OMEGA-3 EMULSION FROM RUBBER (HEVEA BRASILIENSIS) SEEDS

WAN NURAISHA BINTI WAN KAMARUDIN

A report submitted as partial fulfillment of the requirements for the award of the

degree of Bachelor in Engineering (Chemical)

Faculty of Chemical Engineering

Universiti Teknologi Malaysia

JANUARY 2013

v

ABSTRACT

The formulation of omega-3 emulsion from rubber ( Hevea Brasiliensis) seed

oil must be based on the best performance of the emulsion formation in varying the

type and composition of emulsifier used. By choosing rubber seed that has been a

beneficial waste as the main resource in producing emulsion for food purpose, people

can get one of the important sources of omega-3 that normally is supplied by the fish

oil. The difference of them are the absentees of ecosapetanoic acids (EPA) and

docosahaxaneoic acids (DHA) in the rubber seed oil, but still it has a significant

value of linolenic acids (LNA) about 19.22%. Type of the emulsion is determined

according to the HLB value of emulsifier. Emulsifier with a low HLB value lead to

the formation water-in-oil (W/O) emulsion and the oil-in-water (O/W) is in another

way around. The dilution method with water and oil is used for confirmation.

Preparation of rubber seed oil (RSO) involved knocking the kernel, peeling, cutting,

drying the rubber seed and extraction of oil by soxhlet extraction. Then, RSO is

being mixed with distilled water and emulsifier including agents by homogenizer.

Non-ionic emulsifier such as lecithin and span 80 will be chosen to use in the

formulation. The best ratio of emulsifier and type of emulsion are investigated to

produce a stable emulsion based on small droplet size, colour of the emulsion, low

moisture content, moderate viscosity and slightly acidic pH value. From the analysis

conducted, the best formulation selected is E2 with 50% distilled water, 6% lecithin

of the total oil and 47% rubber seed oil (RSO).

vi

ABSTRAK

Formulasi emulsi omega-3 daripada minyak biji getah (Hevea Brasiliensis)

adalah berdasarkan pembentukan emulsi yang stabil di mana beberapa jenis

pengemulsi dan komposisi emulsi yang berbeza telah digunakan. Biji-biji getah

daripada pokok getah yang terbuang boleh memberi manfaat kepada manusia kerana

ia mengandungi omega-3 yang kebiasaannya boleh didapati daripada minyak ikan

yang merupakan makanan tambahan untuk kepentingan tubuh badan. Namun begitu,

biji getah tidak mengandungi ecosapentanoic (EPA) dan asid docosahaxaneoic

(DHA) tetapi masih mempunyai nilai asid linolenik (LNA) yang tinggi sebanyak

19.22%. Jenis emulsi yang akan terhasil ditentukan oleh nilai HLB pada pengemulsi.

Nilai rendah membawa kepada pembentukan air dalam minyak (W/O) dan

sebaliknya untuk pembentukan minyak dalam air (O/W). Namun begitu, ia perlu

dipastikan dengan menggunakan metod pencairan dengan air dan minyak.

Penyediaan minyak buah getah (RSO) merangkumi pemecahan kulit biji getah,

pengupasan, pemotongan, pengeringan biji getah, pengekstrakan minyak dengan

menggunakan soxhlet dan yang terakhir transesterifikasi. Selepas itu, RSO dicampur

bersama air suling dan pengemulsi. Pengemulsi bukan ionik seperti lesitin dan span

80 digunakan dalam penggubalan emulsi. Nisbah kepekatan bahan dan jenis

pengemulsi terbaik dikaji untuk menghasilkan emulsi yang stabil yang mempunyai

karakteristik seperti saiz titisan yang kecil, warna emulsi yang tertentu, kandungan

lembapan yang rendah, kelikatan yang sederhana dan nilai pH yang sederhana

rendah. Daripada analisis yang dijalankan, formulasi yang terbaik adalah formulasi

E2 yang mengandungi 50% air suling, 6% lesitin daripada berat minyak dan 47%

minyak buah getah (RSO).

vii

TABLE OF CONTENTS

CHAPTER TITLE PAGE

TITLE PAGE

DECLARATION

DEDICATION

ACKNOWLEDGEMENT

ABSTRACT

ABSTRAK

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF SYMBOLS AND ABBREVIATIONS

LIST OF APPENDICES

i

ii

iii

iv

v

vi

vii

xi

xii

xiv

xvi

1 INTRODUCTION 1

1.1 Background of Study 1

1.2 Problem Statements 3

1.3 Objectives 3

1.4 Hypothesis 4

1.5 Scope 4

2 LITERATURE REVIEW 5

2.1 Hevea Brasiliensis

2.1.1 The Properties of Rubber Seed oil

5

7

2.2 Important of Omega-3 Polyunsaturated fatty acids 7

viii

2.3 Emulsion

2.3.1 Microemulsion

2.3.2 Multiple Emulsion

8

9

10

2.4 Differences between Types of Emulsion 11

2.5 Purpose of Emulsion 12

2.6 Food emulsion 12

2.7 Food Additive 13

2.7.1 Preservative Omega-3

2.7.2 Antioxidants

2.7.3 Colouring Agent

2.7.4 Flavouring

2.7.5 Emulsifiers

2.7.5.1 Non- ionic Emulsifier

14

14

15

15

16

17

2.8 Major Factors Influencing the Formation and

Stability of Beverage Emulsion

19

2.9 Unstable Emulsion Mechanism 20

2.10 Conventional Methods in Preparing Emulsion

2.10.1 Homogenizer

2.10.2 Comparison between Available Methods

for Emulsion

22

22

23

2.11 Beaker Method in Making Emulsion 24

2.12 Hydrophilic- Lipophilic Balance (HLB) 24

2.13 Bancroft’s Rule 25

2.14 Phase Inversion 26

2.15 The Physical Properties of Emulsion

2.15.1 Type of Emulsion

2.15.2 Particle Size

26

26

27

2.16 Viscosity Properties 28

2.17 Moisture content 29

2.18 pH Measurement 30

2.19 Formulation from the Previous Study 31

3 METHODOLOGY

3.1 Introduction 33

ix

3.2 Materials 33

3.3 Apparatus 34

3.4 Sampling

3.4.1 Preparation of Rubber Seed from Kernel

3.4.2 Extraction of Rubber Seed Oil

35

35

35

3.5 Producing Rubber Seed Oil Emulsion

3.5.1 Preparation of Ingredients

3.5.2 Mixing of Ingredients

3.5.2.1 Homogenizer

36

37

37

39

3.6 Determination Type of Emulsion

3.7 Measurement Stability of Emulsion

3.8 Determination of Physical Characteristic

3.8.1 pH Measurement

3.8.2 Droplet Size Measurement

3.8.3 Measurement of Viscosity

3.8.4 Determination of Moisture Content

3.8.5 Observation Colour of Emulsion

3.9 Statistical Analysis

39

39

40

40

40

41

43

43

43

4 RESULTS AND DISCUSSION

4.1 Preliminary Study

4.1.1 Span 80

4.1.2 Lecithin

4.2 Determination Type of Emulsion

4.3 Stability of Emulsion

4.4 Physical Characteristics of the Emulsion

4.4.1 Effect Concentration of Span 80 on

Viscosity

4.4.2 Effect Concentration of Span 80 on pH

4.4.3 Effect Concentration of Span 80 on

Moisture Content

4.4.4 Effect Concentration of Span 80 on

Droplet Size

4.4.5 Effect Concentration of Span 80 on

45

45

45

49

51

52

53

54

55

56

57

61

x

Colour Observation

4.5 One-Way ANOVA Test

4.6 Selection of Base Formulation

4.6 Experimental errors

62

65

65

5 CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusions

5.2 Recommendations

66

66

67

REFERENCES 68

Appendices A-D 76-77

xi

LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Result of analysis for RSO 7

2.2 Comparison of emulsion and microemulsion. 10

2.3 Types of emulsions 11

2.4 Emulsifier and their regulatory status in US and Europes 16

2.5 Comparison between homogenizer and mechanical stirrer

methods

23

2.6 HLB number ranges and their application 25

2.7 Emulsion effect on the HLB value and phase ratio 27

2.8 Emulsion Preparation in selected previous research for

model emulsion

31

3.1 Material and its function 34

3.2 The apparatus and equipment with its function 34

3.3 How emulsifier, oil base and water base are mixed 37

3.4 Emulsion composition and its code 38

4.1 Preliminary study on ratio of oil to water for span 80 46

4.2 Preliminary study on percentage of span 80 46

4.3 Preliminary study on ratio of oil to water for lecithin 49

4.4 Preliminary study on percentage of lecithin 49

4.5 Effect Percentage of span 80 on degree of creaming 52

4.6 Effect Percentage of lecithin on degree of creaming 52

4.7 Result for colour observation 61

4.8 Analysis of omega-3 emulsion samples for various

formulation

62

xii

LIST OF FIGURES

FIGURE NO. TITLE PAGE

2.1 Rubber seed 5

2.2 Fatty acid composition in MRSO. 6

2.3 Chemical structure of omega-3 and omega-6 6

2.4 Parameters that influence the formation and stability of

beverage emulsion.

20

2.5 The schematic of emulsion destabilization mechanisms 21

2.6 How the homogenizing valve work 22

3.1 Rubber seed before peeled 35

3.2 The arrangement of soxhlet extractor. 36

3.3 Homogenizing step with homogenizer 38

3.4 Microscope used in analyzed droplet size of emulsion 41

3.5 Viscometer for viscosity analysis 42

3.6 The general flow chart of the overall experiment 44

4.1 Emulsion for ratio oil to water for (a) 25: 75 (b) 60:40 47

4.2 Emulsion for ratio oil to water for 50:50 but with the

concentration of emulsifier (a) E17-6% (b) E16-7%

48

4.3 Emulsion for ratio oil to water for 50:50 but with the

concentration of emulsifier (a) E15-8.5% (b) E14-10%

48

4.4 Emulsion with ratio oil to water for (a) 60:40 (b) 25: 75 50

4.5 Emulsion for ratio oil to water for 50:50 with the

concentration of emulsifier (a) E1-3% (b) E2-6% and (c)

E3-9%

51

4.6 Observation during determination type of emulsion. 51

xiii

4.7 The graph of viscosity against the formulation with the

increasing percentage of emulsifier

54

4.8 The effect on pH due to the increasing percentage and

type of emulsifier

55

4.9 The effect of percentage and type of emulsifier on

moisture content

56

4.10 The microstructure of emulsion with different

concentration of span 80 (a) 6%-E7 (b) 7%-E6(c) 8.5 %-

E5 (d) 10%-E4

58

4.11 The microstructure of emulsion with different

concentration of lecithin (a) 3%-E1 (b) 6%-E2(c) 9 %-E3

58

4.12 Frequency of distribution of long term stable emulsion

graph with 4 different formulations

59

4.13 Frequency of distribution of shortterm stable emulsion

graph with 4 different formulations.

59

4.14 Graph for maximum droplet size based on the

concentration of emulsifier.

60

xiv

LIST OF SYMBOLS AND ABBREVIATIONS

AA - Arachidonic acid

ANOVA - Analysis of variance

C - Carbon

CHD - Coronary heart disease

cP - Centipoise

d - Diameter

DHA - Docosahaxaneoic acid

E1-E17 - Emulsion formulation

EPA - Ecasapetanoic acid

fw - fraction of the dispersed phase

g - Gram

GLC - Gas Liquid Chromatography

GMS - Glycerol monostearate

HLB - Hydrophylic-Lipophilic Balance

HPLC - High Performance Liquid Chromatography

kw - Conductivity of continuous phase

LA - Linoleic acid

LNA - Linolenic acid

Lα - Lamellar phase

MG - Monodiglyceride

ml - Millilitres

MRSO - Malaysian rubber seed oil

n - Number of droplet

n-3 - Omega-3

xv

n-6 - Omega-6

NaOH - Sodium Hydroxide

O - Oxygen

O/W - Oil in water emulsion

O/W/O - Oil-in-water-in-oil emulsion

oC - Degree Celsius

PIT - Phase inversion Temperature

PUFA - Polyunsaturated Fatty Acid

R - Alkane chain

RPM - Rotation per minute

RSO - Rubber seed oil

S.D - Standard deviation

TAG - Triacyiglycerol

UV - Ultraviolet

US - United State

V - Volume

W/O - Water in oil emulsion

W/O/W - Water-in-oil-in-water emulsion

μ - Micro (10-6

)

% - Percentage

xvi

LIST OF APPENDICES

APPENDIX TITLE PAGE

A One-way ANOVA analysis for Moisture Content 76

B One-way ANOVA analysis for pH value 76

C One-way ANOVA analysis for Viscosity 77

D One-way ANOVA analysis for Droplet Size 77

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