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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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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)
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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
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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
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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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REFERENCES Bono, A., Farm, I.Y., Yasir, S.M., Arifin, B. and Jasni, M.N. (2011). Production of
Fresh Seaweed Powder Using Spray Drying Technique. Journal of Applied
Sciences. 11, 2340-2345.
Carmen, S. (2007). New Technologies to Synthesis Extract and Encapsulate Natural
Food Colorants. Bulletin of University of Agricultural Sciences and
Veterinary Medicine Cluj-Napoca. 64, 63-64.
Chan, E.S., Z.H. Yim, Phan, S.H., Mansa, R.F. and Ravindra, P. (2011).
Encapsulation of Herbal Aqueous Extract through Absorption with Ca-
Alginate Hydrogel Beads. Food and Bioproducts Processing. 88,195–201.
Desai, K.G.H. and Park, H.J. (2005). Recent Developments in Microencapsulation
of Food Ingredients. Drying Technology. 23, 1361-1394.
Drusch, S. (2007). Sugar Beet Pectin: A Novel Emulsifying Wall Component for
Microencapsulation of Lipophilic Food Ingredients by Spray-Drying. Food
Hydrocolloids. 21, 1223-1228.
Dunstan, D.E., Chen, Y.M.L., Liao R., Salvatore, D.V., Boger, and Pricab, M.
(2001). Structure and Theology of the K-Carrageenan/Locust Bean Gum
Gels. Food Hydrocolloids. 15, 475-484.
Dufosse, L., Galaupa, P., Yaronb, A., Aradb, S.M., Blancc, P., Murthyd , K.C. and
Ravishankard, G. A. (2005). Microorganisms and Microalgae as Sources of
Pigments for Food Use: A Scientific Oddity or an Industrial Reality? Trends
in Food Science and Technology. 16, 389-406.
Dziezak, J.D. (1988). Microencapsulation and encapsulated ingredients. Food
Technology.42, 136-151.
Ersus, S. and Yurdagel, U. (2007). Microencapsulation of Anthocyanin Pigments of
Black Carrot (Daucuscarota L.) by Spray Drier. Journal of Food
Engineering. 80, 805–812.
67
Fathia, M., Mozafarib, M.R. and Mohebbi, M. (2012). Nanoencapsulation of Food
Ingredients using Lipid Based Delivery Systems. Trends in Food Science and
Technology. 23, 13-27.
Gamboa, O.D., Gonçalves, L.G. and Grosso, C.F. (2011). Microencapsulation of
Tocopherols in Lipid Matrix by Spray Chilling Method. Procedia Food
Science. 1, 1732-1739.
Gharsallaoui, A., Roudaut, G., Chambin, O., Voilley, A. and Saurel, R. (2007)
Applications of Spray-Drying In Microencapsulation of Food Ingredients.
Food Research International. 40, 1107-1121.
Grimont, F. and Grimont, P.A.D. (2006). The Genus Serratia. Prokaryotes. 6, 219-
244.
Han, S.B., Kim, H.M., Kim, Y.H., Lee, C.W., Jang, E.S., Son, K.H., Kim, S.U. and
Kim, Y.K. (2000). T Cell Specific Immunosuppression by Prodigiosin
Isolated from Serratia marcescens. International Immunopharmacology
Journal, 20, 1-13.
Iglauer, S. Wu, Y., Shuler, P., Tang, Y. and Goddard, W.A. (2011). Dilute Iota- and
Kappa-Carrageenan Solutions with High Viscosities in High Salinity Brines.
Journal of Petroleum Science and Engineering. 75, 304-311.
James, M.A. and Alger, N.E. (1981). Plasmodium berghei: Effect of Carrageenan on
the Course of Infection in the A/J Mouse. International Journal for
Parasitology. 11, 217-220.
Kenyon, M.M. (1995). Modified Starch, Maltodextrin, and Corn Syrup as Wall
Materials for Food Encapsulation. ACS Symposium Series. 590, 42-50.
Laos, K., Lõugas, T., Mändmets, A. and Vokk, R. (2007). Encapsulation of Carotene
from Sea Buckthorn (Hippophaë Rhamnoides L.) Juice in Furcellaran Beads.
Innovative Food Science and Emerging Technologies. 8, 395-398.
Mohan, J. (2007). Organic Spectroscopy. Principles and Applications. Alpha Science
International. Ltd., U.K.
Nakashima, T., Shimizu, M. and Kukizaki, M. (2000). Particle Control of Emulsion
by Membrane Emulsification and Its Applications. Advanced Drug Delivery
Reviews. 45, 47-56.
Nedovic, V., Kalusevica, A., Manojlovicb, V., Levica, S. and Bugarskib, B. (2011).
An Overview of Encapsulation Technologies for Food Applications. Procedia
Food Science. 1, 1806 -1815.
68
Obón, J.M., Castellar, M.R., Alacid, M. and Fernández-López, J.A. (2009).
Production of a Red-Purple Food Colorant from Opuntia Stricta Fruits by
Spray Drying and It’s Application in Food Model Systems. Journal of Food
Engineering. 90, 471-479.
Patel, R.P., Patel, M.P. and Suthar, A.M. (2009). Spray Drying Technology: An
Overview. Indian Journal of Science and Technology. 2, 44-74.
Peter, S. and Given. J.r. (2009). Encapsulation of Flavours in Emulsions for
Beverages Review Article. Current Opinion in Colloid and Interface
Science.14, 43-47.
Reineccius, G.A. (1989). Flavour encapsulation. Food Reviews International. 5, 147-
176.
Ronald, C.D. (1997). Spray Drying Innovative Use of an Old Process. Design
Elements. 7, 97-113.
Rosenberg, M., Kopelman, I. J. and Talmon, Y. (1990). Factors Affecting Retention
in Spray-Drying Microencapsulation of Volatile Materials. Journal of
Agricultural and Food Chemistry. 38, 1288–1294.
Sadeghi, A., Shahidi, F., Mortazavi, S.A. and Mahalati, M.N. (2008). Evaluation of
Different Parameters Effect on Maltodextrin Production By α-Amylase
Termamyl 2-X. World Applied Sciences Journal. 3, 34-39.
Shahidi, F. and Han, X.Q. (1993). Encapsulation of Food Ingredients. Critical
Review in Food Science and Nutrition. 33, 501-547.
Song, C., Makoto, S., Osamu, J., Shinji, O., Yasunori, N. and Akihiro, Y. (2000).
High production of prodigiosin by Serratia marcescens Grown on Ethanol.
Biotechnology Letters. 22, 1761-1765.
Song, M.J., Bae, J., Lee, D.S., Kim, CH., Kim, J.S., Kim, S.W. and Hong, S.I.
(2006). Purification and Characterization of Prodigiosin Produced by
Integrated Bioreactor from Serratia sp. KH-95. Journal of Bioscience and
Bioengineering, 101, 157-161.
Su, Y.L., Fu, Z.Y., Zhang, J.Y., Wang, W.M., Wang, H., Wang, Y.C., Zhang, Q.J.
(2008). Microencapsulation of Radix Salvia Miltiorrhiza Nanoparticles by
Spray-Drying. Powder Technology. 184, 114-121.
Struchkova, M.I., Dvoryantseva, G.G., Belova, T.P., Sklyar, Y.E. and Evstigneeva,
R.P., (1973). Protonation of Acetyl and Formyl Derivatives of Pyrrole.
Chemistry of Heterocyclic Compounds. 9, 1355-1360.
69
Venil, C.K. and Lakshmanaperumalsamy, P. (2009). Application of Statistical
Design to the Optimization of Culture Medium for Prodigiosin Production by
Serratia marcescens SWML08. Malaysian Journal of Microbiology. 5, 55-
61.
Vos, P., Faas, M.M., Spasojevic, M. and Sikkema, J. (2010). Encapsulation for
Preservation of Functionality and Targeted Delivery of Bioactive Food
Components. International Dairy Journal. 20, 292-302.