EXTRACTION OF SELECTED

BIOACTIVE COMPOUNDS FROM

AVERRHOA BILIMBI FRUITS AND

THEIR THERMAL STABILITY DURING

SPRAY DRYING

NORLIA MUHAMAD

DOCTOR OF PHILOSOPHY

(BIO-PROCESS ENGINEERING)

UNIVERSITI MALAYSIA PAHANG

SUPERVISOR’S DECLARATION

We hereby declare that we have checked this thesis and in our opinion, this thesis is

adequate in terms of scope and quality for the award of the degree of Doctor of

Philosophy (Bio-process Engineering).

Signature :

Name of Supervisor : ASSOC. PROF. DR. JOLIUS GIMBUN

Position : ASSOCIATE PROFESSOR

Date :

Signature :

Name of Supervisor : PROF. DR. MASHITAH MOHD. YUSOFF

Position : PROFESSOR

Date :

STUDENT’S DECLARATION

I hereby declare that the work in this thesis is my own except for the quotations and

summaries which have been duly acknowledged. The thesis has not been accepted for

any degree and is not concurrently submitted for award of other degree.

Signature :

Name : NORLIA BINTI MUHAMAD

ID Number : PKB12001

Date :

EXTRACTION OF SELECTED BIOACTIVE COMPOUNDS

FROM AVERRHOA BILIMBI AND THEIR THERMAL STABILITY

DURING SPRAY DRYING

NORLIA MUHAMAD

Thesis submitted in fulfillment of the requirements

for the award of the degree of

Doctor of Philosophy (Biotechnology Engineering)

Faculty of Chemical and Natural Resources Engineering

UNIVERSITI MALAYSIA PAHANG

JULY 2017

ii

DEDICATION

In the name of Allah, The Most Gracious and The Most Merciful

I humbly dedicated this thesis to…

my dear husband; Ahmad Fakaruddin Md. Salleh

my lovely sons; Ahmad Haikal and Ahmad Haziq

my special encouraging parents; Mak and Abah

my remarkable family members,

my friends,

who gave me everlasting inspiration,

never ending encouragements,

priceless support

and pray without ceasing..

Thank you very much

iii

ACKNOWLEDGEMENTS

In the name of God, the Most Gracious and the Most Merciful.

Above all, thanks to Allah and His messenger Prophet Muhammad SAW for the love,

mercy and guidance that leads me to be a true Muslim scholar.

I would like to take this precious moment to express my sincere and wholehearted

appreciation to my supervisor, Associate Professor Dr. Jolius Gimbun for his helpful

advice, germinal idea, continuous encouragement, constructive guidance and constant

support in making this research possible. He always impressed me with his outstanding

professional conduct, strong conviction for science and her belief that PhD study is only

a start of a life-long learning experience.

I also would like to express very special thanks to my co-supervisor, Professor Dr.

Mashitah Mohd Yusoff for her suggestions and co-operation throughout the study.

I also sincerely thanks for the time spent proofreading and correcting my manuscripts.

My sincere thanks to all my friends and lab members for their support and effort in

respect to technical assistance and continuous guidance throughout the course of this

study. Special thanks to the staffs of Technical Unit Faculty of Chemical and Natural

Resources Engineering (FKKSA) that are so kind hearted sharing their technical

assistance and knowledge.

For my dear husband Ahmad Fakaruddin, very much appreciate with your love,

patience and edgeless support. Not to forget my sons, Ahmad Haikal and Ahmad Haziq

for your passion. I acknowledge my sincere indebtedness and gratitude to my parent,

Mak, Abah, my parent in law and my family for their love, encourage, help and pray.

Last but not least, for my late father as my idol. May Allah bless you father.

Alhamdulillah…

vi

TABLE OF CONTENTS

DECLARATION

TITLE PAGE

ACKNOWLEDGEMENTS iii

ABSTRAK iv

ABSTRACT iv

TABLE OF CONTENTS vi

LIST OF TABLES x

LIST OF FIGURES xi

LIST OF SYMBOLS xiii

LIST OF ABBREVIATIONS xiv

CHAPTER 1 INTRODUCTION 1

1.1 Motivation and Statement of Problem 1

1.2 Objectives 4

1.3 Scope of this Research 5

1.4 Main Contribution of This Work 5

1.5 Organisation of This Thesis 7

CHAPTER 2 LITERATURE REVIEW 9

2.1 Overview 9

2.2 Averrhoa bilimbi 9

2.3 Plant Extraction 10

2.4 Ultrasonic Assisted Extraction 15

2.5 Thermal Degradation Kinetic 18

2.6 Spray Drying 22

2.7 Microencapsulation 25

vii

2.7.1 Effect of Wall Material 29

2.7.2 Effect of Solid Concentration 33

2.8 Liquid Chromatography Mass Spectrometry Quadruple Time-of-Flight 33

2.9 Optimization Strategies 34

2.9.1 One-Factor-at-Time Approach 34

2.9.2 Response Surface Methodology 35

2.10 Summary 37

CHAPTER 3 MATERIALS AND METHODS 38

3.1 Overview 38

3.2 Introduction 38

3.3 Chemicals 40

3.4 Plant Material 40

3.5 Ultrasonic Assisted Extraction 41

3.6 Thermal Degradation Kinetic 43

3.7 Microencapsulation and Spray Drying 44

3.7.1 Encapsulating Agent Solution Preparation 44

3.7.2 Spray Drying Process 45

3.8 Analysis 45

3.8.1 Liquid Chromatography Mass Spectrometry Quadruple Time-of-

Flight 46

3.8.2 High Performance Liquid Chromatography (HPLC) 47

3.8.3 Viscosity Determination 48

3.8.4 Moisture Content Analysis 48

3.8.5 Particle Size Analysis 48

viii

3.8.6 Field Emission Scanning Electron Microscopy (FESEM) 49

3.9 Statistical Analysis 49

3.10 Summary 49

CHAPTER 4 IDENTIFICATION AND ULTRASONIC ASSISTED

EXTRACTION OF BIOACTIVE COMPOUND FROM A. BILIMBI 50

4.1 Overview 50

4.2 Introduction 50

4.3 Identification of Bioactive Compounds 51

4.3.1 Qualification of Bioactive Compounds 51

4.3.2 Quantification of Bioactive Compounds 54

4.4 Ultrasonic assisted extraction 59

4.4.1 Effect of Extraction Temperature 59

4.4.2 Effect of Ultrasonic Power 61

4.4.3 Effect of Extraction Time 63

4.4.4 Effect of Solvent to Solid Ratio 65

4.4.5 Effect of Methanol Concentration 67

4.4.6 Optimization of UAE by Response Surface Methodology 69

4.5 Summary 73

CHAPTER 5 THERMAL DEGRADATION KINETIC OF NICOTINIC

ACID, PANTOTHENIC ACID AND CATECHIN IN A. BILIMBI

EXTRACT 74

5.1 Overview 74

5.2 Introduction 74

ix

5.3 Thermal Degradation Kinetic 75

5.4 Summary 83

CHAPTER 6 MICROENCAPSULATION OF BIOACTIVE

COMPOUNDS FROM A. BILIMBI 84

6.1 Overview 84

6.2 Introduction 84

6.3 Physical Properties of Spray-Dried Powder 86

6.3.1 Moisture Content 86

6.3.2 Particle Size Distribution 88

6.3.3 Particle Morphology 91

6.4 Microencapsulation of Bioactive Compounds 95

6.4.1 Effect of Different Wall Material 95

6.4.2 Effect of Different Concentration of Wall Material 96

6.4.3 Effect of Different Air Inlet Temperature 97

6.5 Summary 98

CHAPTER 7 CONCLUSION AND RECOMMENDATIONS 100

7.1 Conclusion 100

7.2 Future Work 102

REFERENCES 103

APPENDIX A 116

APPENDIX B 118

LIST OF PUBLICATION AND AWARD 139

x

LIST OF TABLES

Table 2.1 Nutrient Composition of A. bilimbi Fruit 11

Table 2.2 Previous Work on A. bilimbi 12

Table 2.3 Thermal Degradation Kinetic Study 21

Table 2.4 Review of Spray Drying Condition 26

Table 2.5 Review in Wall Materials 31

Table 3.1 Variation of Process Factors for Optimization Using CCD 43

Table 3.2 Microencapsulation Parameters 45

Table 4.1 Intra-day and inter-day precision of the method 59

Table 4.2 Three-Factor Central Composite Design Used For RSM with the

Experimental and Predicted Values for the Independent Variables 71

Table 5.1 Rate Constant (k, min-1

) of Pantothenic Acid and Catechin in

A. bilimbi at Various Temperatures 80

Table 6.1 Properties of Powder 86

xi

LIST OF FIGURES

Figure 2.1 Illustration of Scanning Electron Micrographs (100x) of Epimedium

Leaf Samples from Different Extraction Methods. (A) Untreated Leaf;

(B) After UAE; (C) After Soxhlet Extraction 17

Figure 2.2 Illustration of Transmission Electron Micrographs (3000x) of

Epimedium Leaf Samples from Different Extraction Methods.

(A) Untreated Leaf; (B) After UAE; (C) After Soxhlet Extraction 18

Figure 2.3 A Schematic Diagram of the Spray-Drying Process 23

Figure 2.4 Morphology of Different Types of Microcapsules 29

Figure 3.1 Research Plan for Identification of Bioactive Compounds from A.

bilimbi and Its Thermal Stability during Spray Drying 39

Figure 3.2 Averrhoa bilimbi Fruits 40

Figure 3.3 Schematic Diagram of an Ultrasonic Assisted Extraction 42

Figure 3.4 Schematic Diagram of Spray Dryer 46

Figure 4.1 LCMS-Q-TOF Extracted Ion Chromatogram and Mass Spectra of

Nicotinic Acid in A. bilimbi 52

Figure 4.2 LCMS-Q-TOF Extracted Ion Chromatogram and Mass Spectra of

Pantothenic Acid in A. bilimbi 52

Figure 4.3 LCMS-Q-TOF Extracted Ion Chromatogram and Mass Spectra of

Catechin in A. bilimbi 53

Figure 4.4 Graph of Calibration Curve for Nicotinic Acid 55

Figure 4.5 Graph of Calibration Curve for Pantothenic Acid 55

Figure 4.6 Graph of Calibration Curve for Catechin 56

Figure 4.7 Identification of Nicotinic Acid (a), Pantothenic Acid (b) and Catechin

(c) by Matching UV Spectra of A. bilimbi Extract to Standard 57

Figure 4.8 Identification of Nicotinic Acid (a), Pantothenic Acid (b) and Catechin

(c) from A. bilimbi Extract by Comparing Retention Time with

Standard Solution (Recorded at 254 nm, 197 nm and 280 nm,

respectively) 58

Figure 4.9 Effect of UAE Extraction Temperature on Extraction Yield of

Bioactive Compound 60

Figure 4.10 Effect of UAE Extraction Power on Extraction Yield of Bioactive

Compound 62

Figure 4.11 Effect of UAE Extraction Time on Extraction Yield of Bioactive

Compound 64

xii

Figure 4.12 Effect of UAE Solvent to Solid Ratio on Extraction Yield of Bioactive

Compound 66

Figure 4.13 Effect of UAE Methanol Concentration on Extraction Yield of

Bioactive Compound 69

Figure 4.14 Predicted Optimum Condition for Extraction Yield of Bioactive

Compound Using Central Composite Design 72

Figure 5.1 HPLC Chromatograph Profile of A. bilimbi Before and After Heat

Treatment at 100 °C For 0, 4 And 10 Min for Nicotinic Acid

(Recorded at 254 nm) 76

Figure 5.2 HPLC Chromatograph Profile of A. bilimbi Before and After Heat

Treatment at 100 °C For 0, 4 and 10 Min for Pantothenic Acid

(Recorded at 197 nm) 76

Figure 5.3 HPLC Chromatograph Profile of A. bilimbi Before and After Heat

Treatment at 100 °C for 0, 4 and 10 Min for Catechin (Recorded at

280 nm) 77

Figure 5.4 Thermal Degradation Kinetics of Nicotinic Acid During Heating at

90, 100 and 120 °C 78

Figure 5.5 Thermal Degradation Kinetics of Pantothenic Acid During Heating at

90, 100 and 120 °C 78

Figure 5.6 Thermal Degradation Kinetics of Catechin During Heating at 90, 100

and 120 °C 79

Figure 5.7 Arrhenius Plot for Degradation of Nicotinic Acid, Pantothenic Acid

and Catechin in A. bilimbi During Heating 82

Figure 6.1 Particle Size Distribution of Lyophilised Grounded A. bilimbi 88

Figure 6.2 Particle Size Distribution of Spray Dried Powder; (A) Wall Material;

(B) Concentration Of Wall Material; (C) Air Inlet Temperature 90

Figure 6.3 Morphology for Powder Encapsulated With Different Wall Materials 92

Figure 6.4 Morphology for Maltodextrin Encapsulated Powder at Different

Concentration 93

Figure 6.5 Morphology for Maltodextrin Encapsulated Powder at Different Air

Inlet Temperature 94

Figure 6.6 Retention of Nicotinic Acid, Pantothenic Acid and Catechin

Encapsulated With Different Wall Material and Air Inlet Temperature 99

xiii

LIST OF SYMBOLS

% Percent

°C Degree celcius

Ea Arrhenius activation energy

k Reaction rate constant

xiv

LIST OF ABBREVIATIONS

DE Dextrose equivalent

DW Dry weight

FESEM Field emission scanning electron microscopy

GA Arabic gum

GAE Gallic acid equivalent

HPLC High performance liquid chromatography

LCMS-Q-TOF Liquid chromatography mass spectrometry coupled to quadruple

time-of-flight

AE Microwave-assisted extraction

MD Maltodextrin

ME Maceration

OFAT One-factor-at-a-time

RSM Response surface methodology

SE Soxhlet extraction

SFE Supercritical fluid extraction

UAE Ultrasonic-assisted extraction

UHPLC Ultra-high performance liquid chromatography

UV Ultra violet

WPI Whey protein isolate