UNIVERSITI PUTRA MALAYSIA COCOA BUTTER …psasir.upm.edu.my/id/eprint/5746/1/FSTM_2009_18_abstract.pdftemperature and 6) to determine the triacylglycerols composition and fatty acid

UNIVERSITI PUTRA MALAYSIA

COCOA BUTTER EXTRACTION FROM COCOA NIBS

USING SUPERCRITICAL CARBON DIOXIDE

ASEP EDI KUSNADI

FSTM 2009 18

COCOA BUTTER EXTRACTION FROM COCOA NIBS USING

SUPERCRITICAL CARBON DIOXIDE

ASEP EDI KUSNADI

DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA

2009

COCOA BUTTER EXTRACTION FROM COCOA NIBS USING SUPERCRITICAL CARBON DIOXIDE

By

ASEP EDI KUSNADI Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

April 2009

ii

This Thesis is specially dedicated to Mamah and Apa (alayarham), Aih, Ujang, Dadan and Nanang and all their family for the unconditional patient, love and support.

iii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirements for the degree of Doctor of Philosophy

COCOA BUTTER EXTRACTION FROM COCOA NIBS USING SUPERCRITICAL CARBON DIOXIDE

By

ASEP EDI KUSNADI

April 2009 Chairman: Professor Jinap Selamat, PhD

Faculty: Food Science and Technology Cocoa beans consist mainly of cocoa butter (50-55% w/w). High quality cocoa butter

used in food, cosmetic and pharmaceutical products is obtained by mechanical press,

expeller, and solvent extraction using hexane. Supercritical fluid extraction (SFE)

using carbon dioxide as a solvent has provided an excellent alternative to the use of

chemical solvent in the extraction of cocoa butter from different plant matrices. In

comparison with established methods, SFE has some important advantages,

particularly in its ability to yield products that are completely free from processing

residues. SFE is also an alternative from the standpoint of time-saving and for

environmental reasons such as the reduction of the use of large solvent volumes.

Carbon dioxide (CO2) is an ideal solvent for extraction of natural products because it

is nontoxic, odorless, tasteless, non explosive, readily available, and easily

removable from the products.

iv

This work is divided into several part namely; 1) the effect of flow rate with different

pressure on cocoa butter extraction using supercritical carbon dioxide (SC-CO2), 2) a

study on the sample matrix involving: the effect of particle size, degree of

fermentation and alkalization on cocoa butter extraction using supercritical fluid; 3) a

study on the effect of moisture content, roasting time and roasting temperature on

cocoa butter extraction using supercritical fluid, 4) a study on the effect of cosolvents

with types and concentrations of polar cosolvents and non polar cosolvents in cocoa

butter extraction using SC-CO2, 5) to evaluate the mass transfer parameters in cocoa

butter extraction by SFE using the Sovova Lacks plug flow model (SLM) for the

effect of flow rate with different pressure, the effects of polar and non polar

cosolvents, and also using single sphere model (SSM) for the effects of particle size,

degree of fermentation, pH alkalization, moisture content, roasting time and roasting

temperature and 6) to determine the triacylglycerols composition and fatty acid

methyl esters composition of cocoa butter extracted that resulted from extraction

using SC-CO2 and SC-CO2 with cosolvents.

The study on the effect of flow rate and pressure found that the optimum conditions

for flow rate and pressure for cocoa butter extraction using SC-CO2 were at 2 ml/min

and 35 MPa, r espectively. The highest yields were obtained from cocoa nibs sample

with smaller particle size (S1= 0.07 mm) with 92% yield for almost 20 h using SC-

CO2, unfermented nibs (F1) with 100% yield for almost 10 h using ethanol 25% as

cosolvent in SC-CO2 and roasting treatment of 150 oC and 35 min (R6) with 100%

yield for almost 14 h using ethanol 25% (mol %) as cosolvent in SC-CO2. Increasing

roasting time and temperature have resulted in the increase of the yield. Furthermore,

v

the highest yield was produced from high pH (7.5 -7.9) of dark alkalized cocoa

liquor (A1) with 73.70% yield for nearly 18 h extraction time using ethanol 25% as

cosolvent in SC-CO2 and high moisture content (M5 =17.64%) with 60.73% after

nearly 20 h extraction using SC-CO2.

Statistically, the yield (p<0.05) significantly was affected by all the treatment,

however no significant different was observed for both light alkalized cocoa liquor

(pH = 6.8-7.2) and alkalized natural cocoa liquor (pH = 5.0 – 5.9), and both moisture

content of 9.79 and 17.64%, respectively.

Ethanol showed the best cosolvent on the yield in SC-CO2, followed by isopropanol,

acetone, hexane and cyclohexane. The yield increased with an increase in

concentration of cosolvents (25%>15%>5%), except for acetone in which 15%

concentration was higher than 25% and 5%. Extraction of cocoa butter using SC-CO2

with polar cosolvents obtained a yield higher than non polar cosolvents. All

treatment of cosolvents studied statistically significantly different at p<0.05.

The two mathematical models are based on mass transfer were used. First model, the

Sovova’s lack’s plug flow model (SLM) was used to describe the extraction process

of effect of flow rate with different pressure and effect of polar and non polar

cosolvents. The hardly accessible solute xk and the volume mass transfer coefficients

in the fluid phase (F) and solid phase S were used as fitting parameters. The

maximum average deviation between measured and calculated oil yield was 6.861%.

Mass transfer coefficients in the fluid phase and solid phase varied between 6.528.E-

vi

06 to 1.498E-04 s-1 and between 5.185.E-06 to 9.144E-04 s-1, respectively. Second

model, the single sphere model (SSM) was used to describe the extraction process of

effect of particle size, degree of fermentation, pH of alkalization, moisture content,

roasting time and roasting temperature. Adjusting of effective intraparticle diffusion

coefficient (De) and estimation of parameter of coefficient of mass transfer in the

fluid phase (kf) and overall mass transfer coefficient kp and parameters were

evaluated. The result showed that De, kf and kp varied between 8.900E-16 to 1.850E-

09 m2/s, between 1.139E-10 to 2.115E-04 m/s and between 9.581E-12 to 8.992E-05

m/s, respectively. All experimental data results of extraction curves were fitted fairly

well by using both of the SLM model and SSM model with average absolute relative

deviation (AARD) between measured and calculated oil yield were maximum of

6.861.

The results showed that cocoa butter extracted from various treatments had three

main triacylglycerol (TG) namely Glycerol-1,3-Dipalmitate-2-Oleate (POP),

Glycerol-1-Palmitate-2-Oleate-3-Stearate (POS), and Glycerol-1,3-Distearate-2-

Oleate (SOS). In general, POS was the major component with 42.16 to 45.78%

followed by SOS and POP with 27.60 to 31.67% and 20.09 to 22.79%, respectively.

Furthermore, analysis of fatty acid (FA) showed that palmitic acid (C16:0), stearic

acid (C18:0) and oleic acid (C18:1) were the three main fatty acids in the cocoa

butter extracted with stearic acid being the major component with 34.82 to 39.06%,

followed by oleic acid and palmitic acid with 28.48 to 31.72% and 28.27 to 31.33%,

respectively. Statistically, there are differences the effects of all treatment on TG and

FA compositions.

vii

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

PENGEKSTRAKAN LEMAK KOKO DARI PADA NIB KOKO MENGGUNAKAN BENDALIR SUPERKRITIK KARBON DIOKSIDA

Oleh

ASEP EDI KUSNADI

April 2009

Pengerusi: Profesor Jinap Selamat, PhD

Fakulti: Sains dan Teknologi Makanan Sebahagian besar daripada komponen biji koko terdiri daripada lemak koko (50-55%),

dimana lemak koko yang berkualiti tinggi yang digunakan di dalam produk makanan,

kosmetik dan farmaseutikal diperolehi melalui proses pengekstrakan menggunakan

tekanan secara mekanik, penggunaan tekanan dengan kuasa putaran dan pelarut kimia

seperti heksana. Proses pengekstrakan dengan cecair superkritik (SFE) yang

menggunakan gas karbon dioksida sebagai pelarut telah menjadi satu alternatif yang

baik bagi pengekstrakan lemak koko daripada matriks tumbuhan yang berbeza

berbanding pengekstrakan menggunakan pelarut kimia. Dibandingkan dengan cara

yang sedia pakai, SFE mempunyai beberapa kelebihan terutamanya dalam

menghasilkan produk yang bebas dari sisa-sisa pemprosesan. SFE juga lebih baik dari

segi penjimatan masa dan lebih mesra alam sekitar kerana mengurangkan penggunaan

pelarut dalam isi padu yang tinggi. Karbon dioksida pula merupakan pelarut yang

viii

ideal bagi pengekstrakan produk asli kerana ciri-cirinya yang tidak toksik, tidak

berbau, tidak mempunyai rasa, tidak mudah meletup/terbakar, sedia didapati dan

mudah dipisahkan dari produk.

Kajian ini dimulakakan dengan mengkaji, 1) kesan kadar alir dengan tekanan yang

berbeza ke atas pengekstrakan lemak koko menggunakan supercritical karbon

dioksida (SC-CO2), 2) kajian matriks sampel: kesan saiz partikel, darjah penapaian

dan pengalkalian, 3) kajian untuk kesan kandungan kelembapan, masa pemanggangan

dan suhu pemanggangan dan 4) kesan pelarut penyokong/pengubahsuai (CS)

mengikut jenis dan kepekatan dari pada CS yang bersifat polar dan tidak polar semasa

pengekstrakan lemak koko menggunaan SC-CO2, 5) kajian pemindahan jisim

menggunakan model Sovova yang berasaskan model lanjutan aliran palam Lack

(SLM) untuk pengaruh kadar alir dengan tekanan berbeza dan kesan CS yang polar

dan tak polar, dan juga model partikel bulat tunggal (SSM) untuk pengaruh saiz

partikel, tingkatan penapaian, pengalkalian, massa pemanggangan dan suhu

pemanggangan dan 6) kajian komposisi daripada trigliserida dan asid lemak yang

terekstrak menggunakan SC-CO2 dan CS.

Kajian ini mendapati keadaan optimum bagi kadar alir dan tekanan yang digunakan

untuk pengekstrakan lemak koko menggunakan SC-CO2 adalah pada 2 mL/min dan

35 MPa. Hasil tertinggi diperolehi daripada nib koko yang mempunyai saiz partikel

terkecil (S1= 0.07 mm) dengan penghasilan 92% setelah hampir 20 jam menggunakan

SC-CO2, nib koko tertapai (F1) dengan penghasilan 100% setelah hampir 10 jam

menggunakan etanol sebagai CS di dalam SC-CO2 dan juga bagi pemanggangan 150

ix

oC dan 35 min (R6) dengan 100% hasil setelah hampir 14 jam menggunakan 25%

etanol (%mol) sebagai CS di dalam SC-CO2. Peningkatan suhu dan masa

pemanggangan telah menningkatkan hasil yang diperolehi. Hasil yang tinggi juga

diperolehi daripada likur koko hitam yang beralkali pada pH tinggi 7.5-7.9 (A1)

dengan 73.70% hasil setelah pengekstrakan selama 18 jam menggunakan 25% etanol

sebagai CS di dalam SC-CO2 dan juga pada kandungan kelembapan yang tinggi (M5

= 17.64%) dengan 60.73% hasil setelah pengekstrakan menggunakan SC-CO2 selama

20 jam.

Secara statistiknya, hasil yang diperolehi dipengaruhi oleh semua perlakuan

penyelidikan pada p<0.05, tetapi tiada perbezaan yang signifikan diperolehi bagi

kedua-dua koko likur beralkali rendah (pH = 6.8-7.2) dan koko likur beralkali asli (pH

= 5.0 – 5.9) dan juga pada kedua-dua kandungan kelambapan masing-masing 9.79 dan

17.64%.

Etanol telah menunjukkan ciri terbaik sebagai CS bersama SC-CO2, diikuti oleh

isopropanol, aseton, siklohesana dan heksana. Peningkatan hasil diperolehi bersama

peningkatan kepekatan (mol%) daripada CS (25%>15%>5%), kecuali bagi aseton

yang mana pada kepekatan 15% mempunyai hasil yang lebih tinggi dari 25 dan 5%.

Pengekstrakan lemak koko dengan SC-CO2 bersama CS yang polar mempunyai hasil

yang tinggi berbanding CS yang tidak polar. Semua perlakuan dalam penyelidikan

tentang pengaruh CS secara statistik menunjukkan perbezaan yang signifikan pada

p<0.05.

x

Kedua dua model berasaskan pemindahan jisim dapat dilakukan. SLM dapat

digunakan untuk menjelaskan terjadinya pengaruh kadar alir dengan tekanan berbeza

dan efek dari pada jenis dan konsentrasi CS yang polar dan non polar. Parameter dari

xk, F dan S dapat digunakan untuk penilaian parameter pemindahan jisim dalam fasa

bendalir dan padatan dari pada data eksperimen. AARD maksimum adalah 6.861%.

Koefisien pemindahan jisim fasa bendalir dengan padatan adalah beragam masing-

masing diantara 6.528.E-06 - 1.498E-04 s-1 dan 5.185.E-06 - 9.144E-04 s-1. Untuk

model kedua (SSM), hasil kajian menunjukkan bahawa nilai-nilai De, kf dan kp

beragam, dimana masing-masing berada diantara 8.900E-16 - 1.850E-09 m2/s,

1.139E-10 - 2.115E-04 m/s dan 9.581E-12 - 8.992E-05 m/s. Semua rajah dari data

eksperimen memberikan hasil yang bersesuaian dengan model, dengan AARD

maksimum pada 6.861.

Keputusan menunjukkan lemak koko yang diekstrak dari proses yang berbeza

mengandungi tiga jenis trigliserida (TG) utama yaitu 1,3-Dipalmitoyl-2-Oleoyl-

Gglycerol (POP), 1-Palmitoyl-2-Oleoyl-3-Stearoyl-Glycerol (POS) dan 1,3-

Distearoyl-2-Oleoyl-Glycerol (SOS). Secara amnya, POS didapati dalam kandungan

yang tertinggi sebanyak 42.16-45.78% diikuti oleh SOS dan POP masing-masing

sebanyak 27.60-31.67% dan 20.09-22.79%. Analisa asid lemak (FA) menunjukkan

bahawa asid palmitik (C16:0), asid stearik (C18:0) dan asid oleik (C18:1) merupakan

tiga asid lemak utama didalam lemak koko yang diekstrak dengan kandungan tertinggi

asid strearik sebanyak 34.82-39.06%, diikuti oleh asid oleik dan asid palmitik masing-

masing sebanyak 28.48-31.72 dan 28.27-31.33%. Secara statistik terdapt pengaruh

yang berbeda dari semua perlakukan terhadap kandungan TG dan FA lemak.

xi

ACKNOWLEDGEMENTS

Alhamdulillahirabbil'alamin, first of all I would like to express my utmost thanks

and gratitude to the Almighty Allah S.W.T, the Sustainer, the most Gracious and

most Merciful; without whose will no one can achieve anything. My salawat and

salam is addressed to His righteous messenger, prophet Muhammad S.A.W.

I would like to take this opportunity to express my appreciation and gratitude to the

chairman of my supervisory committee, Prof. Dr. Jinap Selamat for her invaluable

guidance, suggestions, discussions and patience throughout the research and

preparation of this thesis. I am also grateful to the members of my supervisory

committee, Prof. Dr. Russly Abdul Rahman, Assoc. Prof. Dr. Harcharan Singh and

Dr. Nazimah Sheikh Abdul Hamid for their constructive comments and suggestions.

My sincere gratitude is also extended to the financial support provided by the

Intensification of Research in Priority Area (IRPA) fund for this research which was

awarded to Prof. Dr. Jinap Selamat. I am also indebted to all the staffs of the

Department of Food Science for their generous cooperation. An acknowledgement

is also due to Prof. Dr. H. Adang Kadarusman (Alayarham) and Prof Dr. H.

Sutarman, Dean Faculty of Industrial Engineering, University of Pasundan,

Bandung.

Special thanks also to all her graduate friends, especially cocoa group members and

food Safety; Tan Teng Ju, Yusep Ikrawan, Willy Pranata Widjaja, Liza bt Mad

xii

Saleh, Muh. Zuhkrufuz Zaman, Bakti Kumara, Misnawi, Rashidah Sukor, Noor

Sofalina, Elham Moazami, Ismail Fitri, and Mohd. Safzan for sharing the literature

and invaluable assistance. The time spent and memorable memories will always be

cherished.

Last but not least, I also wish to express my deepest appreciation to my beloved

parents, sister and brother for all over their moral support, understanding, endless

love, patience, and never ending encouragement and support in anyway during the

many years of my seemingly ending pursue for knowledge.

xiii

I certify that a Thesis Examination Committee has met on 28 April 2009 to conduct the final examination of Asep Edi Kusnadi on his thesis entitled “Cocoa Butter Extraction from Cocoa Nibs Using Supercritical Carbon Dioxide” in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Pertanian Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Doctor of Philosophy. Members of the Thesis Examination Committee were as follows: Roselina Karim, PhD Lecturer Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Robiah Yunus, PhD Associate Professor Faculty of Engineering Universiti Putra Malaysia (Internal Examiner) Lai Oi Ling, PhD Associate Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Internal Examiner) Mohd Omar Abdul Kadir, PhD Professor Faculty of Industrial Technology University of Sains Malaysia Country of Malaysia (External Examiner)

________________________ BUJANG KIM HUAT, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 17 September 2009

xiv

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows: Jinap Selamat, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Russly Abdul Rahman, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Member) Harcharan Singh, PhD Associate Professor Faculty of Engineering Universiti of Malaya (Member)

_________________________________ HASANAH MOHD. GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: 16 October 2009

xv

DECLARATION

I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institutions. ___________________ ASEP EDI KUSNADI Date: 15 September 2009

xvi

TABLE OF CONTENTS

PageABSTRACT iii ABSTRAK vii ACKNOWLEDGEMENTS xi APPROVAL xiii DECLARATION xv LIST OF TABLES xxi LIST OF FIGURES xxv LIST OF ABBREVIATIONS xxix

CHAPTER

1 GENERAL INTRODUCTION 1

2 LITERATURE REVIEW 8 2.1 Origin and Botany of Cocoa 9 2.2 Processing of Cocoa Bean 7 2.2.1 Fermentation 11 2.2.2 Roasting 15 2.2.3 Alkalization 21 2.2.4 Grinding 21 2.3 Cocoa Butter 22 2.4 Extraction Methods 26

2.4.1 The Conventional Liquid Solvent Extraction 26 2.4.2 Advantages of Conventional Solvent

Extraction 27

2.4.3 Disadvantages of Conventional Solvent Extraction

28

2.5 The Supercritical Fluid Extraction 29 2.5.1 The Supercritical Fluid 29 2.5.2 Properties of Supercritical Fluid 33 2.5.3 Historical Background of Supercritical Fluid

Extraction 38

2.5.4 Advantages of Supercritical Fluid Extraction 40 2.6 The Experimental Parameters in Supercritical Fluid

Extraction 42

2.6.1 Extraction Time 43 2.6.2 Supercritical Flow Rate 47 2.6.3 Extraction Cell 49 2.6.4 Supercritical Fluid Flow Direction 50 2.6.5 Pressure 52 2.6.6 Temperature 53 2.6.7 Sample Matrix and Analyte Properties 55 2.6.8 Nature and Composition of Supercritical fluids 57

xvii

2.7 Cosolvent or Modifier in SFE 60 2.8 Modeling on Supercritical Fluid Extraction 62 2.8.1 Sovova’s Lack’s Plug Flow Model (SLM) 64 2.8.2 Single Sphere Model (SSM) 69

3 MATERIALS AND METHODS 84 3.1 Materials 84 3.2 Methods 84 3.2.1 Supercritical Fluid Extraction Method with

Pure Carbon Dioxide (SC-CO2) 84

3.2.2 Supercritical Fluid Carbon Dioxide (SC-CO2) with Cosolvents Method

86

3.2.3 Determination of Yeld 87

3.2.4 Measurement of Real and Bulk Density of Cocoa Nibs Particles

88

3.2.5 Modeling of Supercritical Fluid Extraction with Sovova’s Lacking Plugs Flow Model (SLM)

88

3.2.6 Modeling of Supercritical Fluid Extraction with Single Sphere Model (SSM)

90

3.2.7 Triacylglycerol (TG) Profile Analysis by using High Performance Liquid Chromatography (HPLC)

91

3.2.8 Fatty Acid (FA) Profile Analysis by using Gas Chromatography (GC)

91

3.2.9 Statistical Analysis 92

4 EFFECT OF FLOW RATE WITH DIFFERENT PRESSURE ON COCOA BUTTER EXTRACTION USING SC-CO2

93

4.1 Introduction 93 4.2 Materials and Methods 94 4.2.1 Materials 94 4.2.2 Methods 94 4.2.3 Statistical Analysis 96 4.3 Results and Discussion 97 4.3.1 Effect of Flow Rate with Different Pressure on

the Experimental Yield and Comparison with Calculated Mass Transfer Values

97

4.3.2 Effect of Flow Rate with Different Pressure on Triacylglycerol

106

4.3.3 Effect of Flow Rate with Different Pressure on Fatty Acid Methyl Ester

110

4.4 Summary

113

xviii

5 EFFECT OF PARTICLE SIZE, DEGREE OF FERMENTATION AND ALKALIZATION ON COCOA BUTTER EXTRACTION USING SUPERCRITICAL FLUID

115

5.1 Introduction 115 5.2 Materials and Methods 117 5.2.1 Materials and Treatments 117 5.2.2 Methods 118 5.2.3 Statistical Analysis 120 5.3 Results and Discussion 121 5.3.1 Effect of Particle Size on the Experimental

Yield and Comparison with Calculated Mass Transfer Values

121

5.3.2 Effect of Fermentation Degree on the Experimental Yield and Calculated Mass Transfer Values

127

5.3.3 Effect of Alkalization on the Experimental Yield and Calculated Mass Transfer Values

133

5.3.4 Effect of Particle Size, Degree of Fermentation and Alkalization on Triacylglycerol

138

5.3.5 Effect of Particle Size, Degree of Fermentation and Alkalization on Triacylglycerol Effect of Particle Size, Degree of Fermentation and Alkalization on Fatty Acid Methyl Ester

143

5.4 Summary 149

6 EFFECT OF MOISTURE CONTENT, ROASTING TIME AND ROASTING TEMPERATURE ON COCOA BUTTER EXTRACTION USING SUPERCRITICAL FLUID

151

6.1 Introduction 151 6.2 Materials and Methods 155 6.2.1 Materials and Treatments 155 6.2.2 Methods 156 6.2.3 Statistical Analysis 158 6.3 Results and Discussion 158 6.3.1 Effect of Moisture Content on the

Experimental Yield and Comparison with Calculated Mass Transfer Values

158

6.3.2 Effect of Roasting Time and Roasting Temperature on the Experimental Yield and Calculated Mass Transfer Values

163

6.3.3 Effects of Moisture Content, Roasting Time and Roasting Temperature on Triacylglycerol

169

6.3.4 Effects of Moisture Content, Roasting Time and Roasting Temperature on Fatty Acid Methyl Ester

172

6.4 Summary 176

xix

7 EFFECT OF POLAR COSOLVENTS ON COCOA

BUTTER EXTRACTION USING SC-CO2 177

7.1 Introduction 181 7.2 Materials and Methods 181 7.2.1 Materials 181 7.2.2 Methods 181 7.2.3 Statistical Analysis 185 7.3 Results and Discussion 187 7.3.1 Effects of Ethanol, Isopropanol and Acetone

Concentration on the Experimental Yield and Comparison with Calculated Value of Mass Transfer

187

7.3.2 Effects of Ethanol, Isopropanol and Acetone Concentration Levels on Triacylglycerol Profile and Selectivity (α)

195

7.3.3 Effects of Ethanol, Isopropanol and Acetone Concentration Levels on Fatty Acid Methyl Ester Profile and Selectivity (α)

205

7.4 Summary 213

8 EFFECT OF NON POLAR COSOLVENTS ON COCOA BUTTER EXTRACTION USING SC-CO2

215

8.1 Introduction 215 8.2 Materials and Methods 217 8.2.1 Materials 217 8.2.2 Methods 218 8.2.3 Statistical Analysis 220 8.3 Results and Discussion 221 8.3.1 Effect of Hexane and Cyclohexane

Concentration on the Experimental Yield and Comparison with Calculated Value of Mass Transfer

225

8.3.2 Effect of Hexane and Cyclohexane Concentration on Triacylglycerol Profile and Selectivity (

229

8.3.3 Effects of Hexane and Cyclohexane Concentrations on Fatty Acid Methyl Ester Profile and Selectivity (α)

235

8.4 Summary 241

9 CONCLUSIONS AND RECOMMENDATIONS 244 9.1 Conclusions 244 9.2 Recommendations 250

xx

BIBLIOGRAPHY 252 APPENDICES 273 BIODATA OF STUDENT 282 LIST OF PUBLICATIONS 283

xxi

LIST OF TABLES

Table Page

2.1 Characteristic of three different roasting methods 18

2.2 Lipid compositions (wt %) of cocoa butter 23

2.3 Fatty acids distribution (wt %) of cocoa butter 24

2.4 Triacylglycerol compositions (%) of cocoa butter 25

2.5 Comparison of the properties of supercritical CO2, supercritical fluids, liquids and gases

35

2.6 Critical Conditions for Various Solvents 37

2.7 The model based on differential mass balance and applied to the extraction of natural products with near critical CO2

54

4.1 Calculated and estimated SLM model parameters of mass transfer for cocoa butter extraction using supercritical carbon dioxide at T = 60 oC with different flow rate and pressure

104

4.2 Triacylglycerol composition (area %) of cocoa butter extracted by supercritical carbon dioxide (SC-CO2) for effect of flow rate (f) with different pressure

108

4.3 Fatty acids composition (area %) changes of cocoa butter extracted by supercritical carbon dioxide (SC-CO2) for effect of flow rate (f) with different pressure

112

5.1 Calculated and estimated model (SSM) parameters of mass transfer for cocoa butter extraction using SC-CO2 at T = 60 oC and flow rate = 2 mL/min with different of particle size

126

5.2 Calculated and estimated model (SSM) parameters of mass transfer for cocoa butter extraction using SC-CO2 at T = 60 oC and flow rate = 2 mL/min with different of fermentation

132

5.3 Calculated and estimated model (SSM) parameters of mass transfer for cocoa butter extraction using SC-CO2 at T = 60 oC and flow rate = 2 mL/min with different of alkalization

137

5.4 Triacylglycerol composition (area %) of cocoa butter extracted by supercritical carbon dioxide (SC-CO2) at 35 MPa, 60 oC and 2 mL/min of flow rate with different particle size and extraction time

139

xxii

5.5 Triacylglycerol composition (area %) of cocoa butter extracted by supercritical carbon dioxide (SC-CO2) and 25% ethanol as cosolvent 35 MPa, 60 oC and 2 mL/min of flow rate with different fermentation treatment and extraction time

140

5.6 Triacylglycerol composition (area %) of cocoa butter extracted by supercritical carbon dioxide (SC-CO2) and 25% ethanol as cosolvent 35 MPa, 60 oC and 2 mL/min of flow rate with different pH level of alkalization and extraction time

141

5.7 Fatty acids composition (area %) of cocoa butter extracted by supercritical carbon dioxide (SC-CO2) at 35 MPa, 60 oC and 2 mL/min of flow rate with different particle size and extraction time

146

5.8 Fatty acids composition (area %) of cocoa butter extracted by supercritical carbon dioxide (SC-CO2) and 25% ethanol as cosolvent at 35 MPa, 60 oC and 2mL/min of flow rate with different fermentation treatment and extraction time

147

5.9 Fatty acids composition (area %) of cocoa butter extracted by supercritical carbon dioxide (SC-CO2) and 25% ethanol as cosolvent at 35 MPa, 60 oC and 2mL/min of flow rate different pH level of alkalization and extraction time

148

6.1 Calculated and estimated model (CSM) parameters of mass transfer for cocoa butter extraction using SC-CO2 at T = 60 oC and flow rate = 2 mL/min with different of moisture content

162

6.2 Calculated and estimated model (CSM) parameters of mass transfer for cocoa butter extraction using SC-CO2 at T = 60 oC and flow rate = 2 mL/min with different of roasting

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6.3 Triacylglycerol composition (area %) of cocoa butter extracted by supercritical carbon dioxide (SC-CO2) at 35 MPa, 60 oC and 2 mL/min of flow rate with different of moisture content and extraction time

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6.4 Triacylglycerol composition (area %) of cocoa butter extracted by supercritical carbon dioxide (SC-CO2) and 25% ethanol as cosolvent at 35 MPa, 60 oC and 2 mL/min of flow rate with different of roasting treatment and extraction time

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6.5 Fatty acids composition (area %) changes of cocoa butter extracted by supercritical carbon dioxide (SC-CO2) at 35 MPa, 60 oC and 2 mL/min of flow rate with different of moisture content and extraction time

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6.6 Fatty acids composition (area %) changes of cocoa butter extracted by supercritical carbon dioxide (SC-CO2) and 25% ethanol as cosolvent at 35 MPa, 60 oC and 2 mL/min of flow rate with different of roasting treatment and extraction time

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7.1 Composition (%) and flow rate of solvent mixture based on mol% of polar cosolvents in SC-CO2 at 35 MPa pressure, 60 oC temperature and 2 mL/min flow rate

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7.2 Triacylglycerols and fatty acids composition (%) of cocoa butter obtained by soxhlet method from cocoa liquor

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7.3 Calculated and estimated model (SLM) parameters of mass transfer for cocoa butter extraction using SC-CO2 with cosolvents at T = 60 oC, P = 35 MPa, f = 2 mL/min with different in the concentration of polar cosolvents

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7.4 Triacylglycerols composition (area %) changes of cocoa butter extracted from cocoa liquor with ethanol as cosolvent in SC-CO2 at 35 MPa, 60 oC and 2 mL/min with different ethanol concentration and extraction time

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7.5 Triacylglycerols composition (area %) changes of cocoa butter extracted from cocoa liquor with isopropanol as cosolvent in SC-CO2 at 35 MPa, 60 oC and 2 mL/min with different isopropanol concentration and extraction time

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7.6 Triacylglycerols composition (area %) changes of cocoa butter extracted from cocoa liquor with acetone as cosolvent in SC-CO2 at 35 MPa, 60 oC and 2 mL/min with different acetone concentration and extraction time

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7.7 Fatty acids composition (area %) changes of cocoa butter extracted from cocoa liquor by ethanol as cosolvent in SC-CO2 at 35 MPa, 60oC and 2 mL/min of flow rate with different ethanol concentration and extraction time

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7.8 Fatty acids composition (area %) changes of cocoa butter extracted from cocoa liquor by isopropanol as cosolvent in SC-CO2 at 35 MPa, 60 oC and 2 mL/min of flow rate with different isopropanol concentration and extraction time

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7.9 Fatty acids composition (area %) changes of cocoa butter extracted from cocoa liquor by acetone as cosolvent in SC-CO2 at 35 MPa, 60 oC and 2 mL/min of flow rate with different acetone concentration and extraction time

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8.1 Composition (%) and flow rate of solvent mixture based on mol% of non polar cosolvents in SC-CO2 at 35 MPa pressure, 60 oC temperature and 2 mL/min flow rate

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