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SPRAY DRIED PRODIGIOSIN FROM Serratia marcescens AS A FOOD COLORANT

SHAHLA NAMAZKAR

UNIVERSITI TEKNOLOGI MALAYSIA

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SPRAY DRIED PRODIGIOSIN FROM Serratia marcescens AS A FOOD COLORANT

SHAHLA NAMAZKAR

A dissertation submitted in partial fulfillment of the requirements for the award of the degree of

Master of Science (Chemistry)

Faculty of Science Universiti Teknologi Malaysia

Jan 2013

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To my beloved mother and father

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ACKNOWLEDGEMENT

First and foremost, I would like to acknowledge the lessons, support and

guidance of my supervisor, Prof. Dr. Wan Azlina Ahmad. Her continuous

commitments to research studies ensure not only constant results but also have

granted her the respect of all her students. I thank her for giving me the opportunity

to carry out this research in the fields of biochemistry and biotechnology.

I’d like to thank all my friends in biotechnology laboratory: Kak Diana, Kak

Wan, Kak Rozi, Risha, Kak Shakila, Shuhada, Kak Zaleha, En. Ali, Iezzat and

Riyadh for their valuable experience and opinions along this study. I also would like

to thank all lectures and staffs of Chemistry department especially to En. Jefri, En.

Amin for their help with FESEM, FTIR and UV-Visible analysis.

My sincere appreciation also extends to my parents and my sister for their

support, encouragement, care and love.

Last but not least, for anyone I’ve forgotten that has involved directly or

indirectly in completing this project, thank you.

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ABSTRACT Pigments from microorganisms can serve as an alternative source to

replace synthetic pigments used in the food industry. Natural pigments have

some limitations including solubility, sensitivity and short stability upon

exposure to light, pH and high temperature. Thus, encapsulation via spray drying

can be employed to enhance the pigment’s properties. In this study, spray-dried

microcapsules containing red pigment (prodigiosin) extracted from Serratia

marcescens was produced using κ-carrageenan and maltodextrin as encapsulation

agents. The effect of spray-drying parameters on the encapsulation yield (EY),

particle size, moisture content and colour intensity of the prodigiosin

microcapsules at different ratios of prodigiosin/encapsulation agent were studied.

The most intense colour was obtained for the 1:1 ratio (volume ratio of

prodigiosin in ethyl acetate to κ-carrageenan solution) using 200oC inlet

temperature, feed flow rate of 60 m3/h, air pressure of 1.5 bar and feed rate of 3

mL/min. The encapsulated pigment is most stable in powder form at 0°C when

stored in the dark, and thus has superior stability compared to pigment in its free

form. Characterization of spray-dried prodigiosin using FTIR and FESEM

confirmed that the particles were properly coated with encapsulating agents. The

morphology showed the particles were regular shaped spheres with mean

diameters between 0.5µm and 5µm. Finally, the particles were successfully

applied to milk, yogurt and carbonated drinks. The results suggest that the spray-

dried prodigiosin can be useful as a food colorant under the above optimum

operating conditions.

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ABSTRAK Pigmen yang diekstrak daripada mikroorganisma boleh dijadikan sebagai

sumber alternatif bagi menggantikan pigmen tiruan dalam industri pemakanan.

Pigmen semulajadi mempunyai beberapa kelemahan termasuk keterlarutan,

kepekaan dan kestabilan yang rendah terhadap cahaya, pH dan suhu yang tinggi.

Justeru itu, pengkapsulan melalui pengeringan sembur boleh digunakan bagi

menambahbaik ciri pigmen tersebut. Dalam kajian ini, pemikrokapsulan secara

pengeringan sembur bagi pigmen merah (prodigiosin) yang diekstrak daripada

Serratia marcescens telah dihasilkan dengan menggunakan κ-karagenan dan

maltodekstrin sebagai agen pengkapsulan. Kesan pengeringan sembur terhadap

beberapa parameter seperti hasil pengkapsulan, saiz zarah, kandungan kelembapan

dan keamatan warna prodigiosin yang telah dikapsulkan pada nisbah prodigiosin /

agen pengkapsulan yang berbeza telah dikaji. Keamatan warna yang paling tinggi

diperolehi pada nisbah 1:1 (nisbah isipadu prodigiosin di dalam etil asetat terhadap

larutan κ-karagenan) dengan menggunakan suhu masukan 200°C, kadar aliran

suapan 60 m3 / h, tekanan udara 1.5 bar dan kadar suapan 3 mL / min. Prodigiosin

yang telah dikapsulkan lebih stabil dalam bentuk serbuk pada 0°C apabila disimpan

di tempat yang gelap dan kestabilannya lebih baik berbanding pigmen yang tidak

dikapsulkan. Pencirian serbuk prodigiosin menggunakan FTIR dan FESEM

mengesahkan bahawa zarah prodigiosin telah disalut sepenuhnya dengan agen

pengkapsulan. Analisis morfologi menunjukkan partikel prodigiosin berbentuk sfera

dan berdiameter di antara 0.5μm dan 5μm. Akhir sekali, serbuk prodigiosin tersebut

telah berjaya diaplikasikan pada susu, yogurt dan minuman berkarbonat. Hasil kajian

mendapati serbuk prodigiosin boleh digunakan sebagai pewarna makanan dengan

menggunakan parameter optimum yang tersenarai di atas.

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TABLE OF CONTENTS

CHAPTER

TITLE

PAGE

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENT iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES xi

LIST OF FIGURES xii

LIST OF ABBREVIATIONS/ SYMBOLS xiv

LIST OF APPENDICES xvi

1 INTRODUCTION

1.1 Background of Study 1

1.2 Problem Statement 1

1.3 Objectives of Study 2

1.4 Scope of Study 2

1.5 Significance of Study 3

2 LITERATURE REVIEW

2.1 Natural Pigments 4

2.2 Bacterial Pigments 4

2.2.1 Serratia marcescens 5

2.2.2 Prodigiosin 6

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2.3 Encapsulation Technique 7

2.4 The Spray Drying Method and Process 12

2.4.1 Atomization 13

2.4.2 Air Flow 13

2.4.2.1 Co-Current Flow 13

2.4.2.2 Counter-Current Flow 14

2.4.2.3 Mixed Flow 14

2.4.3 Drying Chamber 15

2.4.4 Powder Separation System 15

2.5 Coating Materials 15

2.5.1 Carrageenan 16

2.5.2 Maltodextrin 18

3 EXPERIMENTAL

3.1 Materials 20

3.1.1 Glassware and Chemicals 20

3.1.2 Prodigiosin 20

3.1.3 Bacteria 20

3.1.4 Growth Media 21

3.1.4.1 Nutrient Broth 21

3.1.4.2 Nutrient Agar 21

3.1.5 Active Culture 21

3.2 Methods 22

3.2.1 Preparation of Encapsulation Agents 22

3.2.1.1 Kappa-carrageenan 22

3.2.1.2 Maltodextrin 22

3.2.2 The Spray Drying Procedure 22

3.2.2.1 Preparation of Feed Solution 22

3.2.2.2 Parameters 23

3.2.3 Melting Point 23

3.2.4 Colour Intensity 24

3.2.5 Moisture Content of the Spray-Dried 24

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Prodigiosin

3.2.6 Stability Tests and Storage Condition 24

3.2.7 Solubility of the Spray-Dried Prodigiosin 25

3.2.8 Field Emission Scanning Electron

Microscopy (FESEM)

25

3.2.9 Fourier Transform Infrared (FTIR)

Spectroscopy

25

3.2.10 UV-VIS Spectroscopy 26

3.3 Antimicrobial Properties of the Spray-Dried Particles 26

3.4 Application of the Spray-Dried Pigments in Food

Products

27

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3.4.1 Milk 27

3.4.2 Yoghurt 27

3.4.3 Colourless Carbonated Drink

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4 RESULS AND DISCUSSION

4.1 Encapsulation of Prodigiosin via Spray Drying

Process

29

4.2 The Effect of Spray Drying Parameters 29

4.3 Moisture Content Determination 32

4.4 Stability and Storage Conditions 33

4.4.1 The Effect of pH on Stability of Encapsulated

Prodigiosin

33

4.4.2 The Effect of Temperature on Stability of

Encapsulated Prodigiosin

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4.4.3 The Effect of Light on Stability of

Encapsulated Prodigiosin

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4.5 Solubility of Encapsulated Prodigiosin 39

4.6 Characterization of Encapsulated and Crude

Prodigiosin

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4.6.1 FTIR Spectroscopy 39

4.6.2 Morphology 41

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4.6.3 UV-Visible Spectroscopy 44

4.7 Antimicrobial Properties of Spray-dried Prodigiosin 45

4.8 Application in Food System 46

5 CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion 47

5.2 Recommendation 47

REFERENCES 48

APPENDIX

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LIST OF TABLES

TABLE

NO.

TITLE PAGE

2.1 Microorganisms and their pigments. 5

2.2 Overview of common microencapsulation processes. 10

2.3 General advantages of encapsulation technique in food

products.

11

2.4 Commonly used coating materials in the food industry. 16

3.1 Spray drying parameters. 23

4.1 The effect of spray drying parameters on encapsulation of

prodigiosin using κ-carrageenan 1% w/v.

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4.2 The effect of spray drying parameters on encapsulation of

prodigiosin using maltodextrin 5% w/v.

30

4.3 Half-life profiles, regression and degradation constant of

spray-dried prodigiosin degradation using zero order reaction

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4.4 FTIR characteristic bands of crude prodigiosin, encapsulated

prodigiosin coated with κ-carrageenan and maltodextrin.

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4.5 Killing percentage of crude and encapsulated prodigiosin. 45

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LIST OF FIGURES

FIGURE

NO.

TITLE PAGE

2.1 Colonies of Serratia marscescens grown on nutrient agar. 6

2.2 Prodigiosin Structure; 4-methoxy-5-[(Z)-(5-methyl-4-pentyl-

2H-pyrrol-2-ylidene) methyl]-1H, 1′H-2, 2′-bipyrrole.

7

2.3 Core and wall materials position in microcapsules. 8

2.4 Types of microcapsules; (a) simple sphere surrounded by a

coating of uniform thickness (b) particle containing an

irregular shape core (c) several core particles embedded in a

continuous matrix of wall material (d) several distinct cores

within the same capsule and multi-walled microcapsules.

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2.5 Schematic of Spray Dryer LU-222(SD-05). 12

2.6 Schematic of co-current flow. 13

2.7 Counter-Current Flow. 14

2.8 Combined Flow. 14

2.9 Chilean Seaweed. 17

2.10 Molecular structure of Carrageenan, R=H, SO3 for κ-

Carrageenan and ι-Carrageenan respectively.

17

2.11 Gelatinization of κ-carrageenan. 18

2.12 Maltodextrin unit. 19

4.1 The effect of inlet temperature on moisture content

percentage of spray dried prodigiosin coated with κ-

carrageenan and maltodextrin.

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4.2 The effect of pH on the stability of spray-dried prodigiosin

encapsulated with κ-carrageenan (a) in the presence of light;

(b) in the absence of light.

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4.3 The effect of temperature on the stability of spray-dried

prodigiosin coated with κ-carrageenan in liquid (l) and

powder (p) form.

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4.4 The effect of light on stability of spray-dried prodigiosin

coated with κ-carrageenan in powder (p) and liquid (l) forms.

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4.5 The solubility of spray-dried prodigiosin in (A) acetone, n-

hexane, ethyl acetate; (B) HCl and NaOH solutions; (C)

distilled water and (D) crude prodigiosin.

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4.6 FESEM images of (1) spray-dried prodigiosin coated with κ-

carrageenan (2) maltodextrin; dried at 200°C (A1 and A2),

170°C, (B1 and B2), 150°C (C1 and C2) inlet temperature.

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4.7 The effect of three inlet temperatures on particle size

distribution of spray-dried prodigiosin coated with κ-

carrageenan and maltodextrin.

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4.8 UV-Vis spectra of crude prodigiosin (red) prodigiosin coated

with κ-carrageenan dried at inlet temperature of 200°C

(green), 170°C (pink) and 150°C (light green); prodigiosin

coated with maltodextrin dried at inlet temperature of 200°C

(brown), 170°C (purple) and 150°C (dark blue).

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4.9 Application of spray-dried prodigiosin powder (1) coated

with κ-carrageenan in (a) yogurt, (b) milk, (c) colourless

carbonated drink, (2) coated with maltodextrin, (a) yogurt,

(b) milk, (c) colourless carbonated drink.

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LIST OF ABBREVIATIONS / SYMBOLS

[A] - Absorption

B.subtilis - Bacillus subtilis

Ch.violaceum - Chromobacterium violaceum

cm - Centimeter

DDW - Dionized Distilled Water

E.coli - Escherichia coli

EY - Encapsulation yield

FESEM - Field Emission Scanning Electron Microscope

FTIR - Fourier Transform Infrared Spectroscopy

g - gram

HCl - Hydrochloric acid

KBr - Potassium Bromide

kV - Kilo voltage

k - Degradation rate

κ-carrageenan - Kappa carrageenan

MC - Moisture content

mL/min - Milliliter per minute

m3/h - Cubic meter per hour

mg - Milligram

NA - Nutrient Agar

NaoH - Sodium Hydroxide

nm - Nanometer

NB - Nutrient Broth

°C - degree Celsius

OD - Optical Density

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OD540 - Optical Density at 540 nm

pH - -log[H+]

S.aureus - Staphylococcus aureus

rpm - Rotation per minute

t1/2 - Half life

UV-VIS - UV Visible spectroscopy

w/v - Weight per volume

µg - microgram

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LIST OF APPENDICES

APPENDICES

TITLE PAGE

A1 Regression plots of spray-dried prodigiosin upon

exposure to different pH in the presence of light.

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A2 Regression plots of spray-dried prodigiosin upon

exposure to different pH without the presence of light.

54

A3 Regression plots of spray-dried prodigiosin upon

exposure to different temperatures in liquid and powder

forms.

56

A4 Regression plots of liquid and powder forms of spray-

dried prodigiosin in the presence and without presence of

light.

58

B1 FTIR spectroscopy of crude prodigiosin in ethyl acetate. 59

B2 FTIR spectroscopy of pure kappa-carrageenan. 60

B3 FTIR spectroscopy of pure maltodextrin. 61

B4 FTIR spectroscopy of spray-dried prodigiosin coated

with maltodextrin.

62

B5 FTIR spectroscopy of spray-dried prodigiosin coated

with κ -carrageenan.

63

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CHAPTER 1

INTRODUCTION

1.1 Background of Study

Natural pigments have been used to replace synthetic dyes in recent decades.

However, undesirable properties of natural pigments such as solubility and short

term stability limit their application in the food industry (Tripathi et al., 2007; Ersus

and Yurdagel, 2007).

In order to make natural pigments widely applicable and comparable to

synthetic pigments, some properties of natural colorants need to be modified. This is

done via techniques such as encapsulation which will improve the properties of

substances. Encapsulation is a technique for preservation of natural pigments by

entrapping the ingredient in coating materials (Nedovic et al., 2011).

Among encapsulation methods, spray drying is the most common process

employed in the food industry due to feasible and economic operation. By

employing the spray drying method the replacement of natural pigments with

synthetic colorants is possible in the food industry (Bono et al., 2012).

1.2 Problem Statement

Pigments from organisms have been shown to have biological activities and

potential health benefits. Prodigiosin is a natural pigment responsible for the red

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colour in Serratia marcescens. Characteristics such as antimicrobial, antitumor and

antibiotic of prodigiosin make this natural pigment appropriate for medical

applications. Besides unique characteristics of prodigiosin, this pigment is applicable

as a colorant in foodstuffs.

However, prodigiosin have some disadvantages such as low solubility and

rapid colour fading upon exposure to sunlight, high temperature and pH. Some

physical methods via forming a barrier for sensitive substances may provide

resistance toward factor that contribute to colour changes. Spray drying is a

frequently applied encapsulation method in food industry. This method offers an

effective procedure to cover an active compound with a protective wall material.

Various advantages of spray-dried natural pigments make their properties sufficient

to be used as food colorants.

1.3 Objectives of Study

The objectives of the research are:

1. To evaluate the use of encapsulating agents for spray drying of prodigiosin.

2. To evaluate the spray-dried pigment in terms of stability and storage.

3. To apply the encapsulated pigments in food (yogurt, milk and carbonated

drink).

1.4 Scope of Study

Encapsulation of prodigiosin from Serratia marcescens using κ-carrageenan

or maltodextrin as coating materials is one of the scopes of this study. The parameter

optimization of spray dryer i.e. inlet and outlet temperature, feed rate, air flow rate

and air pressure will be performed using several spray-drying runs. The encapsulated

prodigiosin will be evaluated based on the Encapsulation Yield (EY), Moisture

Content (MC) and intensity of the colour. This will be followed by characterization

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of the encapsulated pigment using Field Emission Scanning Electron Microscopy

(FESEM) and FTIR spectroscopy. Stability and optimum storage conditions of the

encapsulated pigments will be tested upon exposure to different factors i.e. pH,

temperature and light. The antimicrobial properties of the encapsulated pigments

against gram negative and gram positive bacteria will also be studied using the

spread plate method. Finally, the encapsulated pigment will be applied in food

models such as milk, yogurt and carbonated drinks.

1.5 Significance of Study

The significance of this study is to achieve a more stable and

environmentally friendly food grade pigment from microbial sources. Employing

simple and cheap technique such as spray drying makes the pigment comparable to

synthetic dyes in term of performance and price.

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