Aspergillus flavus - - ++ - Aspergillus fumigatus - - ++ - Aspergillus niger - - ++ - Fusarium solani - - + - Trichophyton rubrum - - + -

IN = Intracellular extract, EX = Extracellular extract, CH = Chloramphenicol, KZ = Ketoconazole, M = Methanol *Score of activity: + = 7.0 - 12.0 mm; ++ = 13.0 - 30.0 mm; - = no activity

A B

-ve +ve -ve +ve

C D

-ve +ve -ve +ve

Figure 3.3: Disc diffusion assay of crude extract (intacellular) against different test microorganisms (A) Staphylococcus aureus (Gram positive); (B) Bacillus cereus (Gram positive); (C) Klebsiella pneumonia (Gram negative); (D) Yersinia enterocolitica (Gram negative) [-ve = Negative control; +ve = Positive control (30 µg chloramphenicol per disc)]

The extracellular extract did not show any antimicrobial activity towards all test microorganisms. Probably the amount of targeted bioactive compound known to possess antimicrobial activity was lesser compared to intracellular extract. The pigments extracted from both intracellular and extracellular indicated different prodigiosin concentrations as stated in the next Section 3.3.3 (Table 3.2), of which the concentration from intracellular was higher than that of extracellular extract. A higher concentration of prodigiosin from extracellular was required to completely inhibit the

test microorganisms. Figure 3.4 shows the color comparison of extracted pigments from supernatant and cell pellets. The difference in color in these extracts could be due to the different extractant solvents used in this study. The 2-propanol used to extract prodigiosin from intracellular based on previous study (Teh Faridah, 2012) exhibited purplish red color, while ethyl acetate used to extract the pigment from extracellular was yellow in color. Ethyl acetate was selected solvent to extract the pigment from supernatant by using the solvent partitioning method (Otsuka, 2008). This method usually involves the use of two immiscible solvents which were ethyl acetate and water in a separating funnel. Kobayashi & Ichikawa, (1991) reported that the prodigiosin from S. marcescens was found secreted extracellularly, or located in cell- associated vesicles or can be found in intracellular granules. The results revealed that prodigiosin extracted from intracellular of S. marcescens IBRL USM84 was more effective than the extracellular.

The production of bacterial pigments intracellularly or extracellularly could be influenced by the requirement of the bacteria to survive at their habitats. Hailei et al., (2012) and Mapari et al., (2005) reported that the pigments were produced intracellular as a response to disadvantageous environmental conditions such as insufficient of nutrient, and harmful of ultraviolet radiation and lethal photo oxidation.

This could be the reason why S. marcescens IBRL USM84 produced intracellular prodigiosin pigment since this strain was isolated from marine environment which was exposed to environmental stress and nutrient limitation. The prodigiosin pigment can also protect the bacterial cells from harmful UV radiation. Similar report with Griffiths et al., (1955) who found the caratenoids has been an important structural component of microbial membranes (Rottem & Markowitz, 1979) which was protects bacterial cells from UV irradiation and photooxidation.

A B

Figure 3.4: Color of extract from intracellular and extracellular, (A) Purplish red color from intracellular extract, 2-propanol as extractant; (B) yellowish orange from extracellular extract, ethyl acetate as extractant

None of the extracts were able to completely inhibit all yeasts and fungi. This can be due to the differences of cell wall structure in yeast and filamentous fungi. The cell wall of yeasts and fungi are primarily composed of chitin, glucans, mannans and glycoprotein (Bowman & Free, 2006). The low susceptibility to antimicrobial agent might be due to the well-defined chitinised cell walls, which are thick and capable of preventing absorption of toxic material into the cells (Aceret et al., 1998). Besides, the specificity of the target site can also influence the susceptibility differences between bacteria and fungi. In bacteria, the antimicrobial agents are targeting more specific structures such as peptidoglycan, 30S or 50S subunit of ribosomes. These antimicrobial agents would not affect the cells comprising 40S and 60S subunit of ribosomes such as fungi (Madigan & Martinko, 2006).

The polarity index (P.I) and water-miscibility characteristics of the solvents are among important factors influencing the extraction capability of certain solute. In this study, 2-propanol was used as the extractant for pellet (intracellular pigment).

According to previous study, 2-propanol was the most effective solvent to extract the intracellular pigment produced by S. marcensens IBRL USM84 based on the pigmentation strength and antimicrobial property (Teh Faridah, 2012). The P.I of 2- propanol was 3.9 and was classified as a moderately polar solvent. The water miscible characteristic has made the pigment extracted from the pellet were conveniently carried out. Furthermore, the red pigment was also reported to be more stable in 2- propanol (Soliev, 2011). However, this solvent cannot be used to extract the pigment from the supernatant due to its water miscible characteristic.

Ethyl acetate was used as the extractant for supernatant (extracellular pigment). Ethyl acetate is moderately polar (P.I = 4.4) with immiscible water characteristic and has been reported to extract a wide range of compounds (both polar and non-polar compounds). Since the compound was in a liquid form, the immiscible solvent such as ethyl acetate was suitably used to allow easy separation between the organic phase containing the extracted solutes and the aqueous phase (medium).

3.3.2 Antioxidant activity of crude 2-propanol extract

Most of natural pigments possess antioxidant properties (Boo et al., 2012;

Powell et al., 2014; Azwanida et al., 2014). However, the oxidative stress due to excessive reactive oxygen (ROS) production destroys the antioxidant capacity, which as a result, causing various chronic diseases to human populations. The presence of

free radical of the ROS, on the other hand, causes the impairment and destruction of cell physiology (Kremer et al., 2012).

The DPPH scavenging of the extract was evaluated where the EC50 of the 2- propanol crude extract was 213.89 µg/mL and the standard EC50 for the quercetin was

47.52 µg/mL (Figure 3.5). From the result, the EC50 value of the extract was approximately four times higher than the EC50 value of quercetin. This indicated that the extract did not possess high antioxidant activity by having low scavenging activity of the DPPH free radicals present. The 2-propanol extract of S. marcescens IBRL

USM84 exhibited a weak hydrogen donor and had lower DPPH scavenging activity thus, the crude extract was a poor antioxidant.

A standard curve of the phenolic content of gallic acid is shown in Figure 3.6.

The phenolic content in the 2-propanol crude extract was 77.30 µg GAE/mg of extract.

The low amount of phenolic content in the 2-propanol crude extract was expected since it was extracted from a marine bacterium S. marcescens IBRL USM84. In plants, phenolic compounds mostly act as multifunctional antioxidants, reducing the free radical content and inhibiting the excessive oxidation, further preventing the cells from dying. High concentration of phenolic compounds in many plants has provided good medicinal values to humans (Kremer et al., 2012).

Scavenging activity activity Scavenging (%) 20

0 0 0.5 1 1.5 2 2.5 3 3.5 Log10 Concentration

2-propanol extract Quercetin

Figure 3.5: DPPH free radicals scavenging activity (%) of quercetin and crude 2- propanol extract (intracellular extract)

2.5

1.5

1 y = 0.001x + 0.180 R² = 0.933

Absorbance at at Absorbancenm 760 0.5

0 0 250 500 750 1000 1250 1500 1750 2000 2250 2500 Concentration (µg GAE/mg of extract)

Figure 3.6: Standard curve of total phenolic content for gallic acid

However, the total antioxidant capacity of prodigiosin from S. marcescens

IBRL USM84 (77.3 µg GAE/mg) was higher than those antioxidant capacity of

prodigiosin from S. marcescens reported by Gulani et al., (2012) i.e. 22.05 µg AE/mg.

Azwanida et al., (2014) reported that DPPH radical scavenging activity of Hylocereus polyrhizus extract from a plant source was higher than S. marcescens IBRL USM84 extract which possibly due to its high phenolic compounds.

Besides, concentration of the phenols in the sample is strongly correlated to antioxidant property of the sample tested (Kosalec et al., 2013; Ifesan et al., 2014;

Prado et al., 2014). Hence, high phenolic content in the sample results in a great antioxidant activity.

3.3.3 Quantitification of prodigiosin from intracellular and extracellular pigment produced by S. marcescens IBRL USM84

The concentration of prodigiosin produced by isolate S. marcescens IBRL

USM84 was calculated by manipulating the linear regression line equation of the standard prodigiosin curve (y = 0.187x + 0.017; R2 = 0.991) (Figure 3.7). The concentration of prodigiosin was expressed as microgram per millilitre (µg/mL).

The comparison of prodigiosin production from intracellular and extracellualar extract produced by S. marcescens IBRL USM84 are listed in Table 3.2.

The yield of 2-propanol extract from intracellular was 7.02 g/L higher than ethyl acetate extract from extracellular which was 0.25 g/L. The prodigiosin production from intracellular pigment of S. marcescens IBRL USM84 in submerged batch culture was 25.13 mg/L (95.01%) also higher than the extracellular pigment which was 1.32 mg/L (4.99%). The prodigiosin production of intracellular pigment from S. marcescens IBRL USM84 was profoundly high compared to several other prodigiosin-producing bacteria including Pseudoalteromonas rubra BF1A IBRL

(3.317 mg/L), Serratia rubidae (8.0 mg/L), Zooshikella rubidus S1-1 (47.8 mg/L),

Serratia sp. BTWJ8 (0.03995 mg/L) and Hahella chejuensis KCTC 2396 (28.0 mg/L)

(Kim et al., 2007; Krishna, 2008; Lee et al., 2011; Siva et al., 2011; Azlinah, 2015).

12 y = 0.187x + 0.017 10 R² = 0.991 8 6 4

2 Absorbance at at Absorbancenm 535 0 0 10 20 30 40 50 60 Concentration of Prodigiosin (µg/mL)

Figure 3.7: Standard prodigiosin calibration curve

Table 3.2: Comparison of quantitfication of intracellular and extracellular pigment from S. marcescens IBRL USM84

Characteristics Intracellular Extracellular (2-propanol extract) (Ethyl acetate extract)

Yield of extract (g/L) 7.02 0.25

Absorbance at 535 nm 4.716 0.264

Concentration of Prodigiosin 25.13 1.32 (mg/L) Concentration of prodigiosin 95.01 4.99 (%)

Based on the results, prodigiosin accumulated at two different sites, namely intra- and extracellular (Wang et al., 2012). The compounds are not entirely secreted by cells and are transferred to the culture medium. Some compounds may remain associated with the cell surface for several reasons such as adsorption, hydrophobicity and biological affinity (Cannell, 1998). In this study, the intracellular prodigiosin showed promising strength pigmentation and also possessed high antibacterial property, while the extracellular prodigiosin exhibited very weak pigmentation strength and no antibacterial property detected (Section 3.3.1; Table 3.1). Figure 3.8 shows the pigmented cells extracted from the S. marcescens IBRL USM84 with intracellular (Figure 3.8A) and extracellular (Figure 3.8B) extracts in a dry paste and after redissolving in 2-propanol (Figure 3.8 C and D). Contrarily, Campas et al.,

(2003) found the extracellular prodigiosin extracted from S. marcescens was reported to have significant bioactivity. Pseudoalteromonas sp. also secreted the antibacterial compound into the culture medium completely as stated by Darabpour et al., (2012) in their finding.

There are different functions of pigments in their role to protect the bacterial cells and help to improve bacterial survival. For example, melanins protect the microorganism cells against environmental stress while, carotenoids protect the cells from harmful UV radiation and against photo oxidation (Mapari et al., 2005). Several

Serratia species produced intracellular prodigiosin pigment such as Serratia sp.

BTWJ8 (Krishna et al., 2008), S. marcescens MSK1 (Bharmal et al., 2012) and S. marcescens (Vijayalakshmi & Jagathy, 2016). In contrast, other Serratia species produced extracellular prodigiosin pigment for example Serratia sp. (Jafarzade et al.,

2013a) and S. marcescens NCIM 5061 (Nadaf et al., 2016). Several bacteria occasionally

secrete the antibacterial compounds as one of the effective methods in controlling their population as well as to prevent the competitors colonizing the adjacent space.

A B

Pigment

C D

Figure 3.8: Pigment extract of S. marcescens IBRL USM84 in a dry form (A) intracellular extract; (B) extracellular extract and after redissolving in 2- propanol at concentration of 5 mg/ml (C) intracellular extract; (D) extracellular

extract

3.3.4 Characterization of prodigiosin pigment produced by S. marcescens

IBRL USM84

Figure 3.9 shows the spectral analysis characterization of intracellular pigment of S. marcescens IBRL USM84. In acidic solution, S. marcescens IBRL

USM84 extract and prodigiosin standard shared the maximum absorption peak which was at 535 nm. The peak shape and maximum absorption spectrum indicated the

presence of chromophore. The pigments would absorb light at some desired wavelength, and therefore the pigment expression was easily monitored using a spectrophotometer.

0.9 535 nm

0.8 -

- 0.7

0.6

- 0.5

- 0.4

- 0.3

0.2 Absorbance (A) Absorbance

0.1

0 -

400 450 500 550 600 650 Wavelength (nm)

Standard prodigiosin Intracellular extract

Figure 3.9: Spectral analysis of intracellular pigment extract of S. marcescens IBRL USM84 and standard prodigiosin

Prodigiosin can exist in two different forms and displays distinct characteristics, depending on hydrogen ions concentration of the solution. The pigment appears as red in color in acidic solution and exhibits a sharp spectral peak at

535 nm (Williams et al., 1955; Teh Faridah, 2012). However, the extracellular pigment of S. marcescens IBRL USM84 showed the maximum absorption peak at 534 nm in acidic solution (Figure 3.10). For extracellular pigment, this finding was similar with previously published results that pigment from supernatant extract of

Pseudoalteromonas rubra BF1A IBRL exhibited the maximal absorption peak at 534 nm in acidic condition (Azlinah, 2015). It might be the prodiginine derivatives which

have some distinct properties among each other but has been classified under the prodiginine group (Gerber, 1969).

0.9 535 nm

0.8 -

0.7 -

- 0.6 -

- 0.5 -

- 0.4 -

- 534 nm -

0.3 -

- Absorbance (A) Absorbance 0.2

- 0.1 -

400 450 500 550 600 650 Wavelength (nm)

Standard prodigiosin Extracellular extract

Figure 3.10: Spectral analysis of extracellular pigment extract of S. marcescens IBRL USM84 and standard prodigiosin

In this research, the prodigiosin from intracellular extract was more dominant compared to the extracellular extract. Furthermore, in an alkaline solution, the pigment from intracellular extract appeared orange-yellow in color and exhibited a broader spectral curve centered at 465 nm (Figure 3.11). This result was supported by

Williams et al., (1955) and Azlinah, (2015) who reported the prodigiosin detection in alkaline solution produced a broader spectral curve centered at 470 nm and 462 nm, respectively. The results indicated the natural red pigment produced by S. marcescens

IBRL USM84 containing prodigiosin. Table 3.3 shows the comparison of property by the intracellular and extracellular extracts of isolate S. marcescens IBRL USM84. In this study, intracellular pigment was selected as the best candidate for further analysis due to its color property, the yield and antibacterial spectrum. 86

0.9 0.8 0.7 535 nm

0.6 -

0.5 -

0.4 465 nm -

0.3 -

- Absorbance (A) Absorbance 0.2 -

0.1 -

0 400 450 500 550 600 650 Wavelength (nm)

Alkaline extract solution Acidic extract solution

Figure 3.11: Spectral analysis of intracellular pigment extract of S. marcescens IBRL USM84 under alkaline and acidic condition

Table 3.3: Property of intracellular and extracellular extracts of isolate S. marcescens IBRL USM84

Pigment Intracellular Extracellular Extractant 2-propanol Ethyl acetate Colour Purplish red Yellow Colour after redissolve Purple Pale red in 2-propanol Yield (g/L) 7.02 0.25 UV/vis peak (nm) 535 534 Susceptible bacteria S. aureus, B.cereus, B. subtilis, MRSA, - A. anitratus - = negative result

Figure 3.12 and Figure 3.13 depict the presumptive test for prodigiosin for intracellular and extracellular extracts, respectively. The formation of a pink colouration in acidic condition (Figure 3.12A and Figure 3.13A) and a yellow colouration in alkaline condition (Figure 3.12C and Figure 3.13C) indicated a positive presumptive test for prodigiosin. The results is similar with Gulani et al., (2012) who

reported the positive presumptive test for prodigiosin extracted from cell pellet of S. marcescens. The extract turned to pink and yellow when added with HCL and ammonia solution, respectively. Picha et al., (2015) also reported the preliminary identification of pigment from S. marcescens indicated the positive presumptive test for prodigiosin when the pigment added with acidified ethanol and alkalinized acetone, respectively.

A B C

Figure 3.12: Presumptive test for prodigiosin from intracellular extract of S. marcescens IBRL USM84. (A) pigment extract added with HCL, (B) control of pigment extract and (C) pigment extract added with NaOH

A B C

Figure 3.13: Presumptive test for prodigiosin from extracellular extract of S. marcescens IBRL USM84. (A) pigment extract added with HCL, (B) control of pigment extract and (C) pigment extract added with NaOH

The protonation of pyrrole group in acidic solution as well as deprotonation of amine group in the chemical structure of prodigiosin in alkaline solution are two main reasons lead to the changes in the coloration of prodigiosin (Struchkova et al.,

1973). Namazkar et al., (2013) also described that in acidic condition, the protonation of pyrrole group occurs at one of the carbon atoms of the second position in the ring and not on nitrogen atom, whereas in alkaline condition, the OH- group deprotonated the amine group in the chemical structure.

Previous study reported that the bioactivity of prodigiosin from Serratia marcescens are due to the diversity of S. marcescens from both marine and terrestrial resources. For example, S. marcescens (Furstner, 2003), S. marcescens (Khanafari et al., 2006) and S. marcescens MSK1 (Bharmal et al., 2012) have been reported to have immunosuppressive, antifungal and antibacterial activity, respectively. Some marine bacteria known to be prodigiosin producers are Vibrio psychroeytrus (D‟Aoust &

Gerber, 1974), Pseudomonas sp. (Gandhi et al., 1976), Vibrio ruber (Shieh et al.,

2003; Wan Norhana, 2004), Zooshikella rubidus (Lee et al., 2011), Hahella chejuensis

(Park et al., 2012), Streptomyces sp. (El-Bondkly et al., 2012) and P. rubra (Priya et al., 2013).

3.3.5 Macroscopic and microscopic analysis of S. marcescens IBRL USM84

3.3.5 (a) Morphological characteristics

S. marcescens IBRL USM84 formed a reddish colony on MA within 24 to 48 hours of incubation at 25oC aerobically. The diameters of the single colony were in the range of 1.0 to 2.0 mm. The colony was circular, convex elevation with dark red pigmentation at the centre and light pink at the edge. The color of the bacterial colony became intensely red as the incubation period increased. Figure 3.14 shows the colony development of S. marcescens IBRL USM84 after 24 hours (Figure 3.14A) and 48 hours (Figure 3.14B) of incubation time on MA. There are two major groups of pigments that produce red color, which are carotenoids and prodigiosin (Nugraheni et al., 2010; Yang et al., 2013). The red pigment exhibits higher antimicrobial activity, followed by orange, yellow and green pigments (Jafarzade et al., 2013a). Figure 3.15 shows the freeze-dried intracellular natural red pigment with prodigiosin produced by

S. marcescens IBRL USM84. Kamble & Hiwarale, (2012) who found the S. marcescens exhibited the brick red colored colonies that indicated the production of prodigiosin as obtained in this study.

A B

Figure 3.14: Colony morphology of isolate S. marcescens IBRL USM84 on Marine agar plates A) after 24 hours of incubation, B) after 48 hours of incubation

Figure 3.15: Freeze dried biomass cells with pigment of S. marcescens IBRL USM84

Actually, S. marcescens can only produce natural red pigment when it was incubated at a temperature of 20 to 30oC. If incubated at a temperature of 37oC it did not produce the red pigment. This is because S. marcescens indicated reduction of pigment production when incubated at higher temperature and exhibited complete block of pigment production when incubated at 42oC (Giri et al., 2004). The effect of various temperatures on the prodigiosin pigment production will be evaluated and discussed in more detail in Chapter 4 to enhance the culture condition for maximal prodigiosin production by S. marcescens IBRL USM84.

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

S. marcescens IBRL USM84 is a Gram negative rod bacterium in the family

Enterobacteriaceae. This isolate has potential to produce a pigment called prodigiosin which range in color from dark red to pale pink. The variety of pigment coloration depending on the species type, incubation period, pH and carbon source (Pandey et al.,

2009).

In this study, the microscopic structure of S. marcescens IBRL USM84 was studied by the aid of phase contrast microscope, SEM and TEM. Under the phase contrast microscope (Figure 3.16) the cells are seen as dark and unflagellated (Figure

3.16A) and also unciliated short rods (Figure 3.16B). Under SEM (Figure 3.17), the micrograph shows and reconfirmed the isolate S. marcescens IBRL USM84 was a short rod bacterium, no flagellum and reproduced by binary fission. Under TEM

(Figure 3.18), the micrographs revealed that isolate S. marcescens IBRL USM84 had a significant amount of pigments, suspected of being responsible for the antibacterial activity which is present intracellularly. These pigments were mainly deposited at the cell envelope of this isolate, indicated by the dark regions around the periphery of the 92

cells (Figure 3.18A). The pigments exist at the cell envelope (Figure 3.18) due to prodigiosin structure characteristic which have conjugated double bond. The electron could not penetrate the structure and become dark-colored region. The cells cultured for 48 hours had higher pigment accumulation at the cell envelope (Figure 3.18B) compared to the cells that were only cultured for 24 hours. Iranshahi et al., (2004) also found that prodigiosin is a non-diffusible red pigment attached to the inner membrane.

However, prodigiosin could also be found in extracellular vesicles and intracellular granules such as in S. marcescens (Kobayashi & Ichikawa, 1991). This finding also supported that the antibacterial activity was from intracellular pigment of isolate S. marcescens IBRL USM84.

A B

Figure 3.16: The observation of S. marcescens IBRL USM84 under the phase contrast microscope at (A) 40X magnification, (B) 100X magnification

Figure 3.17: SEM micrograph of S. marcescens IBRL USM84 with magnification at 10000X

A B

Accumulation of pigment

Figure 3.18: TEM micrographs of S. marcescens IBRL USM84 with magnification at 50000X (A) 24 hours cultivated cell, (B) 48 hours cultivated cell

Prodigiosin ia a secondary metabolite produced by many species of bacteria such as Serratia marcescens, Serratia rubidaea, Vibrio gazogenes, Vibrio psychroerythrous, Pseudomonas magneslorubra, Alteromonas rubra,

Rugamonasrubra, Streptomyces longisporus, Streptomyces spectabilis and

Streptoverticillium rubrireticuli (Variyar et al., 2002). It has also been suggested that prodigiosin probably located in the bacterial cell envelope and may not be released into the medium, a typical characteristic of secondary metabolites (Purkayastha &

Williams, 1960). In the present study, S. marcescens IBRL USM84 isolated from marine sponge produced high prodigiosin from intracellular. It might be due to the function of pigment in protecting the marine bacterium from the environmental stress including nutrition limitation (Hailei et al., 2012), UV radiation and photo oxidation

(Mapari et al., 2005).

3.4 Conclusion

The intracellular natural red pigment extracted from marine bacterial isolate,

S. marcescens IBRL USM84 possessed an antibacterial activity towards the Gram positive and negative bacteria which were S, aureus, B. cereus, B. subtilis, MRSA and

A. anitratus. The prodigiosin from intracellular pigment was more dominant and demonstrated a greater antibacterial activity compared to the extracellular pigment.

Based on the presumptive test results, the pigment from S. marcescens IBRL USM84 indicated a positive presumptive test for prodigiosin.

CHAPTER 4.0: ENHANCEMENT OF PHYSICAL AND CHEMICAL

CULTURE CONDITION FOR PRODUCTION OF PRODIGIOSIN BY

Serratia marcescens IBRL USM84

4.1 Introduction

S. marcescens IBRL USM84 is a marine bacterium that produces a significant natural red pigment prodigiosin with antibacterial activities. The production of prodigiosin is influenced by numerous factors such as bacterial species, physical parameters (incubation time, temperature, pH) and chemical parameters (inorganic salt, carbon and nitrogen sources) (Giri et al., 2004). The cultivation was carried out in a shake flask system. The physical parameters i.e. the effect of culture duration, light, initial pH of medium, temperature, agitation speed, and percentage of agar towards the growth, pigment production and antibacterial activity of S. marcescens IBRL USM84 were studied. The chemical parameters, namely the effect of carbon sources, nitrogen sources, percentage of sodium chloride and the percentage of maltose were also studied. Once the optimal physical and chemical parameters obtained, they would definitely maximize the secretion and synthesis of pigment by the bacterium (Darah et al., 2014). The best culture conditions by physical and chemical parameters were combined to compare the increment of production before and after the enhancement.

This chapter focused on the quantification of the pigment produced by S. marcescens IBRL USM84, bacterial cell growth and also its antibacterial activity. The best culture conditions by physical and chemical parameters for maximal prodigiosin production by S. marcescens IBRL USM84 was also determined.

4.2 Materials and Methods

4.2.1 Enhancement of cultivation conditions in fermentation process for cell growth, antibacterial activity and red pigment production

Before enhancement process, 2.0 % of S. marcescens IBRL USM84 inoculum as a preculture was cultivated into 250.0 mL Erlenmeyer flask containing 0.3 % of agar in 100.0 mL of marine semi-solid medium (Difco, United Kingdom). The initial pH was set at 7.5 and the inoculum was incubated at 25oC with 120 rpm of agitation speed as mentioned in Chapter 3 (Section 3.2.2 a). Several physical and chemical parameters were tested to evaluate their effect on the cell growth, antibacterial activity and pigment production. All the data collected were analyzed to determine the best culture condition for S. marcescens IBRL USM84 in prodigiosin production.

4.2.1 (a) Physical parameters

Physical parameters namely culture incubation durations (0, 8, 16, 24, 32, 40,

48, 56, 64, and 72 hours), light illumination condition (exposed and unexposed to light), initial pH medium (5, 6, 7, 8 and 9), temperature (20, 25, 30, 35 and 40oC), agitation speed (0, 50, 100, 150 and 200 rpm) and agar concentration (0, 0.1, 0.2, 0.3 and 0.4%, w/v) were evaluated. The experiments were done in three replicates. Data were presented as mean ± standard deviation (SD) and were analyzed using ANOVA with p < 0.05 was considered as significant (Darah et al., 2014; Azlinah, 2015). The best culture conditions in every physical parameter were combined to compare the increment of production before and after the enhancement.

4.2.1 (b) Chemical parameters

The chemical parameters evaluated in this study were the different types of carbon sources in the medium formulation (glucose, sucrose, lactose, maltose, fructose, inositol and starch 1% w/v), nitrogen sources (ammonium oxalate, urea, tryptone, yeast extract, peptone, ammonium acetate and casein 1% w/v), percentage of sodium chloride (0, 0.25, 0.5, 1.0, 1.5, 2.0, 3.0 and 4.0 % w/v) and different percentages of maltose (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % w/v) (Gulani et al., 2012).

The best culture conditions in every physical and chemical parameter were combined to compare the increment of production before and after improvement. The experiments were performed in three replicates and the data were presented as mean with standard deviation (SD).

4.2.2 Determination of cell growth, antibacterial activity and pigment production

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

The cell growth was measured at 620 nm using a spectrophotometer

(Spectronic Unicam, Genesys 10UV) (Gulani et al., 2012). The absorbance at 620 nm multiplied by the dilution factor (if any) to express the concentration of cell growth.

The actual concentration was compared to standard curve provided (Appendix 3).

4.2.2 (b) Assay for antibacterial activity

The antibacterial activity in the broth medium was quantitatively determined based on Lorian (1991). Methicillin-resistant Staphylococcus aureus (MRSA) was

selected for antibacterial analysis since this bacteria is the most important and life threatening pathogen. A loopfull colony of 24 hours old MRSA was inoculated in 5.0 mL of nutrient broth in 20.0 mL universal bottle and incubated at 37oC for 24 hours.

The turbidity of MRSA inoculums was visually adjusted equivalent to 0.5 McFarland standards. About 0.1 mL of the MRSA inoculum was inoculated in 7.9 mL nutrient broth, followed by the addition of 2.0 mL of redissolved dried pigment extracted from

S. marcescens IBRL USM84. The mixture was then incubated at 37oC for 18 hours.

The control consisted of the same materials as for the test culture but no addition of the pigment extracted from S. marcescens IBRL USM84, instead it was replaced by addition of another 2.0 mL of nutrient broth. The degree of inhibition of the MRSA was determined based on the decrease in the culture turbidity measured at 560 nm compared to the control. The antibacterial activity of the pigment extracted was defined as one unit (U) of the antibacterial activity which resulted in the reduction or inhibition of 1.0% of the growth of MRSA (Darah et al., 2014).

4.2.2 (c) Extraction and analysis of prodigiosin production

After centrifuged, the pellet was collected and added with acidified 2- propanol (Qrec) (4% of 1M HCL in 1L 2-propanol) until a volume of 30.0 mL was achieved. The mixture was thoroughly vortexed and resuspended. Then, the mixture was centrifuged at 4000 rpm for 30 minutes. The resulting supernatant was collected in a fresh vial. The pellet was re-washed using acidified 2-propanol until a volume of

20.0 mL was achieved. The mixture was vortexed and resuspended. After that, the mixture was centrifuged at 4000 rpm for 30 minutes. The supernatant was transferred into the same vial (Slater et al., 2003). The prodigiosin production was determined at

535 nm using a spectrophotometer (Spectronic Unicam, Genesys 10UV). The actual

concentration was compared to the standard prodigiosin calibration curve (Chapter 3,

Figure 3.7). The rest of the pigment extract was concentrated in a rotary evaporator

(Heidolph, Laborota 4000) and was dried in the glass Petri dish in a fume hood until dried red pigment formed for further analysis.

4.2.2 (d) Statistical analysis

The data analysis was performed using the Scientific Package for Social

Science (SPSS) software and p value of <0.05 was considered as significant. The significant difference of the mean data was analyzed by ANOVA and Duncan test using PASW Statistics version 22.

4.3 Results and Discussion

4.3.1 Enhancement of production by physical and chemical parameters

Several physical parameters such as pH, temperature, agitation speed and percentage of agar affect the production of prodigiosin by bacteria. In this present study, the cultural conditions were improved during the enhancement process to obtain a maximum production of prodigiosin with antibacterial property. According to

Mendez et al., (2011), pH and temperature were among physical parameters that greatly increased the pigment production.

The prodigiosin production also can be influenced by chemical parameter such as carbon sources, nitrogen sources and inorganic salts that were supplemented into the cultivation medium. Giri et al., (2004) stated that carbon sources play an important role in enhancement of the cell growth and prodigiosin production. This is

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precious for industrial scale in aiming to produce high potential products with low wastage (Giri et al., 2004; Gulani et al., 2012).

MRSA was selected as a test bacterial for antibacterial analysis in the enhancement part for both physical and chemical parameters since it is the most important and life threatening pathogen. MRSA is also known as a major nosocomial pathogen and had been proven to be the most common causative agent, exhibiting a resistance to 50% of tested synthetic antibiotics (Darabpour et al., 2011). This pathogen is responsible for the largest outbreak of hospital-acquired infection (HAI) worldwide (Feher et al., 2010). The resistance of MRSA to methicillin is due to the cell acquires a chromosomal gene, mec A, which encodes a methicillin-inducible penicillin-binding-protein, resulting in the decreased affinity to methicillin (De

Olieveira et al., 2013). Therefore, the ability of S. marcescens IBRL USM84 extract to inhibit the MRSA cells was a great progress in eliminating the MRSA infection.

Besides S. marcescens IBRL USM84, another marine bacteria Pseudoalteromonas rubra BF1A IBRL which was also prodigiosin producer was reported to exhibit antibacterial activity against S. aureus, B.cereus, B. subtilis, MRSA and A. anitratus

(Azlinah, 2015).

4.3.1 (a) Effect of culture duration

The effects of varying the incubation periods on the growth, prodigiosin production and antibacterial activity are shown in Figure 4.1. The results showed that the prodigiosin production was initiated and drastically increased during the logarithmic growth phase. The production continued until the late stationary growth phase. The results also displayed that the anti-MRSA activity was highly correlated

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with the pigment production of S. marcescens IBRL USM84. The highest antimicrobial activity was noted at 2.76 µg/mL of the pigment production and at 48 hours of incubation time (p< 0.05).

3 I 40 35 2.5 F G H E 30 2 D 25 1.5 20 C B B 15 1 Growth(g/L) 10 Prodigiosin (µg/mL) Prodigiosin 0.5 A 5 0 0 AntibacterialActivity(U/mL)

0 8 16 24 32 40 48 56 64 72 A A Culture Duration (Hours)

Prodigiosin (ug/ml) Growth (g/L) Antibacterial Activity (U/ml)

Figure 4.1: Effect of culture duration on prodigiosin production, antibacterial activity and growth of S. marcescens IBRL USM84 [MB initial pH 7.5; 2% v/v inoculums of 17 hours old culture (1 x 109 cells/mL); 0.3% of agar; temperature 25oC; 120 rpm of agitation speed; light condition; results were average of triplicates ± SD]

The highest production of prodigiosin was at the end of the stationary growth phase of S. marcescens IBRL USM84. The prodigiosin production by S. rubidae was also maximum at the stationary phase (Siva et al., 2011). Contrarily, prodigiosin production by Serratia sp. and Vibrio sp reported by Alihosseini et al., (2008) and

Bharmal et al., (2012) was maximum at the logarithmic growth phase. Astaxanthin produced by Phaffia rhodozyma also reported was produced in the exponential growth phase (Johnson & Lewis, 1979). Zooshikella rubidus, however, produced the highest prodigiosin after cellular multification ended (Lee et al., 2011). Each prodigiosin

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producer has their own specific characteristics in the prodigiosin production profile.

This production varied among the bacteria since different bacteria produced prodigiosin at different growth phases. Wang et al., (2012) also reported that the similar pattern of prodigiosin production from Serratia marcescens TKU011, where the prodigiosin production was started from logarithmic growth phase and continue until the stationary growth phase as obtained in this study.

The production of prodigiosin and the cell growth increased in parallelly, which suggest that the production of prodigiosin was associated with the cell growth.

Similar finding by Xu et al., (2011) and Wang et al., (2012) described that the prodigiosin production was closely related to the cell growth. After the stationary growth phase, the antibacterial activity subsequently declined following the death of S. marcescens IBRL USM84 as reported by Kim et al., (2007) in their research study.

The production of prodigiosin was highest at 48 hours of incubation time that was at the late stationary growth phase of S. marcescens IBRL USM84. The yield of both pigment and antibacterial activity were maximum at the same time, suggesting that the pigment may be involved in antibacterial activity. Further analysis, therefore, is required to rule out this probability. Figure 4.2 shows the pigment in 2-propanol acquired at different incubation times. The color of 2-propanolic pigment changed according to the time of incubation. The color of pigment became intensely red at 48 and 56 hours of incubation time.

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8 16 24 32 40 48 56 64 72

Figure 4.2: Pigment extract in 2-propanol obtained at different cultivation period of S. marcescens IBRL USM84

4.3.1 (b) Effect of light

The effect of light on prodigiosin production, antibacterial activity and growth of S. marcescens IBRL USM84 are shown in Figure 4.3. Incubation at both light and darkness condition did not significantly affect (p> 0.05) the prodigiosin production. The results showed that after 48 hours incubation time the production of prodigiosin was 2.77 µg/mL in the light condition and 2.76 µg/mL in the dark condition. The cell growth for both light and darkness conditions was 0.61 and 0.60 g/L, respectively. The antibacterial activity of pigment extract in light and darkness condition also did not vary very much where 34.72 U/mL was obtained in light condition while in darkness condition was 34.04 U/mL. Wang, (2012) reported that S. marcescens TKU011 exhibited higher prodigiosin production under illumination in shaking culture conditions compared to under dark conditions. Thus, in this study, incubation under the light illumination condition was selected as the optimum condition for the bioactivity of S. marcescens IBRL USM84. Light illumination was also reported to inhibit and destroy the production and stability of prodigiosin, however, prodigiosin is considered to be able to store the visible light energy which is an important factor for pigment synthesis (Ryazantseva et al., 1995; Someya et al.,

2004).

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4 40 A A 3.5 35 3 30 2.5 25 2 20

1.5 15 Growth (g/L) Growth

1 10 Prodigiosin (µg/mL) Prodigiosin 0.5 5

0 0 (U/mL), Activity Antibacterial Light Dark Light Condition

Prodigiosin (ug/ml) AntibacterailActivity (U/ml) Growth (g/L)

Figure 4.3: Effect of light on prodigiosin production, antibacterial activity and growth of S. marcescens IBRL USM84 [MB initial pH 7.5; 2% v/v inoculums of 17 hours old culture (1 x 109 cells/mL); 0.3% of agar; temperature 25oC; 120 rpm of agitation speed; incubation period 48 hours; results were average of triplicates ± SD]

Suryawanshi et al., (2015) had studied the bacterial pigments such as prodigiosin and violacein are potentially useful in the development of commercial sunscreens. These pigments were able to increase the sunscreen protection factor

(SPF) of commercial sunscreens and exhibited antioxidant and antimicrobial activities as a natural UV-protectants. Lee et al., (2001) also stated that many microorganisms that live in high-UV habitats such as solar salterns and other shallow water are able to produce pigments that provide protection against UV-damage. However these two pigments can be produced in the dark condition. Interestingly, there was no effect of light illumination to the astaxanthin production produced by Phaffia rhodozyma mutants as reported by Meyer & Du Preez, (1994).

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Incubation under light and dark conditions also did not significantly affect

(p> 0.05) the production of prodigiosin and the growth of Pseudoalteromonas rubra

BF1A IBRL (Azlinah, 2015). However, a higher antibacterial activity was noted by the culture incubated in the darkness compared to those incubated under light illumination. Moreover, Velmurugan et al., (2009) described that the total darkness was the most effective incubation condition for pigment production by microorganism.

4.3.1 (c) Effect of initial pH of medium

The pH value plays an important role to enhance the bioactivity of microbial culture. The results revealed significant effect (p> 0.05) of prodigiosin production of

S. marcescens IBRL USM84 when cultivated in different initial pH of medium (Figure

4.4). S. marcescens IBRL USM84 required pH 7.0 to produce the highest pigment production (3.31 µg/mL) and antibacterial activity (37.78 U/mL). Samrot et al., (2011) and Gulani et al., (2012) also found that the maximal amount of prodigiosin pigment were produced by S. marcescens SU-10 and S. marcescens at pH 7.0, respectively.

The pH below and above 7.0 had reduced the pigment production and antibacterial activity of this strain drastically. Krishna, (2008) reported that high acidic and alkaline pH may have inhibitory effect on pigment production. In extreme acidic pH, the protonation of pyrrole group occurs on one of the carbon atoms of the second position in the ring and not on nitrogen atom and therefore become nonaromatic (Struchkova et al., 1973). This protonation causes fading in prodigiosin colour. In extreme alkaline pH, the OH- group deprotonated amine group in the structure forming anion. Both conditions lead to the destruction of the highly conjugated system of double bonds and therefore, responsible for the degradation of the pigment, especially in alkaline pH where the rate of reduction is faster (Namazkar et al., 2013). Hence, pH 7.0 was 106

chosen as the best pH for isolate S. marcescens IBRL USM84. The results showed the cells still can grow at the pH below and above 7.0, but the prodigiosin production decreased due to the destruction and degradation of the pigment.

4 45 E 40 3.5 D 3 35 30 2.5 25 2 20 1.5 C

15 Growth (g/L) Growth 1 B 10

Prodigiosin (µg/mL), Prodigiosin 0.5 5 A 0 0

5 6 7 8 9 (U/mL) Activity Antibacterial Initial pH of medium

Prodigiosin (ug/ml) Growth (g/L) Antibacterial Activity (U/ml)

Figure 4.4: Effect of initial pH of medium on prodigiosin production, antibacterial activity and growth of S. marcescens IBRL USM84 [MB; 2% v/v inoculums of 17 hours old culture (1 x 109 cells/mL); 0.3% of agar; temperature 25oC; 120 rpm of agitation speed; incubation period 48 hours in light condition; results were average of triplicates ± SD]

pH is undeniably one of the important factors in buffering the medium to enhance pigment production (Darah et al., 2014). Tortora et al., (2007) agreed that the activity of the enzymes was hugely influenced by pH. Solieve et al., (2011) also stated that the enzymatic condensation of 2-methyl-3-n-amyl-pyrrole (MAP) and 4-methoxy-

2,2-bipyrrole-5-carbaldehyde (MBC) precursors was initiated at pH 7.0 during prodigiosin biosynthesis. Furthermore, the drastic changes of pH can affect the function of amino acid (proline) responsible in inducing the prodigiosin biosynthesis process by blocking the pigment synthesis (Bharmal et al., 2012). Another factor for S.

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marcescens IBRL USM84 had optimum pH 7.0 was because this isolate was previously isolated from marine environment with the pH of seawater close to neutral pH, ranging from 7.2 to 7.6 (Darah et al., 2014). Therefore this strain required neutral pH for the pigment production.

4.3.1 (d) Effect of temperature

The effect of incubation temperature on prodigiosin production by S. marcescens IBRL USM84 was significant (p< 0.05). A narrow range of incubation temperature was shown to obtain a relatively good pigment production and antibacterial activity (Figure 4.5). The highest production was at temperature of 25oC

(3.31 µg/mL), followed by temperature 20oC (1.56 µg/mL). However, the pigment production was dropped drastically when temperature achieved at 30oc and above.

Hence, the incubation temperature of 25oC was selected as optimum temperature. This finding was supported by Gulani et al., (2012) where the prodigiosin production was maximum at 25oC, whereas, the production was completely blocked when temperature was 35oC and above. Williams, (1973) also stated that maximal pigment production by

S. marcescens was between 24 and 28 oC. Darah et al., (2014) also reported the S. marcescens IBRL USM84 preferred to grow and produce redder color of prodigiosin at 25oC of incubation temperature. Besides, the bacterial growth was detected in broader range of temperature (20 to 40 oC) compared to the range of temperature for pigment production (20 to 30 oC). According to the previous study, the white culture of Serratia marcescens exhibited complete block of pigment production when incubated at 42oC. Nevertheless, the pigment production was re-synthesized when the culture was incubated at 28oC. However, the culture was still viable when temperature reached 42oC (Giri et al., 2004). 108

4 C 45 3.5 40 3 35 30 2.5 25 2 B 20 1.5

15 Growth (g/L) Growth 1 10

Prodigiosin (µg/mL), Prodigiosin 0.5 5 A A A

0 0 Antibacterial Activity (U/mL) Activity Antibacterial 20 25 30 35 40 Temperature (°c) Prodigiosin (ug/ml) Growth (g/L) Antibacterial Activity (U/ml)

Figure 4.5: Effect of temperature on prodigiosin production, antibacterial activity and growth of S. marcescens IBRL USM84 [MB (pH 7.0); 2% v/v inoculums of 17 hours old culture (1 x 109 cells/mL); 0.3% of agar; 120 rpm of agitation speed; incubation period 48 hours in light condition; results were average of triplicates ± SD]

Xu et al., (2014) separated and identified 16 proteins after incubating S. marcescens JNB5-1 at 28oC and 37oC. Among them, O-methyl transferase and oxidoreductase which acted as biosynthesis enzymes of prodigiosin and proteins related with the precursor substances involved in prodigiosin biosynthesis including proline, methionine, serine, 2-Octenal and Malonyl-CoA were obviously down- expressed at 37oC where the levels of mRNA transcriptional of O-methyl transferase, oxidoreductase and transketolase have decreased compared to 28oC. They concluded that S. marcescens JNB5-1 could produce prodigiosin at lower temperature and was blocked at higher temperature. Higher temperature may have inhibitory effect on the expression of enzymes related with prodigiosin biosynthesis.

Figure 4.6 shows the color changes of MB-cultivated strain incubated for 48 hours at various temperatures. The color of MB changed to red at temperatures

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ranging from 20oC to 25oC.The color of MB turned yellowish orange at temperature of

30oC and above. This condition showed that the production of pigment by S. marcescens IBRL USM84 was temperature dependent.

o o o o 20oC 25 C 30 C 35 C 40 C

Figure 4.6: Marine Broth after cultivated with S. marcescens IBRL USM84 for 48 hours at 120 rpm and at different incubation temperature

4.3.1 (e) Effect of agitation speed

Agitation is one of the key factors in enhancing the prodigiosin production via submerged fermentation process. Variety in agitation or shaking would affect the cell growth, antibacterial activity and also had significant effect on prodigiosin production (p< 0.05) (Figure 4.7). S. marcescens IBRL USM84 required 150 rpm of agitation speed to produce the highest pigment production (5.27 µg/mL) and antibacterial activity (41.56 U/mL). The results revealed for both higher and lower agitation speeds than 150 rpm had affected the prodigiosin production.

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6 E 45 40 5 D 35 4 C 30 25 3 B A 20 2 15 Growth (g/L) Growth 10 1 Prodigiosin (µg/mL), Prodigiosin 5 0 0

0 50 100 150 200 (U/mL) Activity Antibacterial Agitation Speed (rpm)

Prodigiosin (ug/mll) Growth (g/L) Antibacterial Activity (U/ml)

Figure 4.7: Effect of agitation speed on prodigiosin production, antibacterial activity and growth of S. marcescens IBRL USM84 [MB (pH 7.0); 2% v/v inoculums of 17 hours old culture (1 x 109 cells/mL); 0.3% of agar; temperature 25oC ; incubation period 48 hours in light condition; results were average of triplicates ± SD]

At a static condition where the oxygen supply was limited, S. marcescens

IBRL USM84 still can produce low amount of antibacterial activity (13.11 U/mL).

Figure 4.8 displays the color changes of MB after being cultivated with the strain for

48 hours at different agitation speeds (Figure 4.8 A). However, the cultivation at the static condition indicated the cultivation medium was obviously separated into double layers (Figure 4.8 B). The double layer formed is because of the hydrophobic nature of prodigiosin, which is due to the resonance of its functional group electrons, non-polar dipole moment and also insoluble in water (Namazkar & Ahmad, 2013). S. marcescens IBRL USM84 preferred to produce the red pigment at the upper layer compared to the bottom layer. The accumulation of the bacterial culture at the upper layer was due to higher level of dissolved oxygen at the surface of medium. This has proven that S. marcescens IBRL USM84 is a facultative anaerobic bacterium where they can survive with less oxygen tension (Darah et al., 2014). Shieh et al., (2003) also

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reported some of the anaerobic and facultative anaerobic bacteria which produced antibacterial red pigment exhibited this kind of conditions. Other researchers also reported the same findings but with different species such as Vibrio ruber (Wan

Norhana & Darah, 2005), S. marcescens (Soliev et al., 2011), Streptomyces coelicolor

(Sevcikova & Kormanec, 2004) and Streptomyces lividan (Rossa et al., 2002).

Static Condition (0 rpm) Upper Layer

0 rpm 50 rpm 100 rpm 150 rpm 200 rpm Bottom layer Figure 4.8: Marine broth after cultivated with S. marcescens IBRL USM84 for 48 hours at 25oC (A) Agitated at different agitation speeds (B) Double layer formed at static condition

Besides, prodigiosin production was found to directly decrease with the agitation speed deceleration from the optimal speed level. The low levels of dissolved oxygen in cultivation medium might be the reason for this condition. Pansuriya &

Singhal, (2011) also reported that incomplete mixing and oxygen transfer might be the main factor of inferior production at lower agitation speeds. Thus, mixing through agitation is very important for aeration and nutrient transfer rate in production of secondary metabolite.

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Cell growth was also influenced by agitation speed. The cell growth increased from 0 rpm (static condition) until 150 rpm (optimal agitation speed), and then slightly dropped in its activity at higher agitation speed than the optimal speed level. Darah et al., (2013a) described that insufficient mixing of the medium cultivation at lower agitation speeds may lead to the later stages of growth, which then affected the secondary metabolite synthesis. Higher agitation speeds would cause shearing effect on the cells and this condition also could be involved in disrupting the synthesis of secondary metabolite. Apart from that, agitation speeds can also affect the morphology of microorganisms which eventually influence secondary metabolite production and growth of the microorganisms (Darah et al., 2011).

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

Figure 4.9 represents the effect of the addition of agar into the medium on the growth, antibacterial activity and prodigiosin production (p< 0.05). The amount of prodigiosin production and antibacterial activity increased as the amount of agar increased, and then dropped after achieving its maximal production. The addition of

0.2% of agar was enough to support the growth of S. marcescens IBRL USM84 by producing the highest yield of prodigiosin (6.94 µg/mL) in a semi-solid condition. The cell growth also showed a similar growth pattern with prodigiosin production and antibacterial activity. Nevertheless, the growth production was slightly decreased after achieving the highest growth production (0.71 g/L) at 0.2% of agar. Hence, the addition of 0.2% of agar into the medium was selected as the best condition for cultivation medium.

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8 50 7 E 45 40 6 35 5 D 30 4 C 25 3 B 20 15 Growth (g/L) Growth 2 A 10

Prodigiosin (µg/mL), Prodigiosin 1 5 0 0

0 0.1 0.2 0.3 0.4 (U/mL) Activity Antibacterial % of agar

Prodigiosin (ug/ml) Growth (g/L) Antibacterial Activity (U/ml)

Figure 4.9: Effect of agar on prodigiosin production, antibacterial activity and growth of S. marcescens IBRL USM84 [MB (pH 7.0); 2% v/v inoculums of 17 hours old culture (1 x 109 cells/mL); temperature 25oC ; agitation 150 rpm; incubation period 48 hours in light condition; results were average of triplicates ± SD]

S. marcescens IBRL USM84 was found to be a facultative anaerobic bacterium. It was previously isolated from a surface of the marine sponge Xetospongia testudinaria. Thus, this strain required a semi-solid agar condition to grow and for the bioactivity production. According to Darah et al., (2014), the cells of S. marcencens

IBRL USM84 still can grow and synthesized prodigiosin pigment in the broth medium

(no agar added) but its more inclined to the semi-solid medium where it mimicks the exact condition of its origin habitat. Besides, this semi-solid medium suggested that the motile bacterial cells require a substance to adhere or to attach in order to grow well and producing prodigiosin effectively. Lemos et al., (1985) and Anand et al.,

(2006) reported that the bacterial strains isolated from the marine organisms such as corals, sponges and seaweeds had recorded higher antibiotic production rates compared to the free living bacteria that live in the marine environment. Furthermore,

Gerardo et al., (2006) who found some of the symbiotic microbes in marine

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invertebrates can significantly impact the ecosystem functioning by altering the phenotype of their hosts. Hentschel et al., (2001) also stated that the sponges can host several marine microorganisms included heterotrophic bacteria, archaeabacteria, cyanobacteria and unicellular algae. Besides that, bacteria and algae mainly play a vital role in nutrition absorption and metabolic transport in sponges as described by

Wilkinson & Garrone, (1980). S. marcescens IBRL USM84 also has capability in producing the significant secondary metabolites since it was isolated from sponge- associated microorganisms.

4.3.1 (g) Comparison of the growth, antibacterial activity and prodigiosin production before and after enhancement for physical parameter

After the enhancement of the various physical parameters, a time-course study was conducted to perceive the cumulative effect of various parameters that had a positive effect on the prodigiosin production, antibacterial activity and growth yield.

The improved physical parameters had been incorporated, and samples were taken at

8-hour intervals. Figure 4.10 shows the results of the time-course study before and after enhancements. The optimal incubation temperature for the prodigiosin production was retained at 25oC in the light illumination exposure for 48 hours even after enhancement. Other parameters had also changed after undergoing enhancement process as shown in Table 4.1.

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8 45 J 7 40 I 35 6 H G 30 5 F E 25 4 D 20 3

Growth (g/L) Growth 15 2 Prodigiosin (µg/mL), Prodigiosin C 10 1 B 5 A (U/mL), Activity Antibacterial 0 0 0 8 16 24 32 40 48 56 64 72 Culture Duration (Hours) Prodigiosin- before optimum condition (ug/ml) Prodigiosin-after optimum condition (ug/ml) Growth- before optimum condition (g/L) Growth- after optimum condition (g/L) Antibacterial Activity- before optimum condition (U/ml) Antibacterial Activity- after optimum condition (U/ml) Figure 4.10: Profile of growth, prodigiosin production and antibacterial activity of S. marcescens IBRL USM84 before and after physical parameter enhancements [MB (pH 7.0); 2% v/v inoculums of 17 hours old culture (1 x 109 cells/mL); 0.2% of agar; temperature 25oC; agitation 150 rpm in light condition; results were average of triplicates ± SD]

Table 4.1: The summary of the culture condition before and after enhancements

CHARACTERISTICS BEFORE AFTER

Incubation Time 48 hours 48 hours

Light Exposure Light Light

Initial pH of medium 7.5 7.0

Incubation Temperature 25oC 25oC

Agitation Speed 120 rpm 150 rpm

Percentage of Agar 0.3% 0.2%

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The highest prodigiosin production, antibacterial activity and growth yield were achieved at 48 hours of incubation time. The percentage of increment achieved after physical parameters enhancement was 151.81% (2.76 µg/mL before and 6.95

µg/mL after enhancements) for prodigiosin production, 23.34% (34.45 U/mL before and 42.49 U/mL after enhancements) for antibacterial activity and 33.87% (0.62 g/L before and 0.83 g/L after enhancements) for growth yield. Darah et al., (2011) also reported the secondary metabolite production increased from 1.096 U/mL to 2.81

U/mL, approximately 156.40% increment after physical parameter improvements.

4.3.1 (h) Effect of carbon sources

The cell growth, antibacterial activity and prodigiosin production were significantly affected (p< 0.05) by the addition of different carbon sources into the cultivation medium (Figure 4.11). The highest amount of prodigiosin (42.05 µg/mL) was obtained when the cultivation medium was supplemented with maltose, followed by slight decrease of pigment production in medium supplemented with sucrose and fructose. Surprisingly, the presence of maltose showed a fifteen fold increment in prodigiosin production compared to the existing media. Hence, maltose was selected as the best carbon source for the isolate S. marcescens IBRL USM84. Gulani et al.,

(2012) also found that the highest yield of prodigiosin production was observed when maltose was added into the cultivation medium, while moderate amount of prodigiosin was achieved by replacing it with sucrose and fructose. Sundaramoorthy et al., (2009) also stated the maximal yield of prodigiosin produced by S. marcescens was achieved when maltose was amended in the medium whereas the pigment production decreased after amended with sucrose and lactose.

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45 G 60 40 F 50 35 30 E 40 25 30 20 D 15 20 Growth (g/L) Growth 10 10 Prodigiosin (µg/mL), Prodigiosin 5 A B C 0 0

Glucose Sucrose Lactose Maltose Fructose Inositol Starch (U/mL) Activity Antibacterial Carbon Sources (1%)

Prodigiosin (ug/ml) Growth (g/L) Antibacterial Activity (U/ml)

Figure 4.11: Effect of carbon sources on prodigiosin production, antibacterial activity and growth of S. marcescens IBRL USM84 [MB (pH 7.0); 2% v/v inoculums of 17 hours old culture (1 x 109 cells/mL); 0.2% of agar; temperature 25oC; agitation 150 rpm; incubation period 48 hours in light condition; results were average of triplicates ± SD]

However, the prodigiosin production decreased drastically when the cultivation medium was supplemented with glucose, lactose or starch. It seems that these carbon sources (glucose, lactose and starch) may have repressive effect on pigment production. Previous study also reported that S. marcescens was observed to produce least prodigiosin when carbohydrate was incorporated in the cultivation medium. Clements-Jewery, (1976) reported the reduction of prodigiosin production when glucose was added into the cultivation medium. Besides, Oller, (2005) and

Samrot et al., (2011) demonstrated that glucose or lactose resulted in the decrease of the prodigiosin production when incorporated in the cultivation medium. This may be due to the role of glucose and lactose as suppressors in prodigiosin production.

Furthermore, S. marcescens exhibited an ability to utilize glucose, and then produced the glucose-6-phosphate dehydrogenase alloenzyme which resulted in a repressive effect on the prodigiosin synthesis pathway (Gargallo et al., 1987).

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Figure 4.12 illustrates the flask containing MB with different carbon sources after being cultivated with S. marcescens IBRL USM84 for 48 hours. The MB color changed from pale yellow to blood-red when the broth was supplemented with maltose, sucrose and fructose. The color changes indicated that high pigment concentration was present in MB. The red color decreased in the medium amended with inositol, lactose and starch while no red color was present in the medium amended with glucose. The pigment extract from isolate cultivated in medium supplemented with maltose, sucrose and fructose also exhibited dark red color (Figure

4.13).

Glucose Starch Lactose Inositol Fructose Maltose Sucrose

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

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Glucose Starch Lactose Inositol Maltose Fructose Sucrose Control

Figure 4.13: Pigment extract in 2-propanol obtained after cultivation of S. marcescens IBRL USM84 in various carbon source

4.3.1 (i) Effect of nitrogen sources

The effect of nitrogen sources on prodigiosin production, antibacterial activity and the growth of S. marcescens IBRL USM84is shown in Figure 4.14. Based on the prodigiosin production, the highest yield was obtained in medium that was supplemented with urea (14.58 µg/mL). The medium containing urea produced the blood-red colour indicating the presence of prodigiosin pigment. However, the yield of prodigiosin pigment in the medium supplemented with urea is still lower than the medium without the addition of nitrogen sources (42.05 µg/mL) as described in Figure

4.11. Ramani et al., (2014) reported that cultivation medium added with urea showed decrease in prodigiosin production by S. marcescens. Hejazi & Falkiner, (1997) also reported that pigmentation of prodigiosin was delayed when medium amended with urea and it may be due to the alkaline conditions in the cultivation medium due to the release of ammonium salts. The alkaline condition is not suitable for prodigiosin production and would reduce the bioactivity of S. marcescens IBRL USM84 as explained in Section 4.3.1 (c).

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16 35 E 14 30 12 25 10 20 8 15 6

Growth (g/L) Growth 10

4 D Prodigiosin (µg/mL), Prodigiosin 2 C 5

B A A B Antibacterial Activity (U/mL) Activity Antibacterial

0 0

Urea

Casein

Peptone

Tryptone

Yeast Extract Yeast

Ammonium Acetate Ammonium Ammonium Oxalate Ammonium Nitrogen Sources (1%)

Prodigiosin (ug/ml) Growth (g/L) Antibacterial Activity (U/ml)

Figure 4.14: Effect of nitrogen sources on prodigiosin production, antibacterial activity and growth of S. marcescens IBRL USM84 [MB (pH 7.0); 2% v/v inoculums of 17 hours old culture (1 x 109 cells/mL); 0.2% of agar; temperature 25oC; agitation 150 rpm; incubation period 48 hours in light condition; 1% of maltose; results were average of triplicates ± SD]

The cultivation medium turned to pale pink coloration when added with ammonium oxalate, whereas no red pigment was present in the medium amended with tryptone, yeast extract, peptone, ammonium acetate and casein as shown in Figure

4.15. This result showed that some of nitrogen sources including tryptone, yeast extract, peptone, ammonium acetate and casein may have inhibitory effect on pigment production by S. marcescens IBRL USM84. However, Gulani et al., (2012) found that their S. marcescens preferred to grow well and produced more prodigiosin pigment in the medium supplemented with peptone compared to urea.

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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

Increased production of prodigiosin may be caused by natural components of the peptone or yeast extracts that supplied nitrogen source to the medium, simultaneously gave support to the bacterial biomass. Calcium and magnesium were also present as trace elements, providing the optimum condition for the biosynthesis of prodigiosin (Frank et al., 1997). Moreover, the production of prodigiosin declined when the concentrations of nitrogen source was below the optimum concentration value. Similarly, the production of prodigiosin decreased when the concentrations of nitrogen source was above the optimum concentration value. The change of carbon or nitrogen ratio in cultivation medium affected the pathways of secondary metabolites, particularly those responsible for the production of prodigiosin as described by (Kim et al., 1999).

In this study, the addition of nitrogen sources into the cultivation medium did not show positive enhancement in prodigiosin production. The nitrogen sources that were incorporated in Marine broth with approximate amount per Liter of 0.5% peptone and 0.1% of yeast extract were already sufficient to support the prodigiosin production. It seems that higher concentrations of peptone and yeast extract, could inhibit the synthesis of prodigiosin in S. marcescens IBRL USM84.

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4.3.1 (j) Effect of inorganic salt

Figure 4.16 shows no positive effect in addition of NaCl into the cultivation medium (Marine Broth, MB) in prodigiosin production. The highest prodigiosin production (42.12 µg/mL) was obtained when there was no addition of NaCl (0%).

The pigment production decreased from 42.12 µg/mL to 0.02 µg/ml when the addition of NaCl was increased from 0% to 4%. The effect was significant for prodigiosin production except for 3% (w/v) and 4% (w/v) of NaCl. The cultivation medium became colorless when 3% and 4% of NaCl was added and it was correlated with the declining antibacterial activity. However, S. marcescens IBRL USM84 still can grow and tolerate up to 4% of NaCl. Microorganisms able to grow and proliferate in different salt concentrations and also could be divided into two groups whether salt tolerant or salt requirement (Larsen, 1962; Kushner, 1978).

There are several categories of halotolerant microorganisms that can be used to classify the microbes according to salt tolerant. Non-tolerant are those microbes that tolerate only a small concentration of NaCl about 1% (w/v). Slightly and moderately tolerant are those microbes that tolerate from zero up to 6-8% and 18-20 % NaCl, respectively while extremely tolerant can grow from zero up to saturation of salt

(Larsen, 1986). The results indicated that no extra addition of NaCl was needed to increase prodigiosin production. The 1.945% of NaCl that was incorporated in MB formulation was enough to enhance the prodigiosin production by S. marcescens IBRL

USM84. However, Allen et al., (1983) reported that S. marcescens strain needs NaCl to grow and synthesize the red pigment prodigiosin. S. marcescens IBRL USM84 was a slightly tolerant bacterium and it can tolerate up to 5.945% (1.945% + 4%) of NaCl.

This marine isolate preferred low concentration of NaCl to produce the maximal pigment production. Kushwaha et al., (1982) also found Haloferax sp., a halotolerant

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bacterium that still required low salinities for pigmentation and became colorless at high salinities. In contrast, some halophilic microbes included S. marcescens KH1R

KM035849 (Vora et al., 2014) and Halobacterium (Kushner, 1993) required higher salinities to enhance the red pigmentation.

45 G 60 40 F 50 35 E 30 D 40 25 30 20 C 15 20 Growth (g/L) Growth 10 B 10 Prodigiosin (µg/mL), Prodigiosin 5 A A 0 0

0 0.25 0.5 1 1.5 2 3 4 (U/mL) Activity Antibacterial % of NaCl

Prodigiosin (ug/ml) Growth (g/L) Antibacterial Activity (U/ml)

Figure 4.16: Effect of inorganic salt on prodigiosin production, antibacterial activity and growth of S. marcescens IBRL USM84 [MB (pH 7.0); 2% v/v inoculums of 17 hours old culture (1 x 109 cells/mL); 0.2% of agar; temperature 25oC; agitation 150 rpm; incubation period 48 hours in light condition; 1% of maltose; results were average of triplicates ± SD]

Inorganic salt plays a vital role in physiology and growth of organism.

Previous research conducted by Suryawanshi et al., (2014) found that no significant increase or inhibition in prodigiosin production of S. marcescens was found with different concentrations of sodium and potassium chloride. However, the concentration of these salts beyond 2.0 g/L (w/v) resulted in inhibitory effect on pigment production.

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4.3.1 (k) Effect of percentage of maltose

Since maltose was the best carbon source for S. marcescens IBRL USM84, the optimal percentage of maltose was then determined in enhancing the maximal prodigiosin production. In order to study the effect of different concentrations of maltose on prodigiosin pigment production, 0% to 3% (w/v) of maltose was separately added into the cultivation medium (MB). The effect was significant for prodigiosin production except for 2.5% (w/v) and 3% (w/v) of maltose. Maltose concentration at

1% was found optimal for the production of prodigiosin by S. marcescens IBRL

USM84 (42.28 µg/mL) (Figure 4.17). Followed by 1.5 and 0.5% concentrations of maltose with the production of about 38.43 µg/mL and 36.39 µg/mL, respectively of prodigiosin. At 1.5% of maltose and above, the prodigiosin production decreased as the percentage of maltose increased. The reduction in prodigiosin production by S. marcescens which is known as catabolic repression could happen in high concentration of maltose due to low pH value of the cultivation medium (Williamson et al., 2006a). Martin & Demain, (1980) stated that in most of the secondary metabolite pathways, carbon plays a crucial role in controlling the enzyme activities in most of the secondary metabolite pathways. Therefore, maltose concentration had an effect in secondary metabolite production by S. marcescens IBRL USM84. Sole et al.,

(1997) found that maltose and fructose were blocking the secondary metabolite pathway. A study by Sole et al., (1994) also reported that low pH medium affected prodigiosin production by non-proliferating cells of S. marcescens.

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45 F 60 40 E D 50 35 C B B 30 40 25 30 20 15 20 Growth (g/L) Growth 10 A 10 Prodigiosin (µg/mL), Prodigiosin 5 0 0

0 0.5 1 1.5 2 2.5 3 (U/mL) Activity Antibacterial % of Maltose

Prodigiosin (ug/ml) Growth (g/L) Antibacterial Activity (U/ml)

Figure 4.17: Effect of percentage of maltose on prodigiosin production, antibacterial activity and growth of S. marcescens IBRL USM84 [MB (pH 7.0); 2% v/v inoculums of 17 hours old culture (1 x 109 cells/mL); 0.2% of agar; temperature 25oC; agitation 150 rpm; incubation period 48 hours in light condition; results were average of triplicates ± SD]

The color of MB changed after being cultivated with the strain for 48 hours in different concentrations of maltose (Figure 4.18). The color changed to bloody red when the MB was added with 0.5% to 1.5% of maltose. The red color decreased and turned to pale pink when the MB was amended with 2% to 3% of maltose. It might be due to catabolic repression activity on pigment production. The result also showed weak pigmentation in the MB with no addition of maltose (0%).

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Figure 4.18: Marine broth after cultivated with S. marcesens IBRL USM84 for 48 hours at 25oC, and added different concentration of maltose

4.3.1 (l) Comparison of the growth, antibacterial activity and prodigiosin production before and after enhancement for chemical parameter

In this part, the improved physical and chemical parameters were incorporated, and a time-course study was conducted to perceive the cumulative effect of various parameters that had a positive effect on the prodigiosin production, antibacterial activity and growth yield. Samples were taken at 8-hour intervals during

72 hours cultivation period. Figure 4.19 shows the results of the time-course study before and after enhancement. There was no addition of nitrogen source and inorganic salt even after enhancement. However, the addition of carbon source gave a big effect in enhancing the bioactivity of S. marcescens IBRL USM84 and the summary of the improvement in cultivation medium is as shown in Table 4.2. The highest prodigiosin production, antibacterial activity and growth yield was achieved and retained at 48 hours of incubation time. The percentage of increment achieved after chemical parameters enhancement was 1444.57% (2.76 µg/ml before and 42.63 µg/ml after enhancements) for prodigiosin production 43.25% (34.45 U/ml before and 49.35 U/ml

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after enhancements) antibacterial activity and 141.94% (0.62 g/L before and 1.50 g/L after enhancements) growth yield.

45 J 60 I H 40 G 50 35 F

30 40

25 30 20

E Growth (g/L) Growth

15 20 Prodigiosin (µg/mL), Prodigiosin

D 10 Antibacterial Activity (U/mL) Activity Antibacterial 5 B C A 0 0 0 8 16 24 32 40 48 56 64 72 Culture Duration (Hours) Prodigiosin- before optimization (ug/ml) Prodigiosin- after optimization (ug/ml) Growth- before optimization (g/L) Growth- after optimization (g/L) Antibacterial Activity- before optimization (U/ml) Antibacterial Activity- after optimization (U/ml)

Figure 4.19: Profile of growth, prodigiosin production and antibacterial activity of S. marcescens IBRL USM84 before and after chemical parameter enhancements

[MB (pH 7.0); 2% v/v inoculums of 17 hours old culture (1 x 109 cells/mL); 0.2% of agar; temperature 25oC; agitation 150 rpm in light condition; 1% of maltose; results were average of triplicates ± SD]

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Table 4.2: The summary of the medium improvement before and after enhancements

CHARACTERISTICS BEFORE AFTER

Carbon Source Not added Maltose

Nitrogen Source Not added Not added

Inorganic salt Not added Not added

Percentage of Maltose 0% 1%

The results revealed that the cultivation medium with addition of maltose consistently yielded a fifteen fold increment in the production of prodigiosin. In contrast, Giri et al., (2004) reported that the addition of maltose to nutrient broth does not support prodigiosin production but, the addition of fatty acid as a carbon source played an important role in enhancing the prodigiosin production and cell growth. The increment of prodigiosin production had achieved a forty fold increase when S. marcescens was cultivated in the powdered peanut seed broth that contained fatty acid compared to existing medium.

4.4 Conclusion

The production of prodigiosin produced by S. marcescens IBRL USM84 had achieved 15-fold increase after the enhancement process from 2.76 µg/mL to 42.63

µg/mL. The best incubation condition was achieved when 2 % (v/v) of inoculums (1 x

109 cells/mL) was cultivated in MB with pH 7.0, added with 0.2 % of agar and 1 % of maltose and incubated at a temperature of 25oC in light condition with 150 rpm of agitation speed for 48 hours.

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CHAPTER 5.0: BIOASSAY ANALYSIS AND CHARACTERIZATION OF

PRODIGIOSIN PIGMENT IN PARTITIONATED EXTRACT OF Serratia marcescens IBRL USM84

5.1 Introduction

Various compounds exist in the crude extract. Therefore, a preliminary separation is needed to separate the compounds. Solvent-solvent partitioning method is the most popular technique used and is achieved by separating the pigments between immiscible solvents. The color, yield, UV/vis absorption spectrum and antibacterial activity of each partition were then evaluated. This preliminary separation of the crude extract is required before proceeding to the analytical methods which includes thin layer chromatography (TLC), bioautography, column chromatography

(CC) and ultra performance liquid chromatography (UPLC) techniques (Chapter 6) to detect and identify any bioactive compounds in the crude extract. Thus, it was impossible to resolve a mixture of all compounds on a single chromatography separation.

In this chapter, the separation of pigments produced by S. marcescens IBRL

USM84 using solvent-solvent partitioning method was performed. Dichloromethane partitionated extract was selected since this partitionated extract showed a notable antibacterial activity compared to the other partitionated extracts. The bioassay analysis that was carried out for the dichloromethane partitionated extract included the disc diffusion assay, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time kill study. Besides, the characterization and stability of the pigment produced by S. marcescens IBRL USM84 on pH, temperature, time and light were also investigated.

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5.2 Materials and Methods

5.2.1 Solvent-solvent partitioning

Solvent-solvent partitioning was done following a method by Kupchan &

Tsou, (1973) and Sarkar et al., (2006) with slight modification. This method was divided into two phases, namely phase I and phase II. The phase I involved the retrieval of crude extract from bacteria, while, in phase II, the methanol solution was prepared which was later partitionated by four solvents with different polarities. For phase I, S. marcescens IBRL USM84 was cultivated in MB (pH 7.0) under submerged fermentation condition in the best condition obtained after enhancement process. The cultivation medium (MB) was added with 0.2% of agar and 1% of maltose and incubated at 25oC and 150 rpm for 48 hours in the light condition as stated in Chapter

4.0 (Section 4.3.1 l). The culture was then centrifuged at 4000 rpm for 20 minutes at

4oC after 2 days of cultivation period to separate the cells and supernatant. According to Slater et al., (2003), acidified 2-propanol (polarity index: 3.9) was used to extract intracellular pigments (from cells) with slight modification. The extraction of pigment from the cell was done as described in Chapter 3.0 (Section 3.2.2 b). After the pigment was extracted twice using acidified 2-propanol, the extract was concentrated using a rotary evaporator and was then poured into a glass Petri dish. The extracted pigment was dried in the fume hood until the pigment paste formed. The dried paste was weighed until a constant weight was obtained and expressed in gram per litre (g/L).

For phase II, the dried paste was re-dissolved in methanol, followed by solvent-solvent partitioning using four different polarities of solvents (Tong, 2014) to obtain four different extracts i.e. hexane, dichloromethane, ethyl acetate and butanol extracts. Initially, the crude extract was partitioned with hexane in a ratio of 1:1

(volume-to-volume). Water was added to form a non-polar upper phase. The organic

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layer was taken and concentrated, while the aqueous phase was re-partitioned with dichloromethane, ethyl acetate and butanol. All partitions were concentrated using a rotary evaporator before pouring into a glass Petri dish and placed in the fume hood to form a dried paste. Each dried paste was weighed until a constant weight was obtained and expressed in gram per litre. Figure 5.1 summarizes the partitioning steps performed in this study.

5.2.1 (a) Spectrophotometric analysis of partitioned extract

The dried partitionated extracts were re-dissolved in acidified 2-propanol.The maximum absorbance was measured at the wavelength of 300 to 600 nm using UV spectrophotometer (Genesys; Thermoscientific). The acidified 2-propanol was used as a blank. The peaks that appeared were compared with the peak of the prodigiosin standard (Sigma-Aldrich) as described by Kulkarni et al., (2012).

5.2.1 (b) Susceptibility test of partitionated extract

The antibacterial susceptibility test against the selected test bacteria was carried out using a disc diffusion assay (Section 3.2.2 e). A total of 20 mg of the dried pigments from each partitionation was scraped and transferred into a 2.0 mL

Eppendorf tube, respectively. The partitionated pigment extracts were re-dissolved in

400 µL of methanol (Qrec) and were thoroughly vortexed to completely dissolve the paste.

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Culture broth (48 hours old)

Centrifuged (4000 rpm, 4oC, 45 minutes)

Pellet (intracellular) Supernatant (extracellular) Acidified 2- propanol added Phase I Discard (Extraction of crude pigment) 2-propanol extract Dried using rotary

evaporator

2-propanol extract paste ______+ Methanol (100 mL) _ _ _ _ _

Methanolic extract

Phase II + Hexane (100 mL) and (Partitioning) distilled water (100 mL)

Aqueous Hexane extract

+ Dichloromethane (100 mL)

Aqueous Dichloromethane extract

+ Ethyl acetate (100 mL)

Aqueous Ethyl acetate extract

+ Butanol (100 mL)

Aqueous Butanol extract

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

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5.2.1 (c) Antibacterial activity using broth micro dilution assay

The values of Minimum inhibitory concentration (MIC) values against five susceptible test bacteria were evaluated by using broth micro-dilution method according to Wiegand et al., (2008). While, double strength Mueller Hinton Broth (Hi- media) was used to cultivate the test bacteria.

The initial stock concentration of dichloromethane partition extract was prepared at 8000 µg/mL by dissolving the paste in 50 µL of methanol and 950µL of

MHB (5% of methanol). Two fold serial dilutions were carried out in MHB to dilute the stock solution to a final volume of 31.25 µg/mL. All stock solutions were diluted and prepared according to Table 5.1. Next, 0.1 mL of each concentration of solution was pipetted to microtitre well containing 0.1 mL of bacterial suspension. Each concentration was tested in triplicates.

Bacterial suspensions were prepared by adjusting its turbidity at 0.5

McFarland standard equivalents to 1 x 108 CFU/mL and were then subjected to dilution (1:1000) to obtain intermediate inoculum suspension (1 x 106 CFU/mL) by adding 50 µL of the bacterial suspension into 4950 µL as an inoculum stock. Next, 100

µL of the intermediate inoculum was added to the 100 µL of the extract previously loaded into the 96-well polypropylene microtitre plate to give a final inoculum concentration of 5 x 105 CFU/mL.

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Table 5.1: Scheme for preparing dilution series of moderate water soluble extract to be used in MIC assay

Stock Volume of stock Volume of MHB + Final concentration of (µL) inoculums (µL) concentration crude extract (µg/mL) (µg/mL) 8000.00 100 100 4000.00

4000.00 100 100 2000.00

2000.00 100 100 1000.00

1000.00 100 100 500.00

500.00 100 100 250.00

250.00 100 100 125.00

125.00 100 100 62.50

62.50 100 100 31.25

31.25 100 100 15.63

The MIC assay was aseptically performed using sterile 96-well polypropylene microtitre plates. At first, 100 µL of extract of each dilution was loaded into different wells, followed by the addition of 100 µL of bacterial suspensions into each well. The microtitre plate was then sealed with parafilm and incubated at 37oC for 16 to 20 hours. All tests were performed in triplicates. The well containing MHB and inoculum

(with 5% of solvent) served as a negative control also referred to as a growth control.

The well containing MHB and MHB with extract (both were uninoculated with bacterial inoculums) served as positive controls. The color reference set (MHB with extract) also prepared as a color reference since the extract used in this study was colored.

The MIC value is the lowest concentration of extract that completely inhibit the growth of microorganism in the well (no turbidity), as detected by naked eye. The

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value of MIC was measured by comparing the turbidity of the whole series of the wells with two positive controls (MHB with extract and MHB without extract) at visual reading.

5.2.1 (d) Minimum Bactericidal Concentration (MBC) assay

MBC assay was carried out after MIC assay. The MBC assay was done by subculturing a loop full of aliquots from wells with no apparent growth (no turbidity) and the last well with apparent growth (presence of turbidity) on Nutrient agar (NA) plates. Then, the plates were incubated at 37oC for 24 hours. MBC values were taken based on the lowest concentration of extracts yielding ≥ 99.99 % of growth reduction

(Darah et al., 2013).

5.2.2 Time kill study

Kill curve study was performed according to the methods in CLSI protocol

(CLSI, 2006) and Kyaw & Lim (2012). The study was aimed to determine the effect of prodigiosin extract towards the growth profile of two bacteria, namely MRSA

(Gram positive bacteria) and A. anitratus (Gram negative bacteria). The effect of the extract was tested at three different concentrations i.e.0.500 mg/mL (2 MIC), 0.250 mg/mL (1 MIC) and 0.125 mg/mL (half MIC). Stock concentrations of extract for all

MIC values were prepared at 50.00 mg/mL, 25.00 mg/mL and 12.5 mg/mL, respectively. The concentration of stock preparation of the extract was prepared at 100 times higher than the desired concentration with the aim to dilute the concentration of methanol from 100% to 1%.

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A total of 18.9 mL of sterile Mueller-Hinton broth was prepared in four 100 mL conical flasks. Each conical flask was labeled as control, 2 MIC, 1 MIC and half

MIC. About 1.0 mL of prodigiosin extract prepared at different concentrations was pipetted into each conical flask. For control, the prodigiosin extract was replaced with

1.0 mL of Mueller-Hinton broth and also containing only 1% of methanol for final concentration. Then, 100 µL of bacterial inoculum (approximately 1 x 108 CFU/mL) was pipetted into the conical flask. The same steps were repeated for both MRSA and

A. anitratus.

The conical flasks were incubated at 37oC and agitated at 150 rpm for 48 hours. Sampling was done at every 4 hours interval. About 1.0 mL of the sample was withdrawn from each flask at every harvesting time, followed by ten-fold dilution with a sterile saline solution. Viable cell count was performed by culturing the diluted sample on the Nutrient agar plate to determine the colony-forming unit (CFUs). The bactericidal activity (≥ 3 log unit reduction in log 10 CFU/ml) or (≥ 99.9% reduction in bacterial cells), whereas the bacteriostatic activity (< 3 log unit reduction in log 10

CFU/mL) (< 99.9% reduction in bacterial cells) were determined (CLSI, 2006; De

Oliveira et al., 2013).

5.2.3 Physical characterization of prodigiosin in dichloromethane partition of

S. marcescens IBRL USM84

5.2.3 (a) Effect of temperature towards stability of prodigiosin

The dichloromethane partitionated extract of prodigiosin was transferred into nine test tubes and subsequently placed in water bath for 20 minutes at nine different temperatures, from 27, 30, 40, 50, 60, 70, 80, 90 and 100oC (Castillo et al., 2008). The

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thermo stability of the pigment related to the spectrum absorption value recorded at

535 nm and the antibacterial activity of the pigment was monitored. To determine relative absorbance, the absorbance of the pigment incubated at certain temperatures was compared with the absorbance of the control pigment (pigment untreated with any temperature supposed to have 100% pigment content).

The treated pigment was evaluated for its antibacterial activity using a disc diffusion method as described in Chapter 3.0 (Section 3.2.2 e). A total of 20 µL of the treated pigment was initially dispensed on 6.0 mm sterile antibiotic disc (Whatman

AA). The disc was then transferred onto MHA agar containing MRSA. Untreated pigment served as a control. Results of the antibacterial activity were determined by comparing the activity of treated pigment with the untreated pigment (assumed to have

100% of antibacterial activity).

5.2.3 (b) Effect of pH towards stability of prodigiosin

The pH stability measurement was conducted as proposed by Wan Norhana

(2004) with slight modification. The dichloromethane partition extract was treated with different pH values, ranging from pH 3 to pH 9. At first, the pigment solution was equally divided into eight portions and dried. Secondly, the solution was dissolved in buffers with different pH, namely citrate (pH 3), acetate (pH 4-5), phosphate (pH 6-

7), Tris-HCL (pH 8) and glycine-NaOH (pH 9). All treated pigments were re-washed with neutral buffer (pH 7) to ensure the results obtained were not influenced by acidic and alkaline residue after treatment. All solutions were vortexed, mixed and left treated at room temperature (27oC ±2) for 30 minutes. For the spectrum absorption value, the absorbance was recorded at the wavelength of 535 nm for control sample

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and also for samples treated with different buffer. Relative absorbance was determined by comparing the absorbance of the pigment (absorbance of pigment at certain pH) with the control pigment (absorbance of pigment which was not treated with any buffer and considered to have 100% of pigment content).

The antibacterial activity of the pigment was evaluated using disc diffusion method as described in Chapter 3.0 (Section 3.2.2 e). Twenty micro litre of the treated pigment was loaded on sterile antibiotic disc (Whatman AA disc, 6mm) and the disc was placed on MHA agar which was seeded with MRSA previously. Untreated pigment served as a control. Results of the antibacterial activity were expressed as relative antibacterial activity that was determined by comparing the activity of treated pigment with the untreated pigment (considered to have 100% of antibacterial activity).

5.2.3 (c) Effect of light towards stability of prodigiosin

Dichloromethane partitionated extract was prepared in two Universal bottles and was kept at a room temperature (27±2oC). One bottle was fully covered with aluminum foil to avoid a direct contact with light, whereas, another bottle was stored under the light. The absorbance of control (freshly made samples at 0 day) and the samples (covered and uncovered) was recorded daily for one week at the wavelength of 535 nm to obtain the spectrum absorption value. Relative absorbance was also determined by comparing the absorbance of the samples (absorbance of pigments exposed and unexposed to the light during a week) with the control pigment

(absorbance of pigment at 0 day, which was considered to have 100% of pigment content).

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The antibacterial activity of the pigment was evaluated using a similar method as described in Chapter 3.0 (Section 3.2.2 e) where 20 µL of the dichloromethane pigment solution was dispensed on a 6 mm sterile antibiotic disc

(Whatman AA), followed by placing the disc on MHA agar previously grown with

MRSA. The pigment solution before incubation period (0 day) served as a control.

The antibacterial activity of the tested pigment was compared with the control pigment

(considered to have 100% of antibacterial activity) to determine its relative activity.

5.2.3 (d) Effect of incubation time towards stability of prodigiosin

The dichloromethane partition extract was kept at 4oC under dark condition at varying incubation times i.e. 0, 15, 30, 45 and 60 days. Later, the absorbance of pigment was recorded at 535 nm at every sampling. Relative absorbance was determined by comparing the absorbance of pigments at different incubation times with absorbance of control pigment at 0 day (assumed to have 100% of pigment content).

The antibacterial activity of the treated pigment was evaluated using a similar method in Chapter 3.0 (Section 3.2.2 e) where 20 µL of the dichloromethane pigment solution was dispensed on a 6.0 mm sterile antibiotic disc (Whatman AA), followed by placing the disc on MHA agar previously grown with MRSA. The pigment solution before incubation period (0 day) served as a control. The antibacterial activity of the tested pigment was compared with the control pigment (considered to have 100% of antibacterial activity) to determine its relative activity.

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5.2.4 Statistical analysis

The data recorded was expressed as mean value ± standard deviation. The significant differences of the mean data were analyzed using One Way Analysis of

Variance (ANOVA) and were considered as significant at p< 0.05. Statistical analysis was carried out using SPSS version 22.

5.3 Results and Discussion

5.3.1 Solvent-solvent partitioning process

Solvent-solvent partitioning was performed, where the evaluation was done in terms of colour, UV/vis absorption spectrum and also antibacterial property of each partition. Based on the colour property of partitionated extract of S. marcescens IBRL

USM84 (Figure 5.2), dichloromethane partitionated extract yielded strongest and attractive coloration (purplish-pink) compared to other partitionated extracts. The red brown coloration produced by both ethyl acetate and butanol partitionated extract is quite similar. However, no coloration of pigment exists in the hexane partitionated extract, indicating that the pigments in the isolate S. marcescens IBRL USM84 were mid-polar and polar type of compounds. There is a probability that various types of pigmented compounds with different polarity characteristics are present in S. marcescens IBRL USM84. However, same pigment could also be possibly distributed into two solvent partitions. Besides, the type of solvents and the original color of the natural pigment used in the partitioning process may also influence the coloration of the partitionated extracts.

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A B C D

Figure 5.2: Partitionated extract from crude extract of S. marcescens IBRL USM84 extracted with different solvents (A) Hexane, (B) Dichloromethane, (C) Ethyl acetate and (D) Butanol

Table 5.2 shows the efficiency of extraction by various organic solvents in partitioning process. The yields were varied and were influenced by the polarity of the compounds. In this study, the dichloromethane partition (0.50 g/L) gave the highest extraction yield for isolate S. marcescens IBRL USM84 followed by butanol (0.45 g/L), ethyl acetate (0.23 g/L) and hexane (0.05 g/L).

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

Extractant 2-P HEX DCM EA BUT (Crude)

Yield (g/L) 7.02 0.05 0.50 0.23 0.45

2-P = 2-propanol, HEX = hexane, DCM = dichloromethane, EA = ethyl acetate and BUT = butanol

5.3.1 (a) Spectrophotometric analysis of partitionated extract

Table 5.3 presents the maximum absorbance property of pigments from different partitions. The UV/vis property of dichloromethane partitionated extract indicated the exact absorption spectrum of the pigment, which was one of the major peaks (535 nm) appeared in the dichloromethane partition extract, was similar with the peak of prodigiosin standard (Sigma-Aldrich) and crude extract (intracellular) of S. marcescens IBRL USM84 as discussed in Chapter 3.0 (Figure 3.9). Therefore, the results of color, yield and UV/vis property indicated that the pigments present in the crude extract can be effectively extracted with mid-polar solvents, such as dichloromethane, which also revealed that the pigment was mid-polar in nature.

Table 5.3: Absorption spectrum of extracts of S. marcescens IBRL USM84 in different partitionation extracts

Extractant HEX DCM EA BUT

UV/vis peak(*) - 5401 5431 5461

5352 (501)

HEX = hexane, DCM = dichloromethane, EA = ethyl acetate and BUT = butanol

*1major peak, 2second major peak, ( ) = shoulder peak and - = negative result

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5.3.1 (b) Antibacterial activity of partitioned extract

Table 5.4 shows the antibacterial property of different partitionated extract of isolate S. marcescens IBRL USM84. The results indicated that the dichloromethane partitionaed extract of S. marcescens IBRL USM84 showed greater inhibition spectrum against the tested bacteria compared to other extracts. The inhibition zone of the dichloromethane partitionated extract ranged from 20.0 to 34.0 mm (Figure 5.3) on

MRSA (Figure 5.3A) and A. anitratus (Figure 5.3B). There were only weak inhibition zone of the ethyl acetate partitionated extract ranged from 7.0 to 11.0 mm on S. aureus, B. subtilis and A. anitratus. However no inhibition zone from the hexane and butanol partitionated extracts observed to all the tested bacteria.

Table 5.4: Antibacterial activity of different partitionated extracts of isolate S. marcescens IBRL USM84

Test Inhibition Zone (mm) Microorganisms HEX DCM EA BUT C

S. aureus - 20.3±0.44 8.4±0.38 - 20.2±0.18

B. cereus - 30.7±2.89 - - 10.6±0.16

B. subtilis - 33.3±2.22 10.4±0.29 - 14.5±0.36

MRSA - 23.7±1.78 - - 20.4±0.11

A. anitratus - 21.8±1.09 7.6±0.38 - 13.2±0.31 HEX = hexane, DCM = dichloromethane, EA = ethyl acetate, BUT = butanol, C = chloramphenicol and - = no inhibition zone

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A B

-ve +ve -ve +ve

Figure 5.3: Disc diffusion assay of dichloromethane partitionated extract of isolate S. marcescens IBRL USM84 against (A) MRSA and (B) A. anitratus

[-ve = Negative control; +ve = Positive control]

Figure 5.4 shows the weak inhibition zone of the ethyl acetate partitionated extract on B. subtilis (Figure 5.4A) and no inhibition zone of butanol partitionated extract on A. anitratus (Figure 5.4B) observed. Some of the colour pigments produced by S. marcescens IBRL USM84 can be extracted by butanol, but no desired bioactive compounds present in the butanol partition extract. Mensor et al., (2001) stated that most of the fats, oils and waxes exist in the mixture of compounds produced by the isolate can be extracted by hexane organic solvent.

The pigment content was correlated with antibacterial activity in dichloromethane partitionated extract of S. marcescens IBRL USM84. It was assumed that the compound in the dichloromethane partitionated extract was responsible for the antibacterial activity. Due to this finding, the dichloromethane partitionated extract was chosen as the potential pigment extract and its MIC and MBC values were evaluated. However, the antibacterial activity of the separated compounds was further

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examined by using thin layer chromatography (TLC) and bioautography techniques in the next chapter.

-ve +ve

Figure 5.4: Disc diffusion assay of various partitionated extract against test microorganisms (A) Ethyl acetate partitionated extract against B. subtilis (weak activity) and (B) Butanol partitionated extract against A. anitratus (no inhibition zone)

[-ve = Negative control; +ve = Positive control]

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In general, the partitioning process provided necessary information concerning the polarity of the bioactive compounds present in the crude extract. The process also facilitated in the separation of bioactive compounds from other undesirable compounds. Results also showed that the bioactive compounds were mid- polar in nature since the extraction solvents of these compounds were dichloromethane and ethyl acetate. From marine resources, Cortes et al., (2014) reported a novel antimicrobial activity of a dichloromethane extract obtained from red seaweed

Ceramiun rubrum against pathogenic microbe, Yersinia ruckeri and Saprolegnia parasitica that cause disease in salmonids. Bansemir et al., (2004) found that the dichloromethane extract of Laurencia chondrioides (2 mg/disc) inhibited

Pseudomonas anguilliseptica with inhibition zone of 15.0 ± 7.3 mm.

5.3.1 (c) Determination of Minimum Inhibitory Concentration (MIC) and

Minimum Bactericidal Concentration (MBC) of dichloromethane partition extract

The antibacterial activity of the dichloromethane partitionated extract of isolate S. marcescens IBRL USM84 was evaluated quantitatively by determining the

MIC value using microdilution method. The MIC is defined as the lowest concentration of antibacterial agent yielding no visible bacterial growth after an overnight incubation (Darshan & Manonmani, 2016). The MBC on the other hand was done by streaking a loop full of aliquot from the treated wells that represented the MIC value and above on to fresh plates of nutrient agar. MBC is defined as the lowest concentration of an antibacterial agent causing ≥99.9% reduction in the initial inoculum on subculture (Pankey & Sabath, 2004).

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The evaluation of antibacterial activity was done in vitro to confirm either the dichloromethane partitionated extract was able to inhibit the test bacteria

(bacteriostatic effect) or potentially killed the test bacteria (bactericidal effect). In order to determine the mechanism of antibiosis of the dichloromethane partitionated extract against the test bacteria, the ratio of MBC to MIC was calculated to determine either dichloromethane partitionated extract gave bacteriostatic or bactericidal effects.

The bacteriostatic effect is referred to the ratio of MBC to MIC of more than 4, while, the bactericidal effect is referred to the ratio of MBC to MIC of 4 and below (Pankey

& Sabath, 2004). The results indicated that all the test bacteria shared the same MIC value which was 250 µg/mL. However, the MBC values are variable for different test bacteria. For example, S. aureus, B. subtilis and A. anitratus exhibited a moderate

MBC value (500 µg/mL), whereas B. cereus and MRSA showed lower and higher

MBC values which were 250 µg/mL and 1000 µg/mL, respectively. Table 5.5 summarizes the MIC and MBC values, including the antibiosis mechanism of the dichloromethane partitionated extract of S. marcescens IBRL USM84 against test bacteria. Based on the results, the partitionated extract exhibited bactericidal effect towards all tested strains as the MBC to MIC ratios were below 4.

Generally, the compounds that target the bacterial cell walls or cytoplasmic membranes are often considered as bactericidal agents because of their action in destructing the cell, causing swelling of the cells, subsequently burst and resulting in death (Anderson et al., 2012). The dichloromethane partitionated extract was assumed to contain compounds that targeted to the cell walls or cytoplasmic membranes of all the tested bacteria. However, the higher concentration of the extract was needed to increase MBC value of MRSA (1000 µg/mL) compared to other strains (with the range within 250 µg/mL to 500 µg/mL). This condition might be due to the

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characteristics of MRSA which is known to be resistant towards most antibiotics and always causes severe morbidity and mortality worldwide (Sakoultas & Moellering,

2008).

Table 5.5: MIC, MBC and mechanism of antibiosis of dichloromethane partitionated extract of S. marcescens IBRL USM84 against test bacteria

Test Extract (µg/mL) MBC:MIC Mechanism of Bacteria MIC MBC Antibiosis

S. aureus 250 500 2 (Bactericidal)

B. cereus 250 250 1 (Bactericidal)

B. subtilis 250 500 2 (Bactericidal)

MRSA 250 1000 4 (Bactericidal)

A. anitratus 250 500 2 (Bactericidal)

Most of the marine bacteria have ability in synthesising the secondary metabolites that have inhibitory effect against microorganism. The active compounds might originate from the pigments produced by the isolate or from other non- pigmented compounds. For examples, prodigiosin (Gulani et al., 2012), carotenoid

(El-Refai et al., 2010) and violecein (Duran et al., 2007) were reported to be the common pigments recognized to possess inhibitory effect towards microorganism.

Meanwhile, oleic acid (Leyton et al., 2011), pentabromopseudilin (Feher et al., 2010)

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and 2-n-Penthyl-4-quinolinol (Long et al., 2003) are the non-pigmented compounds which poses antibacterial activity.

5.3.2 Time kill study

The time kill study was tested on MRSA (Gram positive bacteria) and A. anitratus (Gram negative bacteria). The selected bacteria were treated with dichloromethane partitionated extract of S. marcescens IBRL USM84 up to 48 hours at the concentrations of 0.5 x, 1.0 x and 2.0 x MICs. Time kill study was conducted to determine the killing pattern and kinetic of killing by the dichloromethane partitionated extract against MRSA and A. anitratus.

5.3.2 (a) Time kill study of MRSA

Figure 5.5 shows the time kill curve of dichloromethane partitionated extract of S. marcescens IBRL USM84 for MRSA. The control (no extract added) curve demonstrated a continuous and long exponential phase before it reached a stationary phase at 28 hours of incubation time. When treated with half MIC of extract, the growth of cells was delayed but in a later stage (after 12 to 24 hour of incubation time) it managed to achieve the growth as high as in control. After 24 hours of incubation time, the viable cell counts in half MIC were found lower than control and continued until the end of incubation. For the killing curve of at the MIC value, lower viable cell counts were found compared to control and half MIC along the exponential phase.

However, the cell count began to increase after 16 hours of incubation, following the cell growth pattern of half MIC. Viable cells showed a significant reduction in 2 MIC concentrations compared to the control, half MIC and at the MIC. However, the

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growth rate of 2 MIC exhibited a similar cell count with both half MIC and MIC particularly at the beginning of the 36th hours of sampling and onwards.

Log10 (CFU/mL) Log10 2

0 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (Hours)

Control 1/2 MIC 1 MIC 2 MIC

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

Note: Control = untreated; ½ MIC = 125 µg/mL; 1 MIC = 250 µg/mL; 2 MIC = 500 µg/mL

The effect of the extract concentration on MRSA was observed at the 4th hour of sampling where the viable cell count of MIC and 2 MIC were significantly different from both control and half MIC. The 2 MIC concentrations of extract showed bacteriostatic effect because it was achieved in less than 3 log unit reductions in log 10

CFU/mL (or < 99.9% reduction in viable cells). It was obtained between the untreated sample (control) and treated sample as fast as 4 hour exposure time. However, the strain regrew and achieved to the same level as the half MIC and at MIC after 36 hour of exposure time. Similarly, Sim et al., (2014) reported that the active gold compounds at the MIC concentration showed rapid bacteriostatic effect towards P. vulgaris after 4

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hours at exposure time, but the strain grew back to the same level as the control after

24 hour. Meanwhile, the regrowth occurred in E. coli after 4 hours in contact with the same active compounds at MIC and half MIC concentrations. According to Tam et al.,

(2005), the regrowth phenomenon was due to two different subpopulations with dissimilar susceptibility in which the selective growing of resistant sub-population take over the priority killing of the susceptible sub-population at a particular time of interaction. Besides, the rapid bactericidal activity against MRSA occurred after 9 hours in contact to oritavancin (Vidaillac et al., 2011) and more rapid bactericidal activity occurred within 1 hour of contact toward MRSA when treated with active gold compound (Sim et al., 2014). The results revealed that the effectiveness of dichloromethane partitionated extract was more effective for a short time or known as a rapid antimicrobial susceptibility.

5.3.2 (b) Time kill study of A. anitratus

Figure 5.6 shows the time kill curve of dichloromethane partitionated extract of S. marcescens IBRL USM84 for A. anitratus. The growth curves of control and half

MIC were quite similar in term of growth pattern. However, the growth of cells for half MIC concentration was inhibited at the early phase of growth. The differences found between these two curves were the maximum viable cell count achieved from the early stage of growth and during the exponential phase. The viable cell count of half MIC was lower than control at the early stage of growth until 32 hours of incubation time, where was then capable to achieve growth as high as in control. The

MIC showed a moderate growth of bacterial cells and the viable cell count was lower than control and half MIC during the whole incubation time. The 2 MIC displayed slow and obvious inhibition in growth where, the viable cell count was lower than

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control and two others concentration (MIC and half MIC) during the whole incubation time.

10 9 8 7 6 5 4 3

Log10 (CFU/ml) Log10 2 1 0 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (Hours)

Control 1/2 MIC 1 MIC 2 MIC

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

Note: Control = untreated; ½ MIC = 125 µg/mL; 1 MIC = 250 µg/mL; 2 MIC = 500 µg/mL

The effect on growth pattern of A. anitratus can be observed clearly when treated with 2 MIC of the extract. This concentration of extract showed bactericidal effect, because 3 log unit reduction in log 10 CFU/ml (or ≥ 99.9% reduction in viable cells) was obtained between the control and treated sample starting at 16 hours up to

36 hours of incubation time. After that, a slow gradual cell regrowth was observed until at the end of incubation time. The same observation was made by White et al.,

(1996), who reported the regrowth occurred in P. aeruginosa when treated with 2 MIC concentration of meropenem. The regrowth occurred might be due to the extract degradation which affected the effectiveness of the extract throughout the test.

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Friedrich et al., (1995) also reported that the degradation of imipenem and meropenem was detected during the time-kill study.

5.3.3 Stability of prodigiosin pigment in dichloromethane partitionated extract of S. marcescens IBRL USM84

According to Shalinimol & Annadurai, (2012) the stability of pigment is the most important characteristics in the application of pigments in industry. Thus, the extracted pigment containing prodigiosin was treated at various temperature, pH, light and incubation time. The effect of the prodigiosin towards physical parameter was performed by studying the relationship of both prodigiosin readings and its antibacterial activity. Figure 5.7 shows the effect of temperature towards prodigiosin and anti-MRSA activity. The pigment was stable up to 100oC for 20 minutes treatment time period. Similar to the pigment readings, the antibacterial activity of the pigment extract was relatively stable during heat treatment up to 100oC, where the relative antibacterial activity was maintained at 93 to 100 %. The antibacterial activity results indicated that treatment of the pigment at 27oC to 100oC did not show significant difference from the control. This finding revealed that the prodigiosin pigment is to be thermostable pigment and suitable to be applied in high temperature treatments such as in cosmetics formulations and procedures. According to Vaidyanathan et al., (2012) the red pigment extract that they used in their research was stable even at 100°C.

Similarly, Gulani et al., (2012) also stated that prodigiosin pigment is a thermo-stable pigment and relatively stable during heat treatments at 30, 50, 80 and 121oC.

Namazkar et al., (2013) also reported that the thermo-stability of prodigiosin might be contributed by the arrangement of electrons in the prodigiosin structure especially in

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the pyrrole group of the structure since the red coloration of the pigment contributed by the pyrrole group.

120 A A A A A A A A 100 A

(%) 40

27 30 40 50 60 70 80 90 100 Relative Antibacterial Relative (%)Activity

Relative Pigment Relative Absorbance 535 at nm Temperature (°C)

Pigment Antibacterial Activity

Figure 5.7: Stability of prodigiosin pigment at different temperature

Figure 5.8 shows the effect of pH towards the stability of prodigiosin. The pigment was stable at pH range of 4 to 7. The prodigiosin reading was higher at pH range of 4 to 7, then slightly dropped at pH 8 and 9. The relative antibacterial activity was maintained at 93 to 100 % during pH treatment from 4 to 7, with no significant difference from the control. There was a slight decrease in antibacterial activity observed on the pigment extract treated at pH 3 (83.66 %), 8 (82.02 %) and 9 (78.74

%), respectively, where the pigment reading decreased at these points. Figure 5.9 shows the disc diffusion assay plate for antibacterial activity. The inhibition zone by the pigment extract was observed after treated with different pH for 30 minutes.

Gulani et al., (2012) reported that the prodigiosin exhibited better antibacterial activity at the acidic pH compared to the alkaline pH. This is supported with finding from

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Namazkar et al., (2013) who demonstrated that the prodigiosin showed stability at pH

5 was gradually decreased at pH 8 and above. Their findings were almost similar as the results obtained in this study. They also described about the destruction and degradation of the pigment in higher acidic and alkaline conditions and these conditions were contributed by the protonation of pyrrole group and deprotonation of amine group that exists in prodigiosin structure. The results also indicated that the pigment resistant to certain acidic pH which ranged from 4 to 6. Gulani et al., (2012) also reported that the prodigiosin showed higher antibacterial activity at the acidic pH than the basic pH. This property can be contributed by the unique tripyrrole chemical structure in the prodigiosin structure as stated by Cassulo de Araujo et al., (2010). It might be related to the UV/vis property of the prodigiosin as obtained in Chapter 3.3.4

(Figure 3.9). The maximum absorption was achieved at 535 nm in acidic condition with exact prodigiosin color (red) compared to yellow in alkaline condition with maximum absorption at 465 nm (broader spectral curve). This might be the reason why the crude prodigiosin more stable at the acidic pH compared to the basic pH.

120 B B B 100 B A A A 80 60

40 nm (%) nm 20 0

3 4 5 6 7 8 9 Relative Antibacterial Relative (%)Activity Relative Pigment Relative Absorbance 535 at pH

Prodigiosin Antibacterial Activity

Figure 5.8: Stability of prodigiosin pigment at different pH

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5 4 3

6 7 8

Figure: 5.9: Disc diffusion assay by the dichloromethane partitionated extract after treated with different pH for 30 minutes [(C) control without any treatment, (3) pH 3, (4) pH 4, (5) pH 5, (6) pH 6, (7) pH 7, (8) pH 8, (9) pH 9]

Light illumination is another important factor in determining the stability of pigment. From Chapter 4.0 (Section 4.3.1 b) S. marcescens IBRL USM84 was found not to show significant effect on prodigiosin production, cell growth and antibacterial activity when incubated at both light and dark conditions. Figure 5.10 shows the effect of light illumination towards the stability of prodigiosin for one week incubation time.

The pigment seems to be stable at both light and dark conditions during the 7 days of incubation time. Similar to the pigment readings, the antibacterial activity of the pigment extract was relatively stable during incubation time in both conditions, where the relative antibacterial activity was maintained at 94 to 100 %. The results also indicated that treatment of the pigment at light and dark conditions for 7 days did not show significant difference from the control (p> 0.05). However, the antibacterial activity was slightly decreased at day 5 (98.62 %), at day 6 (95.83 %) and at day 7

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(94.72 %), of incubation in the light condition. This condition might be due to the extracted pigment was exposed to the light illumination for a longer period of time and was incubated at room temperature (27±2) oC. Park et al., (2012) reported that prodigiosin is more stable at 4 oC (98 % of stability) compared to at room temperature

27 oC (80 % of stability). Farhan, (2010) also found that a red pigment produced by

Monascus ruber is more stable when stored at dark condition. This finding suggested it is better keeping the pigment extract in the dark condition for long term storage.

Besides, the encapsulation method was also recommended which can be a great alternative to improve the light sensitivity property of the natural pigment as described by Namazkar et al., (2013).

120 120 A A A A A A A A 100 100 a a a a a a a a 80 80

60 60

40 40 Relative Antibacterial Relative (%)Activity 20 20

Relative Pigment Relative Absorbance 535 at (%)nm 0 0 0 1 2 3 4 5 6 7 Time (Day)

Pigment (Exposed) Pigment (Unexposed)

Antibacterial Activity (Exposed) Antibacterial Activity (Unexposed)

Figure 5.10: Stability of prodigiosin pigment towards light illumination

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Figure 5.11 shows the effect of incubation time on stability of the prodigiosin extract. The prodigiosin pigment extract was kept at 4 oC for 60 days in dark condition since the antibacterial activity from S. marcescens IBRL USM84 was noticeably slightly less stable towards the light illumination as the results obtained previously in

Figure 5.10. The results revealed that the prodigiosin was a stable pigment, as more than 96 % of prodigiosin in the dichloromethane partitionated extract remained for up to 60 days at the temperature of 4 oC in the dark condition. The relative antibacterial activity was maintained at 100% until 60 days of incubation time. There was no significant difference of antibacterial activity between the treated pigment and the control (p> 0.05). Table 5.6 shows the stability of the prodigiosin present in dichloromethane partitionated extract at different characteristics.

120 A A A A A 100

20 Relative Antibacterial Relative (%)Activity

Relative Pigment Relative Absorbance 535 at (%)nm 0 0 15 30 45 60 Time (Day)

Pigment Antibacterial Activity

Figure 5.11: Stability of prodigiosin pigment towards incubation time

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Table 5.6: Stability of the prodigiosin pigment in dichloromethane partitionated extract at different characteristics

Characteristics Stability Range

Temperature (oC) 27 - 100

pH pH 4 – 7

Light Condition Day 0 – 7

Dark Condition Day 0 – 7

Incubation Time (days) Day 0 – 60

5.4 Conclusion

The preliminary separation was carried out in this chapter to separate the crude extract of S. marcescens IBRL USM84 into four partitionations which were hexane, dichloromethane, ethyl acetate and butanol partitionated extract. Among these, dichloromethane partitionated extract was selected as potential extract due to its ability to extract the attractive and strong pigment coloration and exhibited broader spectrum of antibacterial activity compared to other partitionations. Solvent-solvent partitioning also provided great information about the polarity of the desired bioactive compound which were mid-polar compound. The red pigment produced by S. marcescens IBRL

USM84 demonstrated multiphasic killing rates and also exhibited both bactericidal and bacteriostatic activities depending on the tested strains and concentrations of extract that has been used. Based on the physical characteristics, the prodigiosin was a thermo stable pigment, stable in neutral to moderate acidic condition, long shelf life

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and stable towards light illumination. But the antibacterial activity was slightly dropped when incubated at room temperature (27oC) under light illumination for a longer time. Overall, the intense and attractive coloration, broad stability range and potential of antibacterial activity of the prodigiosin produced by S. marcescens IBRL

USM84 may have vast applications in industrial technologies.

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CHAPTER 6.0: BIOASSAY GUIDED SEPARATION OF PIGMENT

EXTRACTED FROM Serratia marcescens IBRL USM84

6.1 Introduction

Natural red pigment with prodigiosin activity produced by S. marcescens

IBRL USM84 consisted of more than one bioactive compound. The separation, purification, characterization, and identification are very important to determine a particular compound which is responsible for the antibacterial activity. Thin layer chromatography (TLC), bioautography and column chromatography (CC) techniques were used as the analytical methods to detect the bioactive compounds which existed in the crude extract. After that, the identification of the bioactive compounds was conducted by ultra performance liquid chromatography (UPLC) technique of which the result can be presented in chromatographic fingerprints for rapid characterization of bioactive compounds. Chromatographic techniques are more economical, easy to conduct and used minimal of equipments required for identification of natural compounds (Patra et al., 2012; Mueen-Ahmed, (2008).

This part of study was aimed to isolate, purify and characterize a compound particularly responsible for the antibacterial activity which exists in the crude prodigiosin extract. The bioassay guided analysis that was carried out for the bioactive compound included the disc diffusion assay, MIC, MBC and toxicity study.

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6.2 Materials and Methods

6.2.1 Thin Layer Chromatography (TLC)

Dichloromethane partition extract provided a better pigment quality and also greater antimicrobial activity hence this partition extract was subjected for further separation using TLC plate. The TLC plates (Merck, silica gel 60 F254) were cut of 2 cm x 11 cm strips using a sharp penknife. The strips were scored using a pencil to lightly mark the baseline of the spots at a distance of 1.0 cm of the lower edge of the plate (Rajauria & Abu-Ghannam, 2013). An aliquot of the dichloromethane extract was re-dissolved in methanol (1 mg/mL).By using a 10 µL tip, about 1.0 µL of the extract was spotted on the strips. The spots were left in a fume hood and were dried under a moving air stream for 5 minutes. After that, the strips were developed in an unsaturated glass chamber containing solvents and were run until it reached 0.5 cm from the top of the strips.

The TLC strips were developed under saturated condition in individual solvent consisting of ethanol, dichloromethane, 2-propanol, methanol, chloroform, ethyl acetate, acetone and hexane. Two solvents that gave the best separation were then combined in different ratios. The combination of the solvent systems tested were as follows: (1) 2-propanol: methanol, (2) 2-propanol: ethyl acetate, (3) 2-propanol: ethanol, (4) ethyl acetate: methanol, (5) dichloromethane: methanol and (6) ethanol: methanol. Three different ratios of the solvent mixtures, namely 7:3, 5:5 and 3:7 were used. After that, the best combination of solvent system was tested in ratios of 10:0,

9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10. The best separation of compounds was compared with previous crude extract and prodigiosin standard that were separated by using the same solvent systems.

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The separation pattern (distances) and the color of spots were observed under a normal visual light and Ultraviolet (UV) lamp at 366 nm (long wave) and 254 nm

(short wave) (Tong et al., 2012). The Rf (Retention factor) values of separated compounds were calculated according to the equation below;

Distance of spot from the starting point Rf = Distance of solvent from the starting point

6.2.1 (a) Bioautography assay using agar overlay method

The dichloromethane partition extract was run on TLC strip in ethanol: 2- propanol (8:2) (v/v). The strip was air dried in a fume hood for 30 minutes and was left under the UV lamp for 60 minutes as a plate pre-sterilization step. The strip was placed on the solidified MHA agar plate (Valgas et al., 2007). A molten MHA (10 ml) was inoculated with 1 ml of bacterial suspension (106 CFU/ml), poured over the MHA agar containing TLC plate and was left solidified. The plate was inverted and then incubated for 24 hours at 37oC. Clear areas against a purple background representing inhibition zones around the chromatographic spots on the TLC strip were recorded.

The viability of the cells was determined by spraying the surface with 2 mg/ml of INT

(p-iodonitrotetrazolium) violet salt solution. Zone of inhibition were observed as clear zones against a purple background. The antibacterial activity was measured based on the presence or absence of bacterial growth on the appearance of inhibition zone around the developed spots.

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6.2.2 Column Chromatography (CC) technique

6.2.2 (a) Column packing and development

The bottom of a chromatography glass column (47 cm of length and 2.2 cm of diameter) was plugged with a glass wool (Salituro & Dufresne, 1998). The column was rinsed with methanol and was left to dry. Approximately, 35 g of silica gel

(Acros, Organic) with a diameter of 0.03-0.20 mm was weighed, conditioned with ethanol: 2-propanol (8:2) and stirred with glass rod to obtain a slurry. The slurry was carefully poured to fill three-fourths of the column. The wall of the column was tapped with a rubber pipe and the column was agitated to eliminate the trapped air bubbles.

The column was then left overnight before the sample was loaded. The next day, 0.5 g of dichloromethane extract (previously dissolved in methanol) was applied on the wall using a pasture pipette. Afterwards, the mobile phase (ethanol: 2-propanol) was carefully filled. The exit valve was opened to elute the sample. Finally, the remaining fractions in the silica were collected by washing the column with methanol. The separated fraction was collected in the pre-weighed beaker based on the color produced. All collected fractions were dried in a fume hood for further analysis.

6.2.2 (b) Spectrophotometric analysis of fractions

The pigment present in collected fractions was quantitatively analyzed by using a spectroscopic method. All collected fractions were re-dissolved in acidified 2- propanol and the absorbance of the propanolic solutions was determined at 535 nm using UV-vis spectrophotometer.

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6.2.2 (c) Thin Layer Chromatography analysis of fraction

All fractions were spotted on a TLC plate along with the prodigiosin standard. Rf values of all collected fractions were then determined. TLC analysis was performed as described in Section 6.2.1.

6.2.2 (d) Antimicrobial activity test of fraction

Fraction 4 (light pink fraction) was subjected to sensitivity test. Fraction 4 was chosen due to the highest prodigiosin content detected through spectroscopic analysis (Section 6.2.2 b). The antibacterial activity of Fraction 4 was evaluated using a disc diffusion method (Section 3.2.2 e). The final concentration, 1 mg/disc of the fraction was used. The bacteria used in the test were S. aureus, B. cereus, B. subtilis,

MRSA and A. anitratus.

The initial stock concentration, 8000 µg/ml of Fraction 4 was prepared by dissolving the extract paste in 50 µl of methanol and 950µl of MHB (5 % of methanol). Two-fold serial dilutions were employed to this stock solution in MHB up to 31.25 µg/ml. All stock solutions were further diluted according to Table 6.1. About

100 µl of each concentration of solution were added to microtitre well containing 100

µl of inoculum suspension. Each concentration was tested in triplicates. Methods for inocula preparation, the addition of extract, and inocula in microtitre plate and determination of MIC value were described as in Section 5.2.1 (c).

MBC was then determined according to MIC value by subculturing an aliquot from each well showing no turbidity and the last well showing turbidity on NA plate.

The NA plates were then incubated for 24 hours at 37oC. The lowest concentration of fraction indicating no visible colony growth on NA plate was considered as MBC.

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Table 6.1: Scheme for preparing dilution series of moderate water soluble extract (pink fraction) to be used in MIC assay

Stock Volume of stock Volume of MHB + Final concentration of (µL) inoculums (µL) concentration crude extract (µg/mL) (µg/mL)

8000.00 100 100 4000.00

4000.00 100 100 2000.00

2000.00 100 100 1000.00

1000.00 100 100 500.00

500.00 100 100 250.00

250.00 100 100 125.00

125.00 100 100 62.50

62.50 100 100 31.25

31.25 100 100 15.63

According to MIC results value, the MBC was then determined by subculturing an aliquot from each well showing no turbidity and also the last well that showed growth (turbidity) on NA plate. The NA plates were then incubated at 37oC for 24 hours. The lowest concentration of fraction indicating no visible colony growth on NA plate was taken as MBC.

6.2.3 Preparative TLC for purification

In order to collect the desired compounds in higher amount, the preparative

TLC technique was applied following the method of Rajauria & Abu-Ghannam,

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(2013). Several spots of active fraction were applied on an aluminium-backed TLC plate (Merck, silica gel 60 F254) with prodigiosin standard. The plates were then air- dried. The spots were developed in ethanol: 2-propanol (8:2; v/v). The spots with the

Rf value of 0.72 (Rf value of prodigiosin standard) were gently scrapped from the plate and transferred into 2 ml appendoff tube. The scratched sample was dissolved in methanol (UPLC grade) and was centrifuged at 4000 rpm for 5 minutes to remove silica particles. Then, the collected supernatant was filtered through 0.2 µm filter paper

(Millipore, Whatman).

6.2.4 Ultra Performance Liquid Chromatography (UPLC)

The partitionated extract and preparative TLC purified compound were analyzed by using UPLC (Water Acquity) to check the purity of extract after separation and purification steps. The UPLC system consists of Symmetry BEH C18 column (1.7 µm; 2.1 x 150 mm), 2545/2525 Binary Gradient Module, Waters 717 plus auto-sampler and PDA detector (Waters) coupled with Empower 2 software. The mobile phase was methanol (UPLC grade) and 0.1% (v/v) of Trifluoroacetic acid

(TFA) in the ratio of 8:2 (v/v). The solvent were filtered through a 0.45 µm Sartorius

PTEE membrane filter (47 mm in diameter), while, the buffer was filtered through

0.45 µm Nylon membrane disc filter (47 mm in diameter). Separation was carried out on the UPLC system at room temperature (25 ± 2oC). The system was equilibrated under isocratic condition. The mobile phase flow rate was 0.1 mL/minutes. The sample was dissolved in methanol (UPLC grade) with the injection volume of 5.0 µL.

The sample was detected at the wavelength of 254 nm. The retention time (RT) between the prodigiosin standard and the sample was compared (Juin et al., 2015).

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6.2.5 In vitro toxicity study

The brine shrimp lethality test was done by using the method elaborated by

Sharadamma et al., (2011). The test consisted of four steps, namely preparation of artificial seawater (ASW), hatching of brine shrimp (Artemia salina), preparation of pigment extract and exposure of the brine shrimp to the extract solution at various concentrations.

6.2.5 (a) Preparation of artificial seawater (ASW) and hatching of brine shrimp

(Artemia salina)

The growth medium for A. salina i.e. ASW was prepared by dissolving 38.0 g of sea salts (Sigma) in 1.0 L of distilled water. The solution was heated on a hot plate and stirred using a magnetic stirrer until the salts fully dissolved. The solution was then filtered using Whatman No. 1 filter paper. About 500 mL of the solution was poured into a beaker (1.0 L). Eggs of A. salina (purchased from fish aquarium shop at

Bayan Baru, Penang) were hatched in the beaker containing the sea salt solution, oxygenated from an aquarium pump and incubated under continuous illumination for

24 hours at 28±2 oC. Hatched A. salina cysts were transferred to fresh artificial seawater and incubated for further 24 hours under the light with air sparging to develop the larval stage. Finally, the larvae (nauplii) were collected using a Pasteur pipette and were transferred into universal bottle containing seawater.

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6.2.5 (b) Preparation of pigment extract

The toxicity of the crude extract (dichloromethane partitioning extract) and

Fraction 4 on aquatic species, A. salina was carried out. The initial stock concentrations were separately prepared by dissolving 50 mg/mL of partition extract and Fraction 4 in 500 µL of DMSO respectively, followed by the addition of 500 µL of artificial sea water to yield a stock concentration of 50 mg/mL. The extract was further diluted with artificial seawater into a series of concentrations, ranging from 10

- 2000 µg/mL with a final volume of 5000 µL (Table 6.2). DMSO (2%) in 5000 µL of artificial seawater served as a control. Each concentration was tested in triplicates.

Table 6.2: Preparation of extract for toxicity test

Concentration Volume of stock Volume of artificial Final volume extract (µL) seawater (µL) (µL) (µg/mL)

10 1 4999 5000

50 5 4995 5000

100 10 4990 5000

500 50 4950 5000

1000 100 4900 5000

2000 200 4800 5000

Control Replaced with 200 µL 4800 5000 DMSO (50%)

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6.2.5 (c) Brine shrimp lethality test (BSLT)

The BSLT was conducted in universal bottle containing 5000 µL of seawater with 10 to 2000 µg/mL concentrations of the crude extract and Fraction 4. About 10 to

15 nauplii were added in each universal bottle, and were exposed to the light. All bottles containing the nauplii were left overnight.

. The number of dead (non-motile) and live nauplii was counted under light microscope after 6 hours (acute toxicity) and 24 hours (chronic toxicity) of incubation time. The percentage of mortality was calculated as follows:

Percentage of mortality (%) = Number of dead nauplii x 100 Initial number of live nauplii

Data was corrected if dead nauplii were found in control by deducting the percentage of mortality in control (Mojica & Micor, 2007). The concentration yielding

50% of lethality (LC50) was calculated by using a graph of mean percentage mortality versus log of concentration from linear regression equation. The extract was considered as non-toxic if LC50 value was greater than 1000 µg/mL (Bastos et al.,

(2009).

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6.3 Results and Discussion

6.3.1 Thin Layer Chromatography (TLC)

Ethanol and 2-propanol at ratio 8:2 was selected as a suitable solvent system for dichloromethane extract of S. marcescens IBRL USM84 because it gave the best separation in all the mobile phase tested. The successful separation of compounds by chromatographic method is influenced by suitable solvent system (Patra et al., 2012).

In this study, ethanol and 2-propanol (8:2) was the most suitable solvent system that provided the best compound separation among the tested solvents. The mobile phase successfully separated five distinct spots under visible and UV lights. The spots had different polarities due to their variable travelling distance among each other (Figure

6.1). Three spots with the Rf values of 0.78, 0.72 and 0.66 had pigmented properties which were orange, light pink and pale purple, respectively, since these spots can be observed under visible light (Figure 6.1A). All spots can be visualized under the long wave of UV light, except for the spot with Rf value of 0.66 (pale purple under visible light) (Figure 6.1C). However, only one dark spot with Rf value of 0.72 (light pink under visible light) can be detected under short wave UV light (Figure 6.1E). Figures

6.1B, D and F show the graphical illustration of the chromatograms under different lights.

Table 6.3 shows the TLC analysis of the dichloromethane partition extract of

S. marcescens IBRL USM84 in mobile phase of ethanol and 2-propanol (8:2). Similar results were obtained by Gulani et al., (2012), who developed the chromatogram of methanolic extract of prodigiosin which was separated into 3 bands characterized by purple, red and orange at the Rf value of 0.27, 0.64 and 0.82, respectively. Results of the chromatogram demonstrated that the purple band was the first to be separated, followed by red and orange bands, similar with the results obtained. The red band was

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probably the same light pink spot reported in this study. Williams et al., (1956) separated four spots from prodigiosin acetone extract i.e. blue, red, red and orange with the Rf values of 0.18, 0.48. 0.70 and 0.89. Gulani et al., (2012) also stated that the purple band that they found in their chromatograms of prodigiosin was probably the same blue component as described by William et al., (1956) previously.

A B C D E F Figure 6.1: Chromatograms of the dichloromethane partition extract of S. marcescens IBRL USM84 developed using ethanol: 2-propanol (8:2) and graphical illustration, observed under (A) visible light, (B) visible light (illustration), (C) long UV, (D) long UV (illustration), (E) short UV, (F) short UV (illustration)

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Table 6.3: TLC analysis of the dichloromethane partition extract of S. marcescens IBRL USM84 in mobile phase of ethanol: 2-propanol (8:2)

No Rf Visible light Long UV Short UV

1 0.78 Orange spot Yellowish spot -

2 0.72 Light pink spot Pale pink spot Dark spot

3 0.66 Pale purple spot - -

4 0.60 - Bluish spot -

5 0.45 - Bluish spot -

According to Kumar et al., (2013) the TLC plate is considered polar since the silica gel is used as the stationary phase. The separation of compounds on the stationary phase is influenced by the interaction between the solute and the mobile phase. Thus, more polar compounds move slower on the TLC plate than the less polar compounds. However, Bele & Khale, (2011) also reported when a more polar solvent or mixture of solvents used as the mobile phase, resulting all compounds on the TLC plate move faster and higher up the plate. For example, when more ethyl acetate was added in a mixture of ethyl acetate and heptane used as a mobile phase, higher Rf values were noted for all the compounds. Therefore, the separated compounds (orange, light pink and pale purple) would move faster and travel longer distance and this could be due to the more polar solvent mixture used in this study which was ethanol: 2- propanol (8:2), subsequently resulting in higher Rf values.

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6.3.1 (a) Bioautography analysis of dichloromethane partition extract

The presence of substances with antibacterial compounds can be observed qualitatively on agar overlay assay. The dichloromethane partition extract produced 5 separated spots which were orange, light pink, pale purple (under visible light) and two bluish spots (under long UV light). Only light pink spot with Rf value of 0.72 exhibited inhibitory activity on MRSA (Figure 6.2), S. aureus, B. cereus, B. subtilis and A. anitratus. The appearance of clear zone surrounded by the purple area on the chromatograms indicated the inhibition zone where the bioactive compound had inhibited the bacterial growth. However, all of the tested bacteria were not susceptible to the other separated spots. William et al., (1956) also observed and reported the presence of prodigiosin which was extracted from S. marcescens in their chromatogram at the Rf value of 0.70. This part of study suggested that the light pink spot need to be focused for further analysis.

There were some cases where the antimicrobial activity in the separated compounds disappeared even though the mixture of the compounds in crude extract exhibited good antimicrobial activity before. Masoko & Eloff, (2005) described that the separated compounds probably lost their antimicrobial property due to the active compounds had evaporated from the TLC plate, inadequate amount of active compound, photo-oxidation or the synergism effect between the separated compounds.

The synergism effect between the separated compounds also can be seen more clearly after the mixture of compounds subjected to the next separation part which is column chromatography. The bioactivity of the compound can be reduced after the fractionation if they have synergistic effect between the separated compounds.

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Light pink spot (bioactive compound)

with Rf value at 0.72

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

6.3.2 Column Chromatography

A total of six fractions were collected based on the band colours produced during the fractionation of the active pigment compound from the dichloromethane partition extract. Since the silica gel (polar) was used as a solid phase in this column chromatography, the non-polar compounds were eluted earlier because the polar compounds were observed, ranging from orange and pink to purple. The band colours of the eluted fractions ranged from orange and pink to purple. Each of the collected fractions was then evaluated with UV/vis spectrophotometer and TLC silica gel chromatographic analysis.

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6.3.2 (a) Spectroscopic analysis of fraction

The elution pattern of fractions obtained from column chromatography with ethanol: 2-propanol (8:2) as a mobile phase resulted in the separation of light orange, orange, orange-pink, light pink, pale pink and pale purple. All the collected fractions were analysed by spectroscopic analysis to determine the absorbance value of pigment at 535 nm of wavelength. Figure 6.3 shows that Fraction 4 contains the highest concentration of prodigiosin compared to the other fractions where this light pink fraction (Fraction 4) exhibited the highest absorbance value of prodigiosin pigment which was 0.82A at 535 nm of wavelength.

0.9 0.8 0.7 0.6 0.5 0.4

OD 535 nm 535 OD (A) 0.3 0.2 0.1 0 1 2 3 4 5 6 Fractions

Figure 6.3: The absorbance of different fractions collected from column chromatography of dichloromethane partition extract of S. marcescens IBRL USM84

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A previous study conducted by Williams et al., (1956) reported that the red pigment produced by S. marcescens consists of four fractions. Further separation was done by Lynch et al., (1968) who found a total of six fractions that were separated using column chromatography technique as obtained in this study. They also reported that the various colour of fractions obtained from their research which were purple, orange-red, pink, pink, pink and yellow, differing in absorption spectra which is similar with the various colour of fractions collected in this study.

6.3.2 (b) Thin Layer Chromatography analysis of fraction

The elution pattern of fractions from column chromatography was similar with the previous pattern of partition extract in TLC where the separation resulted in light orange, orange, orange-pink, light pink, pale pink and pale purple. The collected fractions were analyzed on TLC strips by using the same mobile phase, ethanol: 2- propanol (8:2). Table 6.4 shows the color and Rf values of fractions derived from column chromatography. Different Rf values for prodigiosin was previously reported since the TLC strips were developed in the different mobile phase. Nadaf et al., (2016) used methanol: ethyl acetate: chloroform (6:3:1) as a mobile phase and found that the

Rf value was 0.84. Meanwhile, Lins et al., (2014) developed the TLC strips in methanol: chloroform: acetone (2:3:4) and reported the Rf value was 0.59.

Suryawanshi et al., (2014) used butanol: hexane (2:1) and found that the Rf value was

0.86. Besides, Bharmal et al., (2012) also reported the Rf value of the prodigiosin was

0.73 when used petroleum ether: acetone (7:3) as a mobile phase which was near to the Rf value (0.72) obtained in this study.

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Table 6.4: The TLC analysis of fractions collected from column chromatography

Fraction Colour of Rf value Visible light UV light fractions F1 Light orange 0.80 Not detected Yellowish

F2 Orange 0.78 Light orange Yellowish

F3 Orange-pink 0.78 Orange Yellowish 0.72 Not detected Pale pink

F4 Light pink 0.72 Light pink Pale pink

F5 Pale pink 0.66 Pale purple Not detected 0.72 Not detected Pale pink

F6 Pale purple 0.66 Pale purple Not detected

Figure 6.4 shows the TLC strips of standard prodigiosin (Figure 6.4A), dichloromethane partition extract (before fractionation) (Figure 6.4B) and Fraction 4 which was compound with antibacterial activity (Figure 6.4C). The Rf value of light pink fraction (Fraction 4) was similar with the Rf value of prodigiosin standard, which was 0.72, when similar mobile phase ethanol: 2-propanol (8:2) was used. A single spot was detected in Fraction 4, implying that the fraction consisted of a purified compound. However, further confirmation using Ultra Performance Liquid

Chromatography (UPLC) is needed because more than one compound may be present within this single spot.

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A B C

Figure 6.4: Chromatograms developed using ethanol: 2-propanol (8:2) of (A) standard prodigiosin, (B) dichloromethane partitionated extract (before fractionation) and (C) Fraction 4 (compound with antibacterial activity)

6.3.2 (c) Bioassay analysis of fraction from S. marcescens IBRL USM84

From the fractionation part, Fraction 4 (light pink fraction) indicated the highest concentration of prodigiosin pigment and can be considered as an active fraction. Hence, this fraction was selected and subjected to bioassays. Furthermore, the

Fraction 4 and prodigiosin standard showed similar Rf value (0.72) when developed using the same mobile phase which was ethanol: 2-propanol (8:2).

Sensitivity test of the active fraction (Fraction 4) was done to determine the capacity of Fraction 4 against S. aureus, B. cereus, B. subtilis, MRSA and A. anitratus.

Table 6.5 shows the result of sensitivity test of the Fraction 4 against five bacteria in comparison with inhibition zones of dichloromethane partitioned extract. The dichloromethane partitioned extract provided a greater antibacterial activity after

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partitioning process. The antimicrobial activity is potentially more effective after the extract was separated from other accumulated compounds with no antimicrobial activity such as carbohydrates, fat and proteins (De Oleveira et al., 2013). However, the antibacterial activity of Fraction 4 towards all the tested bacteria decreased after fractionation process.

Table 6.5: Sensitivity test results of fractionated extract in comparison with inhibition zones of dichloromethane partitionated extract of S. marcescens IBRL USM84

Test Inhibition zone (mm) Microorganisms DCM Extract (1mg/disc) Fraction 4 (1mg/disc)

S. aureus 20.3±0.44 12.4±0.17

B. cereus 30.7±2.89 14.8±0.98

B. subtilis 33.3±2.22 15.3±0.16

MRSA 23.7±1.78 12.9±0.16

A. anitratus 21.8±1.09 13.5±0.98

The results exhibited that the inhibition zone of the dichloromethane partitioned extract ranged from 20.0 to 34.0 mm and decreased to 12.0 to 16.0 mm after fractionation. Figure 6.5 shows the inhibition zone of Fraction 4 against MRSA

(Figure 6.5C) and B. subtilis (Figure 6.5D), where the diameter sizes of inhibition zone decreased after fractionation compared to dichloromethane partitionated extract

(before fractionation) against MRSA (Figure 6.5A) and B. subtilis (Figure 6.5B).

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A B

C D

-ve +ve +ve -ve

Figure 6.5: Inhibition zone of dichloromethane partitionated extract against (A) MRSA and (B) B. subtilis, Fraction 4 extract against (C) MRSA and (D) B. subtilis

[-ve = Negative control; +ve = Positive control]

A synergistic activity among different compounds in the extract before fractionation was probably the reason for this phenomenon. Awouafack et al., (2013) reported that a purified compound exhibited a lower antibacterial activity compared to the extract (before purification). Meanwhile, Cai et al., (2016) reported that the anti-

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inflammatory effect is contributed by synergistic effect of the individual compounds.

They also speculated that each compound present in the crude extract has their own role to inhibit inflammation, thus the combination of these individual compounds contributed a good synergistic effect in preventing inflammatory. In contrast, Rajauria

& Abu-Ghannam, (2013) reported that purified compound showed higher antimicrobial activity compared to extract. The absence of synergistic effect among different compounds was the probable factor influencing higher antibacterial activity of the purified compound. Meanwhile, the anti yeast activity of both purified and crude extract was insignificant as previously demonstrated by Tong et al., (2012).

Table 6.6 shows the MIC and MBC values of Fraction 4 against five test bacteria (Gram positive and Gram negative bacteria). The results indicated that all test bacteria sharing the same MIC and MBC values which were 1000 µg/mL and 2000

µg/mL, respectively. Fraction 4 exhibited bactericidal activities towards all the tested bacteria as the MBC values were less than four-fold of MIC values. However, the results exhibited that the antibacterial activity was not propelled when the extract was separated using column chromatography as the MIC value was higher compared to the partitionated extract. This finding suggested that there are more than one compounds present in the partitioned extract of S. marcescens IBRL USM84, where in combination might have synergy in their efficacy.

According to Rasoanaivo et al., (2011), synergy means that the effect of the combination is better than the individual effects. Fidock et al., (2004) stated that the significant synergy is at least a two-fold increase in activity, as obtained in this study where the MBC value of the partitioned extract is up to eight times greater than the fraction. This result is similar with previous study reported by Fivelman et al., (2004) where the antimalarial activity of the combination compounds is eight times more 183

effective compared to individual compounds. Soltanian et al., (2016) also reported that the crude extract was more effective than the fractions, since it generated lower MIC value and bacterial inhibition in lower concentrations. Meanwhile, Asano et al., (1999) who found the supernatant produced by S. marcescens possessed synergistic effect on larvicidal activity of Bacillus thuringiensis delta-endotoxin (Cry1C) against the common cutworm, Spodoptera litura.

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

Dichloromethane Fraction 4 Test partitionated extract extract Bacteria (µg/mL) (µg/mL) MIC MBC MIC MBC S. aureus 250 500 1000 2000

B. cereus 250 250 1000 2000

B. subtilis 250 500 1000 2000

MRSA 250 1000 1000 2000

A. anitratus 250 500 1000 2000

S. marcescens IBRL USM84 showed lower MIC values of the crude form

(before purification) against the tested bacteria compared to other Serratia species.

The MIC values of crude methanol extract of S. marcescens PTCC 1111 on S. aureus and E. coli (Zarif et al., 2017), was higher than the S. marcescens IBRL USM84 used in this study. In contrast, the antibacterial activity of prodigiosin from S. marcescens

UFPEDA 398 was higher than S. marcescens IBRL USM84 (Lapenda et al., 2015).

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However, the antibacterial activity of S. marcescens IBRL USM84 was greater than pigmented bacteria from terrestrial microorganism such as Aeromonas sp., E. coli and

Pseudomonas sp. of which the MIC values were in the range of 1500 to 4000 µg/mL

(Rashid et al., 2014). This is because distinctive structures of metabolites derived from the marine bacteria possess a greater bioactivity compared to the terrestrial sources

(Carte, 1996).

Thus, the results revealed that the intracellular prodigiosin (before purification) produced by S. marcescens IBRL USM84 had a greater bioactivity compared to the fractionated extract. It is widely believed that other compounds present in S. marcescens IBRL USM84 may act synergistically to enhance the antibacterial activity. Karthick et al., (2016) reported the fractioned extract of the

Serratia sp. was analyzed in a Gas Chromatography Mass Spectrometry (GC-MS) showed the presence of certain metabolites such as octadecanoic acid, phenol, 2, 4-bis

(1, 1-dimethyl ethyl), nonanoic acid-9 oxo methyl ester which could be responsible for the antibacterial and antifungal activities. This finding further implied that the extract rich in prodigiosin could be a potential natural preservative and antibacterial agent.

There are some advantages when the crude form is chosen as an antibacterial agent, where the crude form is often more economical to be produced, save time and energy and also affordable to produce in high amount. In contrast, the pure compound are often more expensive to produce and also involve complicated parts in purification.

6.3.3 Preparative TLC

Based on the characterization of the non-purified extract using spectroscopic analysis in preliminary identification, the active compound present in S. marcescens

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IBRL USM84 could be prodigiosin (Chapter 3.0; Section 3.3.4). In this part of the study, a preparative TLC-purified compounds was evaluated using spectroscopic analysis to further confirm the characteristics of the active pigment compound. Figure

6.6 shows similar spectroscopic profile between the samples (purified compound) and the prodigiosin standard in term of maximum absorption, confirming that the active pigment produced by S. marcescens IBRL USM84 belonged to prodigiosin-like family. The maximum absorption of both standard and sample was at 535 nm, further verifying that the pigment present in the sample corresponded to prodigiosin. Results of this study were in accordance with several past studies where the maximum UV absorbance of the prodigiosin was at 535 nm (Williams et al., 1956; Ryazantseva &

Andreyeva, 2014; Darshan & Manonmani, 2015).

0.9 0.8 0.7 0.6 0.5 0.4 0.3

Absorbance (A) Absorbance 0.2 0.1 0 400 450 500 550 600 650 Wavelength (nm) Standard prodigiosin Purified compound

Figure 6.6: Characteristic UV-visible of standard prodigiosin and preparative TLC purified compound from S. marcescens IBRL USM84

Previous studies reported that the different maximum absorbance of prodigiosin, ranging from 530 nm up to 537 nm, depended on the bacterial species and the solvents used. Feng et al., (1982) reported the maximum absorption of prodigiosin

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was at 537 nm when the pigment produced by S. marcescens O8 was extracted using acetone. Meanwhile, Alihosseini et al., (2008) reported the prodigiosin of Vibro sp. exhibited maximum absorption of pigment at 530 nm when extracted with methanol.

Nadaf et al., (2016) also reported the prodigiosin producer, S. marcescens NCIM 5061 had a maximum absorption of pigment at 534 nm when ethyl acetate was used as an extractant.

Alihosseini et al., (2008) also used the preparative TLC method to purify prodigiosin. They analyzed a major dark pink color band using HPLC system and identified the major peak corresponded to prodigiosin. Besides, Rajauria & Abu-

Ghanam (2013) who scraped an orange spot from the TLC plate and evaluated the compound using UV/vis spectrophotometer and found that the compound contained high fucoxanthin pigment by comparing the major peak with standard fucoxanthin.

The TLC-purified prodigiosin was further subjected to UPLC analysis for further separation, purification and identification.

6.3.4 Ultra performance of Liquid Chromatography (UPLC)

Basically, HPLC and UPLC analysis have similar function which was used to determine the purity of targeted compound. In the last decade, HPLC became researcher‟s preference as the best standard analytical tool in identification of phytoplankton pigments from seawater because of its sensitivity, rapidity and resolution (Jeffreyet al., 1999; Pasquet et al., 2011; Juin et al., 2015). HPLC is more effective in detection of major pigments compared to minor pigment. Improvement efficiency in HPLC is needed to reduce separation times in HPLC without reducing the quality of the separation by generating higher resolving power. As a solution,

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UPLC performance demonstrated commercial system capable of generating much higher pressures (1000 bar) than used in standard HPLC (Wren & Tchelitcheff, 2006).

The major and minor pigments can be easily identified by its separation quality, separation speed, data processing times, and data quality, which offered significant advantages better than HPLC (Juin et al., 2015).

Ten peaks (two major and eight minor peaks) were obtained when UPLC was used (Figure 6.7), validating the fact that UPLC was more effective in compound separation. The retention times of the two major peaks were almost similar which were

1.637 and 1.666, respectively. Lee et al., (2011) reported the same finding where the prodigiosin and cycloprodigiosin were two major metabolites produced by Zooshikella rubidus S1-1. However, the antibacterial activity of the extract decreased after further separation and purification steps. This could be the reason as to why the extract from

S. marcescens IBRL USM84 exhibited MIC values lower than the purified compound.

1.851

2.178

2.389

3.207

3.587

3.987

4.809 4.467

Figure 6.7: UPLC chromatogram of dichloromethane partitionated extract of S. marcescens IBRL USM84

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UPLC analysis of the preparative TLC exhibited only 1 major peak and 1 minor peak (Figure 6.8). The retention time of the major peak was 1.655, relative to the retention time of prodigiosin standard (1.626) (Figure 6.9). The UPLC separation of preparative TLC revealed that the purity of prodigiosin was increased by column chromatography and preparative TLC since only one major peak was detected. In

HPLC system, the retention time of red pigment produced by S marcescens were 2 to

4 minutes (Yang et al.,2013), two times longer than the retention time in UPLC system. This finding was well-correlated with the research of Wren & Tchelitcheff,

(2006) who stated the separation product using UPLC was faster than HPLC.

1.655

2.409

Figure 6.8: UPLC chromatogram of TLC-preparative purified compounds of S. marcescens IBRL USM84

Figure 6.9 shows the UPLC profile of a standard prodigiosin from S. marcescens (Sigma, Aldrich). The maximum absorption (λmax) in spectrophotometer analysis of the TLC-preparative purified compound was at 535 nm and the retention

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time (r/t) was 1.655 in UPLC, coinciding with the prodigiosin standard (λmax = 535 nm; r/t = 1.626) further confirming that the separated pigment was prodigiosin.

Figure 6.9: UPLC chromatogram of standard prodigiosin

The elution patterns of prodigiosin from S. marcescens and S. marcescens

IBRL USM84 were complementary to each other (Figure 6.10), with a slight difference in the retention time. Mostly, the antimicrobial compound isolated from

Serratia sp. belongs to prodiginine group. Several types of prodiginine pigment were identified such as prodigiosin, cycloprodigiosin, cyclononylprodigiosin, undecylprodigiosin, and butyl-meta-cyclo-heptylprodiginine (Williamson et al., 2006).

However, prodigiosin is frequently produced by environmental isolates of S. marcescens, but hardly ever found as human pathogens or clinical isolates

(Williamson et al., 2007). Thus, it was strongly believed that S. marcescens IBRL

USM84 also produced prodigiosin since this strain was isolated from marine

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environment. Furthermore, the retention time of the purified sample was similar with the prodigiosin standard.

1.626

1.655

: Standard prodigiosin

: TLC-prep purified compound

Figure 6.10: UPLC chromatogram of standard prodigiosin and TLC-preparative purified compounds of S. marcescens IBRL USM84

Besides, different types of prodigiosin are produced by different bacteria. For examples, cycloprodigiosin is produced by Vibrio gazogenes (Alihosseini et al., 2008), heptyl-prodigiosin is produced by Pseudovibrio denitrificans (Sertan-De Guzman et al., 2007) and undecycloprodigiosin is produced by Streptoverticillium rubrireticuli

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(Gerber & Stahly, 1975). Gauthier (1976) also reported that prodigiosin was also produced by marine bacteria, Alteromonas sp. but was non-identical to S. marcescens prodigiosin due to a number of dissimilarities and heterogeneity in the peak position through the HPLC analysis.

6.3.5 In vitro cytotoxicity of extract S. marcescens IBRL USM84

As quoted by Williamson et al., (2007), most of the S. marcescens are known as a major producer of the natural red pigment prodigiosin and rarely found as human pathogens. However, since S. marcescens indicates a close relationship to a clinical strain, further tests on its cytotoxicity is necessary before producing it at a large scale and applies it in industry (Li et al., 2015). In this study, the brine shrimp A. salina was used for cytotoxicity tests to evaluate the cytotoxicity level of S. marcescens IBRL

USM84 extract. The brine shrimp was a suitable test organism since it has a widespread distribution, non-selective grazing, short life cycle and highly sensitive to toxic substances (Faimali et al., 2012). A. salina is selected as bioassay organism because it seems to be an appropriate model species to evaluate the cytotoxicity of marine substances and can be used as a convenient monitor for screening and detecting the cytotoxicity level of various biological compounds.

The LC50 (50% lethality concentration) value was determined using brine shrimp lethality test (BSLT). Figure 6.11 and Figure 6.12 record the LC50 values of brine shrimp nauplii treated with dichloromethane partitionated extract of S. marcescens IBRL USM84 for 6 hours (acute cytotoxicity) and 24 hours (chronic cytotoxicity). The obtained LC50 values for both acute and chronic toxicity were 53.79

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mg/mL and 2.75 mg/mL. These values were greater than1000 µg/mL, thus, the extract was considered as non-toxic (Bastos et al., 2009).

LC50 = 53.79 mg/mL 40 35 30 25

20 Mortality (%) Mortality 15 y = 14.15x - 16.94 10 R² = 0.941 5 0 -5 0 0.5 1 1.5 2 2.5 3 3.5

Log10 Concentration

Figure 6.11: Cytotoxicity result of dichloromethane partition of S. marcescens IBRL USM84 against brine shrimp after 6 hours of exposure time (for acute cytotoxicity test)

LC50 = 2.75 mg/mL 70 60 50 40 30

20 y = 23.68x - 31.45 R² = 0.856 Mortality (5%) Mortality 10 0 -10 0 0.5 1 1.5 2 2.5 3 3.5 -20 Log10 Concentration

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)

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Meanwhile, the LC50 value of Fraction 4 was 189.63 mg/mL for acute cytotoxicity (Figure 6.13) and 15.09 mg/mL for chronic cytotoxicity (Figure 6.14).

These values indicated that Fraction 4 was also non-toxic. Based on the results obtained, both extracts were non-toxic even after the extract was subjected to separation and purification process. The cytotoxicity level decreased when the prodigiosin undergo separation using fractionation. This finding coincided with the antibacterial test where the MIC value was increased after prodigiosin underwent the separation process, indicating that the purified compound was less toxic to all tested bacteria.

LC50 = 189.63 mg/mL 40 35 30 25 20 15

Mortality (%) Mortality 10 y = 12.09x - 13.81 R² = 0.852 5 0 -5 0 0.5 1 1.5 2 2.5 3 3.5

Log10 Concentration

Figure 6.13: Toxicity result of Fraction 4 of S. marcescens IBRL USM84 against brine shrimp after 6 hours of exposure time (for acute cytotoxicity test)

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LC50 = 15.09 mg/mL 50 45 40 35 30 25 20 y = 15.83x - 16.15 15 Mortality (%) Mortality R² = 0.873 10 5 0 -5 0 0.5 1 1.5 2 2.5 3 3.5

Log10 Concentration

Figure 6.14: Toxicity result of Fraction 4 of S. marcescens IBRL USM84 against brine shrimp after 24 hours of exposure time (for chronic cytotoxicity test)

Previous studies reported that the prodigiosin produced by S. marcescens exhibited a lower value of LC50 which considered high toxic (Montaner et al., 2000;

Patil et al., 2011; Lins et al., 2014). However, the prodigiosin extract of marine isolate in this study appeared as non toxic. The most possible reason could be due to the symbiotic relationship between the bacteria and its host that limit the toxin production.

S. marcescens IBRL USM84 was found living symbiotically with the marine sponge

(Xetospongia testudinaria) which existed as a eukaryotic system. This strain may produce secondary metabolites with low toxicity to prevent the host tissue from diseases or harmful effects. This finding is related with the previous study conducted by Strobel & Daisy, (2003). They reported that most of the endophytic strains which live as symbiotic relationship exhibited low toxic level and live harmoniously in the host tissue. Furthermore, Shalinimol & Annadurai, (2012) also reported that the pink pigment prodigiosin extracted from S. marcesces was not toxic to fishes. There was no adverse effect detected in the protein, carbohydrate and lipid content of the test fishes.

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Moreover, there was not much difference between the control and pigment treated fishes. Hence, they concluded that the prodigiosin pigment was not toxic to aquatic species.

According to the antimicrobial test carried out in the early part of this study,

S. marcescens IBRL USM 84 produced natural red pigment intracellularly and exhibited significant inhibition activity against Gram positive compared to Gram negative bacteria. However, there were no inhibition activity showed against yeasts and fungi. These results indicated that prodigiosin is more effective against prokaryote but not eukaryote. This could be the reason why the pigment from S. marcescens

IBRL USM 84 did not show any cytotoxicity activity against brine shrimp

(eukaryotic). Besides, synergistic effect between the compounds present in the extract might be a factor causing reduction of toxicity level in the more purified compound.

Even though prodigiosin has been reported to have cytotoxicity effects against eukaryotic cells especially on haematopoietic cancer cell lines (Montaner et al., 2000), human melanoma cells (Lee et al., 2011), insects (Patil et al., 2011) and microalgae (Yang et al., 2013), but this study verified that prodigiosin from S. marcescens IBRL USM84 was non-toxic. These findings were also supported by the earlier study conducted by Shalinimol & Annadurai, (2012). According to Wang et al.,

(2012a), the cytotoxicity is due to the side chain length of the prodigiosin. A shorter length of alkyl chain may contribute to a higher effect of pigment cytotoxicity. The cytotoxic properties enable the compound to possess a wide variety of bioactivities such as antibacterial activity (Darah et al., 2014), antifouling activity (Priya et al.,

2013), antitumor (Pandey et al., 2007) and antimalarial activity (Papireddy et al.,

2011). Prodigiosin shows little or considered as non-toxic effects towards normal cell lines but an effective proapoptotic agent against various cancer cell lines, including

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cells that are resistant to multi-drugs (Darshan & Manonmani, 2015). They also found no genotoxic effect towards animals and the genotoxicity test exhibited prodigiosin had no significant induction of micronuclei in polychromic erythrocytes of animals at all concentrations.

Table 6.7 summarizes the cytotoxicity levels of prodigiosin at different purity.

Non-toxic properties demonstrated by the extract of S. marcescens IBRL USM84 postulated that it was safe to be used in industrial application to serve as a natural colour with antibacterial and preservative properties. Nevertheless, further study in term of in vivo and clinical test towards higher organism is needed to evaluate the cytotoxicity.

Table 6.7: Summary of cytotoxicity levels of extracts obtained from S. marcescens IBRL USM84

Concentration (mg/mL) Samples 6 hours 24 hours Cytotoxicity (acute cytotoxicity) (chronic cytotoxicity) level DCM partition 53.79 2.75 Not toxic extract Active fraction 189.63 15.09 Not toxic (Fraction 4)

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6.4 Conclusion

The prodigiosin pigment from S. marcescens IBRL USM84 was subjected to separation and purification using column chromatography and preparative-TLC. A single peak which was composite to the peak of prodigiosin standard was detected through UPLC confirming that the purified compounds belong to prodigiosin. Further identification analysis was required to exactly determine the structure of prodigiosin produced by isolate S. marcescens IBRL USM84. Hence the prodigiosin extract of S. marcescens IBRL USM84 was toxic to prokaryotic (bacteria) and non toxic to eukaryotic (brine shrimp) cells. Therefore it is a potential candidate to be used as natural color in the food, textile and cosmetic industries with antibacterial properties and natural preservative.

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CHAPTER 7.0: APPLICATION OF PRODIGIOSIN PIGMENT FROM Serratia marcescens IBRL USM84 AS COLORING AGENT AND ANTIMICROBIAL

AGENT IN LIPSTICK FORMULATION

7.1 Introduction

Pigment or colorants play a vital role in the lipstick formulation which the aesthetic value of the lipstick will be determined by this component (Anju et al.,

2017). The use of synthetic colorants in the lipstick formulation is dangerous to human health. In mild effects, the coal tars contained in the synthetic colorants can cause adverse effects like allergy, nausea, dermatitis and drying of lips whereas in a more risky form they can be carcinogenic and even death (Avinash et al., 2011). Since 2009, a small survey was conducted by U. S Food and Drug Administration (FDA) chemists in analyzing the lead level in the lipsticks sold in the U.S market. They reported that the exposure to lead in long term usage might be hazardous (Hepp, 2012). Therefore, natural colorants are very much sought for to replace synthetic colorants. The natural prodigiosin synthesized by S. marcescens IBRL USM84 through a submerged fermentation process is ecologically safe compared to chemically synthesized colorants. Prodigiosin is an alkaloid bioactive pigment that is known to have several biological properties like antibacterial, antifungal and anticancer activities (Venil &

Lakshmanaperumalsamy, 2009).

An excellent tinctorial effect and antibacterial properties have been exhibited by prodigiosin from S. marcescens IBRL USM84. It can be an added value in the cosmetic formulation to develop an antimicrobial lipstick with natural preservative.

Thus, the present chapter highlighted the potential of prodigiosin from S. marcescens

IBRL USM84 as a sustainable colorant for producing a natural lipstick and its role as an antibacterial agent. A scientific evaluation on the coloring potential of this marine

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species isolate has not been performed in the cosmetic industry. The effectiveness of prodigiosin as an antibacterial agent in the lipstick formulation was investigated and discussed in this chapter. Next, a consumer acceptance survey was conducted in order to investigate the best pigment shade accepted in lipstick formulation.

7.2 Materials and Methods

7.2.1 Lipstick formulation

Five different lipsticks composition were prepared to evaluate the best amount of ingredient in the lipstick formulation. Table 7.1 shows the ingredient with their prescribed quantity in the formulation of lipsticks. The castor oil and shea butter were heated and mixed in a 250 mL beaker using a microwave. Then, the pigment was added to the mixture, heated and stirred until mixed properly. After that, the beeswax was added to the mixture and heated until melting. The mixture was then homogenized as shown in Figure 7.1 and poured into lipstick containers. The lipsticks were left about one hour to solidify.

7.2.2 Evaluation of lipstick

All the formulated lipstick were evaluated on their melting point and surface anomalies as described by Kasparaviciene et al., (2016) for melting point and Anju et al., (2017) for surface anomalies.

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Table 7.1: Ingredients with their prescribed quantity in the lipstick formulation

Quantity (grams) Lipstick Castor oil Shea butter Bees wax Pigment formulation extract (prodigiosin)

F1 15 1 5 2.5

F2 17 2 2 2.5

F3 15 3 3 2.5

F4 13 4 4 2.5

F5 15 5 1 2.5

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

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7.2.2 (a) Melting point

One gram of each lipstick formulation was placed into a glass tube, respectively. All the glass tubes were incubated and heated in a water bath for 5 minutes for each prescribed temperature starting from 50oC. The temperature at which the mixture of lipstick turned to a liquid drop was considered its melting point. The operation was repeated three times.

7.2.2 (b) Surface anomalies

Observation on the lipsticks surface defects such as formation of crystals, the presence of fungi or moulds were done. The textures of the lipsticks were also evaluated to see whether it was smooth, rough, soft, hard or oily.

7.2.3 Antibacterial evaluation test of prodigiosin-formulated lipstick

The susceptible bacteria obtained from earlier analysis of antibacterial activity were used as test bacteria for this part of the study which were S. aureus, B. cereus, B. subtilis, MRSA and A. anitratus.

Five susceptible bacteria, S. aureus, B. cereus, B. subtilis, MRSA and A. anitratus were used as test bacteria. The prodigiosin-formulated lipstick (F3) was chosen for antibacterial evaluation. The quantitative method described by Ortiz,

(2016) and Alihosseini et al., (2008) was performed for antibacterial activity evaluation with slight modifications. One gram of prodigiosin lipstick was cut and transferred into a 250 mL shake flask. Next, 1 mL of bacterial suspension

(approximately 106 CFU/mL) grown in 10 mL nutrient broth was added onto the lipstick surface. Subsequent to the inoculation, the lipstick was incubated for 16 hours 202

at 37oC. Then, 10 mL of sterile distilled water was poured onto the lipstick surface and was agitated at 150 rpm for 5 minutes. The solution was subjected to a serial dilution up to 105 using a 10-fold dilution. Each diluted suspension (100 µL) was plated onto nutrient agar plates and were incubated for 24 hours at 37oC. The control consisted the same materials as for the test lipstick (dyed lipstick) but no addition of prodigiosin pigment (undyed lipstick). The solution of control was subjected to a serial dilution up to 1010 using 10-fold dilution. The dilution suspension (100 µL) from 105 to 1010 were plated onto nutrient agar plates and incubated for 24 hours at 37oC. The numbers of

CFU were enumerated between dyed and undyed lipsticks. The percentage reduction of test bacteria was calculated using the equation below:

Reduction (%) = (X1 – X2) X 100 X1

(Where X1 and X2 are the number of bacteria counted from the undyed and dyed lipstick, respectively)

7.2.4 Lipstick formulation for pre-market research

In this study, both natural and synthetic lipsticks formulations used were as written in Table 7.2. For natural colorant lipstick, various amounts of S. marcescens

IBRL USM84 extract which was 1.0, 2.0 and 3.0 g were used in the formulation

(Figure 7.2), while the synthetic colorants were prepared based on its best match of the naturals. The best composition of lipstick (F3) was applied to produce the lipsticks and the preparation method of lipstick formulation is as stated in Section 7.2.1.

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Table 7.2: Lipstick formulation

Lipstick formulation Importance Weight in gram (g) material

Castor oil Blending agent 15

*Natural/ synthetic color Coloring agent 1.0*/ adequate

Shea butter Softening agent 3

Bees wax Glossy and hardness 3

*Natural Red-1.0 g, Natural Reddish Purple-2.0 g, Natural Purple-3.0 g

A B C

Natural Reddish Purple

Natural Red Natural Purple

Figure 7.2: The various color of lipstick with different quantity of pigment, (A) Natural Red (1.0 g), (B) Natural Reddish Purple (2.0 g), (C) Natural Purple (3.0 g) and (D) The natural colorant lipsticks were dyed on a filter paper

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7.2.5 Pre-market survey

The survey was conducted by questionnaire form method. Lipstick samples were provided to the respondents for reference purpose regarding the color preferences and for the skin irritation test. This research involved 50 female respondents aged between 17 to 52 years old.

7.2.5 (a) Consumer acceptance investigation

The questionnaire survey regarding lipstick sources preferences by consumer whether prefer natural or synthetic sources. The irritation effects caused by the commercial lipsticks currently in-use by the consumer was also investigated to know whether it was chapped, wrinkled, dry or peeled on their lips.

7.2.5 (b) Skin irritation test

The natural colorant lipstick was applied on the skin and left in contact for 10 minutes. The reaction on the skin was observed if any (Avinash et al., 2011).

7.2.5 (c) Ranking Test

A consumer acceptance was done using the Ranking test method as described by (Fisher & Rothamstead, 1982). All the data collected from the Ranking test were analyzed using the Scientific Package for Social Science (SPSS) software in order to determine the best pigment accepted in lipstick formulation. The p value of < 0.05 was considered as significant. Six lipstick samples were prepared, namely Natural Red,

Natural Reddish Purple, Natural Purple (natural colorant), Synthetic Red, Synthetic

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Reddish Purple and Synthetic Purple (synthetic colorant). Likert scale below was used for color parameters.

Preferred 1 2 3 4 5 Color : Not Somewhat Neutral Somewhat Interested interested uninterested interested

7.3 Results and Discussion

7.3.1 Lipsticks evaluation

Most women used the lipstick to enhance the beauty of their lips (Anju et al.,

2017). There are some dominant characteristics for lipstick evaluation which includes texture, melting point and hardness in order to produce a good lipstick and be acceptable to consumers (Deshmukh et al., 2013). The modification of the lipstick formulation is required to produce the best lipstick composition which contained the prodigiosin pigment as a natural colorant. The quantity of ingredients adjusted to get high in melting point and no defect on the surface of the lipsticks. Table 7.3 shows the evaluation of the lipstick formulations and it was found that the natural colorant lipstick formulation (F3) was the best among the five formulations. Both of the lipstick formulations of F1 and F4 showed higher value of melting point which were 68.0oC and 67.0oC, respectively compared to F3 (64oC). There was no defect on the surface of the lipsticks. However the textures of F1 and F4 were harder and a bit rough which might be due to the higher content of bees wax in the formulations. Meanwhile, the lipstick formulation F2 and F5 exhibited the same melting point (60oC) where the value was lower than F3. The surfaces of these lipsticks were too oily and softer because they contained more castor oil and lack of bees wax. 206

Table 7.3: Evaluation of natural colorant lipsticks

Evaluation Natural colorant lipstick formulation parameter F1 F2 F3 F4 F5 Color Purple Purple Purple Purple Purple

Melting 68.0 60.0 65.0 67.0 60.0 point (oC) Texture Hard Soft and Smooth Rough Soft and oily oily Surface No defect No defect No defect No defect No defect anomalies

A good lipstick must be high in melting point to avoid technical downturn during preparation and application. Rajin et al., (2007) reported that the melting point of lipsticks to be in the 60.6 to 64.0oC acceptable limited by the consumer as obtained in the lipstick formulation (F3). The results also revealed that the higher amount of beeswax increased the value of melting point. Previous studies also found that the melting point and hardness of the lipsticks had strong correlation with the amount of waxes (Kamairudin et al., 2014). The lipstick formulation (F3) was selected as the best composition to produce the various lipsticks for pre-market survey, subsequently.

7.3.2 Antibacterial evaluation test of prodigiosin-formulated lipstick

The capability of pathogenic microbes to contaminate the cosmetic products, cross-infections and also the consumers craving to use the natural sources and hygienic cosmetic have led to the need for the establishment of an antimicrobial lipstick. Since the prodigiosin pigment from S. marcescens IBRL USM84 is known to

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poses antibacterial activity, hence the lipstick dyed with the prodigiosin pigment was evaluated for the antibacterial activity. Bacterial densities on lipsticks inoculated with

S. aureus, B. cereus, B. subtilis, MRSA and A. anitratus after 16 hours of incubation are shown in Table 7.4.

Table 7.4: Antibacterial evaluation of lipsticks dyed with antibacterial prodigiosin pigment against different bacteria

No. bacteria after 16 hours (CFU/mL) Percentage of Test bacteria Undyed Dyed bacterial reduction (control lipstick) (prodigiosin pigment) (R% ± SD)

S. aureus 2.96 x 108 2.02 x 105 99.93 ± 0.01 B. cereus 1.57 x 108 1.06 x 105 99.93 ± 0.00 B. subtilis 2.73 x 108 1.04 x 106 99.62 ± 0.01 MRSA 1.12 x 108 1.05 x 105 99.91 ± 0.00 A. anitratus 2.51 x 108 1.52 x 106 99.39 ± 0.02

The enumeration density of 5 test bacteria on dyed lipstick showed a remarkable decrease compared to the control (undyed lipstick). All the tested bacteria showed more than 99.0% of bacterial reduction. The ability of the dyed lipstick to inhibit 99.9% of S. aureus and MRSA are a great finding. S. aureus is the most common isolate from cosmetics and cosmetic tools (Birteksoz et al., 2013). The increase in antibiotic resistance by S. aureus is a growing concern which makes this pathogen becomes an important public health issue (Chen & Tsao, 2013). Meanwhile,

MRSA is a major nosocomial pathogen and potentially life threatening to human

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populations (Darabpour et al., 2011). Lucerna et al., (2015) reported that the MRSA was infected a lip by mimicking angioedema. Wrong identification of MRSA lip infection for angioedema which was not receiving appropriate treatment can cause mortality.

Asrul et al., (2017) reported that the lipstick dyed with plant pigment extract obtained from dragon fruit had the ability to kill S. aureus. However, B. cereus showed a profound resistance towards the extract. The lipstick dyed with prodigiosin produced by S. marcescens IBRL USM84 exhibited 99.9% of B. cereus reduction, indicating a better antibacterial activity of this pigment. Furthermore, the antibacterial activity of prodigiosin from S. marcescens IBRL USM84 was somewhat comparable to commercially-available cosmetic cleaners that were applied on cosmetic products

(Ortiz, 2016). The wipes and spray (70% isopropanol alcohol) used as the cleaning agent for lipstick can kill 99.77% and 99.56% of S. aureus, respectively.

Cosmetic products have high probability to be contaminated with pathogenic microbes at the first moment they are opened. Microorganisms in cosmetics not only affect consumer health, but they can also lead to spoilage or downgrade of the product quality (Birteksoz et al., 2013). Besides, the misconception by consumers like sharing the cosmetic products, the addition of water to thin out the cosmetic and improper storage will greatly increase the contamination which can cause infections (Pack et al.,

2008). Therefore, the addition of preservatives in cosmetics product is needed to regulate microbial contamination during the production, storage, and use of the product (Herman et al., 2013). The main preservatives found in cosmetic products are parabens group which included methylparaben, propylparaben, butylparaben, and ethylparaben that are produced synthetically (Lundov et al., 2009). However, these synthetic preservatives have much controversy in health issue. Birteksoz et al., (2013)

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suggested that parabens can cause reproductive and endocrine dysfunctions. The uses of synthetic ingredients on the lips often caused irritation like dry, chapped and wrinkled lips to the consumers (Azwanida et al., 2014). Thus, the prodigiosin pigment with antibacterial activity produced by S. marcescens IBRL USM84 can be natural alternative preservatives in the cosmetic products.

Due to its ability to inhibit high number of S. aureus, hygienic cosmetic tools such as brushes and sponges made from prodigiosin dyed material can also be proposed. Naz et al. (2012) who tested the microbial contamination of cosmetic brushes and sponges used in beauty salons found these tools were contaminated with

S. aureus. The average contamination of these tools was 105 CFU/mL. S. aureus which was also can cause skin infections such as conjunctivitis, impetigo, boils, and folliculitis (Birteksoz et al., 2013).

7.3.3 Pre-market research

From the survey‟s results that involved 50 female respondents, there were more than 50% cases of irritation on lips after applying the commercialize lipsticks from the current market (Figure 7.3). The dry or peeled lips problem (38%) was the highest percentage of irritation followed by chapped lips (8%) and wrinkled lips (6%).

The dry lips problem could be due to the dyes which contained coal tars that contribute to the color of the lipstick (Swati et al., 2013). Moreover, the lipsticks are often eaten away by the user when applied it on their lips. This not only affects the lips but subsequently threaten the consumer health. Azwanida et al., (2015) reported that more than 50 % of female respondent faced irritation on lips when used the lipstick bought from current market. The dry lips problem also reached the highest case of

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irritation. More than 90% of women preferred natural color source of lipstick rather than synthetic lipstick. The results showed the consumers out there are already realized the dangerous of synthetic ingredient contained in the lipstick. Most of them hesitated the chemical base products and prefer the natural products to enhance their beauty and for their personal care. Previ ous studies also reported the natural color lipsticks prepared from Bixa orellana seeds (Swati et al., 2013) and lycopene extract of

Solanum lycopersicum L (Bhagwat et al., 2017). The research objective is to use the natural pigment as a coloring agent in the lipstick formulation for having no or minimum side effects.

100 6 90 80 48 70 None 60 50 Chapped Lips 8 94 40 6

Percentage(%) 30 Wrinkled Lips/ No 20 38 10 Dry/ Peeled Lips/ Yes 0 Skin Irritation Prefer Natural

Figure 7.3: Consumer acceptance survey

[Bar chart showed the percentages of cases according to consumer‟s responses where the skin irritation bar inclusive of different irritation cases as indicated in the legend and the preference towards natural lipstick]

The skin irritation test was done on respondent with their agreement. Table

7.5 shows no side effect of the natural colorant lipstick when applying it on the skin.

The results showed no irritation effect, itchy or reddish mark appeared on the skin

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after 10 minutes applying the natural colorant lipstick on the respondent skin (Figure

7.4). The natural colorant lipstick has no skin irritation effects could be due to the extract from S. marcescens IBRL USM84 was not toxic as obtained in the cytotoxicity test results from Chapter 6.0 (Section 6.3.5)

Table 7.5: Evaluation of skin irritation test

Inference

Test Natural Red Natural Reddish Natural Purple Purple

Skin Irritation No effect No effect No effect

A B

Figure 7.4: Skin irritation test on the skin, (A) natural colorant lipstick was applied on the skin, (B) no effect on the skin after 10 minutes

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Figure 7.5 shows the Ranking Test results score of six lipstick samples for color preference. The natural red lipstick reached the highest score (224 frequency) followed by natural reddish purple (203 frequency) and synthetic reddish purple (141 frequency). Azwanida et al., (2015) also reported that the natural red lipstick prepared from the extract of Hylocereus polyrhizus obtained the highest score. This is because the red color is a common lipstick color known by community since long ago.

Meanwhile, the purple color was less preferred and it might be due to the not so common in lipsticks industry.

250

200

150 Natural red Natural reddish purple 100 Frequency Natural purple 50 Synthetic red Synthetic reddish purple 0 Synthetic purple red reddish purple red reddish purple purple purple

Natural Synthetic

Figure: 7.5: Consumer acceptance based on color

[Bar chart showed the frequency of consumer acceptance towards Lipstick‟s color of different colorant. Highest bar indicates highest ranked lipstick, where its color is the best accepted]

Overall, the consumers preferred the natural colorant lipstick compared to the synthetic colorant lipstick. The two highest score were natural colorant lipsticks namely natural red and natural reddish purple revealed that society has comprehension

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and awareness to choose the natural base sources. Figure 7.6 shows the various concentration of pigment from S. marcescens IBRL USM84 used in the lipstick formulation and the best matching of synthetic colorant lipstick for pre-market survey.

However, the range of natural color spectrum is still limited compared to synthetic dyes (Rajguru et al., 2016). Further studies on the lipstick production using natural pigment as a coloring agent need to be expanded to meet consumer demand that want the different shades of color. Microorganisms are one of the abundant pigment sources that can contribute to cosmetic industry.

Natural Red Natural Reddish Natural Purple Purple

N1 N2 N3

A B C

Synthetic Red Synthetic Synthetic Reddish Purple Purple S1 S2 S3

D E F

Figure 7.6: The various concentration of lipstick for pre-market survey, formulated from natural colorant (A) Natural Red, (B) Natural Reddish Purple, (C) Natural Purple while from synthetic colorant (D) Synthetic Red, (E) Synthetic Reddish Purple, (E) Synthetic Purple

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7.4 Conclusion

The prodigiosin extract has a potential to be used as a coloring agent in lipstick for different shades (red, reddish purple and purple). This natural pigment of S. marcescens IBRL USM84 can be commercially produced by fermentation process in high scale industry. The prodigiosin dyed lipstick showed an ability to inhibit all the tested bacteria which provided a broader application range such as in cosmetic, food and therapeutic industries.

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CHAPTER 8.0 GENERAL DISCUSSION

There is an abundance of natural pigment sources from marine environment that still remain largely unexplored, understudied, and underexploited rather than terrestrial ecosystems and organisms (Joint et al., 2010). Symbiotic bacteria is one of the promising source of the natural antibacterial compounds that living symbiotically or transiently on the surface of marine invertebrates (Konig et al., 2006;

Brinkmannet al., 2017). In chapter 3.0, a marine bacterium S. marcescens IBRL

USM84 was isolated from marine sponge Xetospongia testudinaria from Pulau

Bidong, Terengganu exhibited the antibacterial activity from intracellular was greater than extracellular. 2-propanol was the most effective extractant for the intracellular pigment produced by S. marcescens IBRL USM84, where the effectiveness of the solvent was evaluated in term of pigmentation strength and antimicrobial property

(Teh Faridah, 2012). From the results obtained, the prodigiosin from intracellular extract was more dominant compared to extracellular extract. The spectral analysis characterization of intracellular pigment extract from S. marcescens IBRL USM84 appeared red in color and showed a sharp spectral peak at 535 nm in an acidic condition. Meanwhile, the pigment extract appeared orange-yellow in colour and exhibited a broader spectral curve centered at 465 nm in an alkaline condition. The results indicated the natural red pigment produced by this strain containing prodigiosin as described by Williams et al., (1955). Pigments are synthesized intracellular by microorganisms always play a vital role in protecting themselves from disadvantageous environmental conditions such as lack of nutrient (Haileiet al.,

2012), ultraviolet radiation and lethal photo oxidation (Mapari et al., 2005).

The enhancement process revealed that culture duration, pH, temperature, agitation speed, percentage of agar, carbon sources, nitrogen sources and inorganic

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salt significantly affected the production of prodigiosin by S. marcescens IBRL

USM84. Meanwhile, light illumination had no significant effect on the pigment production. However, the pigment yield and antibacterial activity did not increase when the cultivation medium was supplemented with nitrogen sources and inorganic salt. The results showed that the addition of maltose into the cultivation medium consistently yielded a fifteen fold increment in prodigiosin production over the existing medium.

Based on the results obtained in chapter 5.0, dichloromethane partitionated extract was selected as potential extract since this partitionated extract demonstrated intense purplish-pink pigmentation with greater antibacterial activity. The prodigiosin from S. marcescens IBRL USM84 remarkably inhibited the growth of

Gram positive and negative bacteria, including S aureus, B. cereus, B. subtilis,

MRSA and A. anitratus. According to physical characteristics, prodigiosin exhibited broad stability range in temperature, neutral to acidic condition, shelf life and light illumination. These special characteristic can be high opportunity for this pigment to be used in industrial technologies.

The red pigment produced by S. marcescens IBRL USM84 was isolated and characterized. The pigment corresponded well to prodigiosin where the maximum

UV/vis absorption was at 535 nm. The presence of prodigiosin in the extract of S. marcescens IBRL USM84 was further confirmed when a combination of column chromatography, preparative TLC and UPLC methods were used. A high correlation between the pigmentation and antibacterial activity was also confirmed. The cytotoxicity test also found that the prodigiosin was not toxic to higher organisms, especially aquatic spesies (Shalinimol & Annadurai, 2012).

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A reasonable scientific justification for pigment extract application in cosmetic industry was also given. The prodigiosin dyed lipstick has potential in reducing acceptable number of colonies on its surface. Therefore, the results indicated that the bacterial pigment from S. marcescens IBRL USM84 could be used as an alternative natural colorant resource for cosmetic dyeing and natural preservatives with antibacterial activity. The natural color from prodigiosin has high potential as a coloring agent for lipstick to give different shades of color from red to purple depending on the amount of pigment used in the lipstick formulation.

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CHAPTER 9.0 GENERAL CONCLUSION AND RECOMMENDATIONS FOR

FUTURE STUDY

9.1 General conclusion

In this study, S. marcescens IBRL USM84 isolated from marine sponges,

Xetospongia testudinaria was shown to be rich in antibacterial properties. Prodigiosin, a pigment derived from this bacterium noted a high correlation with the antagonistic activity. The antibacterial prodigiosin produced by this strain acts as biological weapon to protect itself and its host form pathogenic attack or marine predators. Some conclusions can be made based on the results obtained from the present study:

1. The marine bacterium isolate, S. marcescens IBRL USM84 produced the natural red pigment prodigiosin intracellularly, where the pigments were mainly deposited at the cell envelope of this isolate.

2. The yield of prodigiosin and antibacterial activity influenced by some physical cultivation factors including culture duration, pH, temperature, agitation speed, percentage of agar but less affected by light illumination. Meanwhile, the addition of maltose in the cultivation medium highly increased the pigment production.

3. Dichloromethane was selected as the best extractant for extracting the prodigiosin pigment since this extract exhibited greater antibacterial activity against four Gram positive bacteria (S. aureus, B, cereus, B, subtilis and MRSA) and one Gram negative bacteria (A. anitratus) . The MIC values obtained for this extract was same towards all

219

the tested bacteria which were 250 µg/mL. Meanwhile, the mechanisms of antibiosis were bactericidal since the ratios of MBC to MIC were below 4.

4. From the separation and purification of pigment analysis indicated the pigment produced by S. marcescens IBRL USM84 had similar characteristic with standard prodigiosin. Further confirmation using UPLC detected a single peak which was composite to the peak of prodigiosin standard confirming that the purified compound was belongs to prodigiosin.

5. An attractive color of natural pigment from S. marcescens IBRL USM84 not only can be used as a colouring agent in cosmetic products but as natural preservatives with antibacterial activity which had broad application in cosmetic, food and pharmaceutical industries.

9.2 Recommendation for future study

Based on the findings obtained from this research study, there are some recommendations that can be suggested in order to fully utilise the potential of natural red pigment produced by S. marcescens IBRL USM84 as source of a colouring agent with antibacterial compounds. The high consumer demand in getting the different shades of color for cosmetic products, suggested the extraction of this pigment using different types of solvent. The colour of extract can be influenced by the solvent that has been used in the extraction process. However, the effectiveness of the extract towards antibacterial properties must be evaluated and attainable.

Based on the cytotoxicity test of prodigiosin extract of S. marcescens IBRL

USM84 indicated that this pigment was not toxic to aquatic species and higher

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organism (eukaryote). Before the pigment could be accepted for industrial application, detailed cytotoxicity study is required towards test animals such as mice, rabbits or monkeys to confirm either the pigment confirmed toxic to higher organisms or safe to use. Besides that, further clinical tests are needed to access the cosmetic formulation for better efficacy. On the other hand, the prodigiosin pigment from S. marcescens

IBRL USM84 had broad application as a natural colouring agent and natural preservatives in foods and beverages industries since the cytotoxicity test results considered this pigment was not toxic to higher organism. To date, studies on natural coloring agent for lipstick products using prodigiosin extract from S. marcescens

IBRL USM84 strain have not been carried out elsewhere, thus, this study has created a novelty by producing a pigment from natural resources.

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APPENDICES

Appendix 1: Standard curve of total phenolic content for gallic acid

2.5

1.5

1 y = 0.001x + 0.180 R² = 0.933

Absorbance (atAbsorbancenm) 760 0.5

0 0 250 500 750 1000 1250 1500 1750 2000 2250 2500 Concentration (µg GAE/mg of extract)

Appendix 2: Standard prodigiosin calibration curve

12 y = 0.187x + 0.017 10 R² = 0.991 8 6 4 2

0 Absorbance at 535nm at Absorbance 0 10 20 30 40 50 60 Concentration of Prodigiosin (µg/mL)

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Appendix 3: Standard curve for growth profile

35 y = 5.998x - 1.273 R² = 0.991 30

15 OD (620nm) OD 10

0 0 1 2 3 4 5 6 7 Dry Cell Weight (g/L)

Total Dry Cell Weight Linear (Total Dry Cell Weight)

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Appendix 4: SPSS - Enhancement

1) Culture duration

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2) Light illumination

3) Initial pH

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4) Incubation temperature

5) Agitation speed

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6) Percentage of agar

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7) After enhancement (physical parameters)

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8) Carbon sources

*NOTE: Glucose-1, Sucrose-2, Lactose-3, Maltose-4, Fructose-5, Inositol-6, Starch-7

9) Nitrogen sources

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*NOTE: Ammonium Oxalate-1, Urea-2, Tryptone-3, Yeast Extract-4, Peptone-5, Ammonium Acetate-6, Casein-7

10) Percentage of NaCl

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11) Percentage of maltose

12) After enhancement (physical and chemical parameters)

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Appendix 3: SPSS – Characteristics

1) Temperature

273

2) pH

274

3) Light illumination i) Dark (unexposed)

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ii) Light (exposed)

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4) Incubation time

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Appendix 5: QUESTIONNAIRE

AGE: _____ GENDER: ______OCCUPATION:______Please spare a few minutes of your valuable time to answer this simple questionnaire 1. Which sources of color that you prefer in your lipstick formulation? A) Natural B) Synthetic

2. Do you have skin irritation problem on your lips after applying the lipstick bought from current market? A) Yes B) No If yes, please answer the question number 3. 3. What type of the skin irritation? A) Wrinkled Lips B) Chapped Lips C) Dry/Peeled Lips 4. Which lipstick colors listed below to be your preference? Give your answers base on the likert scale provided.

Preferred 1 2 3 4 5 Color : Not Somewhat Neutral Somewhat Interested interested uninterested interested

Natural Natural Natural Red Reddish purple Purple

N1 N2 N3

Synthetic Synthetic Synthetic Red Reddish purple Purple

S1 S2 S3

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LIST OF PUBLICATIONS

PROCEEDINGS AND CONFERENCES

Teh Faridah, N., Wan Norhana, N., Jain. K. and Darah, I. (2016). Antibacterial Activity of Various Extracts of Serratia marcescens IBRL USM84 from Marine Isolate. In: Friday Seminar, October 2016, School of Biological Sciences, Universiti Sains Malaysia (USM), Pulau Pinang, Malaysia.

Teh Faridah, N., Wan Norhana, N., Jain. K. and Darah, I. (2016). Antibacterial Activity of Serratia marcescens IBRL USM84 Extracts. In: 10th Indonesia Malaysia Thailand Golden Triangle (IMTGT) UNINET Conference, December 2016, Faculty of Natural Resources, Prince of Songkhla University, Hat Yai, Songkhla, Thailand.

Teh Faridah, N., Wan Norhana, N., Jain. K. and Darah, I. (2016). Colour Stability Evaluation of Pigment Extracted from Serratia marcescens IBRL USM84. In: 10th Indonesia Malaysia Thailand Golden Triangle (IMTGT) UNINET Conference, December 2016, Faculty of Natural Resources, Prince of Songkhla University, Hat Yai, Songkhla, Thailand.

Teh Faridah, N., Wan Norhana, N., Jain. K. and Darah, I (2014). Antibacterial and Toxicity Studies of the Natural Red Pigment Produced by a Local Strain of Serratia marcescens IBRL USM84. In: 9th Indonesia Malaysia Thailand Golden Triangle (IMTGT) UNINET Conference, November 2014, Gurney Hotel, Pulau Pinang, Malaysia.

Teh Faridah, N., Wan Norhana, N., Jain. K. and Darah, I. (2014). Optimization of physical and chemical parameters for the production of prodigiosin natural pigment by a marine bacterium Serratia marcescens IBRL USM84 in submerged fermentation system.In: 9th Indonesia Malaysia Thailand Golden Triangle (IMTGT) UNINET Conference, November 2014, Gurney Hotel, Pulau Pinang, Malaysia.

Teh Faridah, N. and Darah, I. (2013). Development Production of a Natural Red Pigment with Antibacterial Prodigiosin Compounds by a Local Strain of Serratia marcescens USM84. In: Proceedings of the 8th Annual PPSKH Postgraduate Biocolloqium, June 2013, School of Biological Sciences, Universiti Sains Malaysia (USM), Pulau Pinang, Malaysia.

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JOURNALS

Darah, I., Teh Faridah, N., Jain, K. and Lim, S. H. (2018). Influence of physical and chemical parameters for prodigiosin natural red pigment production by Serratia marcescens IBRL USM84. Process Biochemistry (under review).

Ibrahim, D., Nazari, T. F., Kasim, J. and Lim, S. -H. (2014). Prodigiosin - An antibacterial red pigment produced by Serratia marcescens IBRL USM 84 associated with a marine sponge Xestospongia testudinaria. Journal of Applied Pharmaceutical Science, 4 (10): 001-006.

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