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UNIVERSITI PUTRA MALAYSIA

AQUEOUS ENZYMATIC EXTRACTION AND BASIC QUALITY ASSESSMENT OF RICE (Oryza sativa L.) BRAN OIL

MOHAMED REZA HUSSAIN

FSTM 2011 13

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AQUEOUS ENZYMATIC EXTRACTION AND BASIC QUALITY ASSESSMENT OF

RICE (Oryza sativa L.) BRAN OIL

MOHAMED REZA BIN HUSSAIN

MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2011

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AQUEOUS ENZYMATIC EXTRACTION AND BASIC QUALITY ASSESSMENT OF

RICE (Oryza sativa L.) BRAN OIL


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia in

Fulfillment of the Requirement for the Degree of Master of Science.

June 2011

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This dissertation is dedicated to

Norihan Zakaria

for endurance, inspiration, encouragement, understanding,

sacrifices and doa’s

during the completion of this project.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of

the requirement for the degree of Master of Science.

AQUEOUS ENZYMATIC EXTRACTION AND BASIC QUALITY ASSESSMENT

OF RICE (Oryza sativa L.) BRAN OIL

By


June 2011

Chairperson : Associate Professor Azizah Hj Abd Hamid, PhD

Faculty : Faculty of Food Science and Technology

Rice bran, an industrial by-product of rice milling is rich in proteins, lipids, dietary fibers

and antioxidant compounds, such as tocopherol, tocotrienol and oryzanol. This study

evaluated the chemical composition of rice bran obtained from 4 Malaysian rice varieties.

Results indicated that there is no significant (P>0.05) difference in chemical composition

of all varieties analyzed. The effect of individual and mixtures of enzymes, namely

cellulase (Celluclast 1.5L), protease (Alcalase), amylase (Termamyl), polygalacturonase

(Viscozyme), and pectinase (Pectinex ULTRA SP-L) combined with other process

parameters - dilution ratio, aqueous media, homogenization, mixing equipment, and

centrifugation speed at different level on rice bran oil yield was evaluated. Results of the

study showed that these enzymes had successfully extracted the rice bran oil from rice

bran (75.66%) compared to that without enzymes (18.97%). Results also showed that

using Alcalase resulted in higher oil yield compared to other enzymes. The maximum oil

recovery of 70.3% was achieved at pH 9 and 70oC with <750 m particle size bran, 2.0%

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enzyme concentration and 3-hour extraction times. Iodine value (104.7± 0.3 g

iodine/100 g oil), peroxide value (0.5± 0.2 meq/kg) and anisidine value (24.8± 0.2) of

enzyme extracted oil obtained was comparable to that of commercially available rice bran

oil. The oil however, was found to contain appreciable free fatty acid (2.6± 0.3 as % oleic

acid), although still within the acceptable level for edible oils. It is encouraging to note

that the enzyme extracted oil consisted of significantly higher concentration of both total

and individual isomers (α-, β+γ, δ-) of tocopherol and tocotrienol compared SE oil. High

level of oryzanol (2344 ppm) and carotenoids (-carotene and lycopene) (58.72 and

12.74 ug/100g) were also found in the oil. The result showed that enzymatic extracted

rice bran oil exhibited appreciable antioxidative activity that was significantly (P<0.05)

highest compared to that of solvent extracted as measured by DPPH radical scavenging

method, ferric thiocyanate (FTC) and thiobarbituric acid (TBA) tests. The study revealed

that RB from Malaysian rice consisted of excellent level of nutrient especially many

health promoting constituents and which could be considered as the most valuable rice

milling process. The study also demonstrated that extracts of the RBO are a viable source

of natural antioxidants or as value–added products in the preparation of specialty oil and

for enrichment of certain products such as salad oil or functional ingredient in the

development of functional food.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains.

PENGEKSTRAKAN AKUAS BERENZIM DAN PENILAIAN KUALITI ASAS

MINYAK DEDAK BERAS (Oryza sativa L.)

Oleh


Jun 2011

Pengerusi : Professor Madya Azizah Hj Abd Hamid, PhD

Fakulti : Fakulti Sains dan Teknologi Makanan

Dedak beras merupakan satu hasil sampingan pengilangan beras yang kaya dengan

sumber protein, lemak, serat diet dan bahan antioksidan seperti tokoferol, tokotrienol dan

orizanol. Kajian ini dijalankan untuk menentukan komposisi kimia dedak 4 varieti beras

di Malaysia. Keputusan menunjukkan bahawa tiada perbezaan yang signifikan (P>0.05)

di dalam komposisi kimia untuk semua varieti beras yang dianalisis. Kesan enzim secara

individu dan campuran enzim iaitu enzim selulase (Celluclast 1.5L), protease (Alcalase),

amilase (Termamyl), poligalakturonase (Viscozyme), dan pektinase (Pectinex SP-L ultra)

untuk mengekstrak minyak dedak beras telah digabungkan dengan lain-lain parameter

iaitu nisbah pencairan, jenis media berair, kaedah penghomogenan, peralatan

pencampuran dan kelajuan pengemparan pada tahap yang berbeza telah dinilai.

Keputusan kajian menunjukkan bahawa enzim tersebut telah berjaya mengekstrak

minyak dedak beras (75.66%) berbanding tanpa penggunaan sebarang enzim semasa

pengekstrakan (18.97%). Dari keputusan analisis, didapati penggunaan Alcalase telah

menghasilkan kandungan minyak yang lebih tinggi berbanding penggunaan enzim yang

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lain. Perolehan minyak yang maksimum sebanyak 70.3% telah dicapai pada pH 9 dan

70oC dengan < 750m saiz zarah dedak, kepekatan 2.0% enzim dan 3-jam masa untuk

pengekstrakan. Nilai iodin (104.7± 0.3 g iodine/100 g minyak), nilai peroksida (0.5± 0.2

meq/kg) dan nilai anisidin (24.8± 0.2) minyak pengekstrakan enzim adalah setanding

dengan minyak dedak beras yang didapati di pasaran. Walaubagaimanapun, minyak

dedak beras didapati mengandungi asid lemak bebas yang memadai (2.6± 0.3 sebagai %

asid sebagai % oleik), walaupun ianya masih berada pada tahap yang boleh diterima

sebagai minyak makan. Hal ini mendorong untuk diberi perhatian bahawa pengekstrakan

enzim menghasilkan minyak yang mengandungi kepekatan tokoferol dan tocotrienol

beserta individu isomernya (α-, β + γ, δ-) jauh lebih tinggi. Kandungan orizanol (2344

ppm) dan karotenoid (-karotena and likopena) (58.72 and 12.74 ug/100g) yang dikesan

juga adalah tinggi berbanding pengekstrakan mengunakan pelarut. Keputusan kajian

menunjukkan bahawa ekstrak berenzim minyak dedak mempunyai aktiviti antioksidan

yang lebih tinggi secara signifikan (P <0.05) berbanding dengan ekstrak berpelarut yang

diukur dengan kaedah pemerangkapan radikel DPPH, ferik tiosianat (FTC) dan ujian asid

tiobarbiturik (TBA). Keputusan kajian menunjukkan bahawa varieti beras Malaysia

mengandungi kandungan nutrien yang sangat baik terutamanya mengandungi juzuk yang

boleh meningkatkan kesihatan dan boleh dianggap sebagai proses pengilangan padi yang

paling berharga. Kajian juga menunjukkan bahawa ekstrak dari minyak dedak beras

adalah sumber antioksidan semulajadi yang tinggi dan sebagai nilai tambah produk dalam

penyediaan minyak khusus dan untuk memperkayakan produk tertentu seperti minyak

salad atau sebagai ramuan fungsian dalam pembangunan makanan berfungsi.

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ACKNOWLEDGEMENTS

Syukur Alhamdullillah to the Almighty Allah s.w.t for giving me strength, patience and

capability to complete this project and thesis write up and salawat and salam to his

righteous messenger, prophet Muhammad s.a.w.

First of all, I would like to express my deepest thanks and appreciation to the Chairman

of Supervisory Committee, Associate Professor Dr. Azizah Binti Abdul Hamid of the

Department of Food Science, Faculty of Food Science and Technology, UPM for her

guidance, patience, advice, constructive criticisms and encouragement throughout this

interesting research and most importantly her kindness to give me a chance to gained

knowledge and experience through the seminar that I have attended. All the valuable

experiences that I gained as one of her graduate students and her continuous commitment

and willingness to help towards the success of my study will always be remembered deep

in my heart.

My deep sense of gratitude and respect is quoted to my co-supervisors, Professor Dr.

Yaakob Bin Che Man, Professor Dr. Azizah Binti Osman and Professor Dr. Nazamid Bin

Saari, for their sincere advice, support and encouraging interest in my study. My sincere

thank also extended to all the highly dedicated, friendly and helpful staffs of Faculty of

Food Science and Biotechnology, UPM and to all my fellow graduate students especially

in the Nutraceutical and Enzymology Lab. Sincere gratitude is also dedicated to

Malaysian Government for the financial support provided through IRPA fund.

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Paramount gratitude is also owed to my beloved wife Norihan Binti Zakaria for her

patience and encouragement to give me strength to complete this research. Finally, my

deepest gratitude and appreciation to my parents, Norliah Binti Abdullah for their

continuous support, and all my brothers and sisters, thank you so much for their love and

support who have been inspiring my life.

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I certify that an Examination Committee met on 9 Jun 2011 to conduct the final

examination of Mohamed Reza Bin Hussain on his Master of Science thesis entitled

“Aqueous Enzymatic Extraction and Basic Quality Assessment of Rice (Oryza sativa)

Bran Oil” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980

and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The committee

recommends that the candidate be awarded the relevant degree. Members of the

Examination Committee are as follows:

SEYED HAMED MIRHOSSEINI, Ph.D.

Food Technology Department,

Faculty of Food Science and Technology,

Universiti Putra Malaysia.

(Chairman)

BADLISHAH SHAM BAHARIN

Associate Professor,

Department of Food Science,

Faculty of Food Science and Technology,


(Internal Examiner)

MAZNAH ISMAIL, PhD

Professor

Department of Nutrition and Dietetics,

Faculty of Medicine and Health Sciences,


(Internal Examiner)

MAMOT SAID, PhD


Universiti Kebangsaan Malaysia.

(External Examiner)

__________________________________

NORITAH OMAR, PhD

Associate Professor/Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date :

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This thesis was submitted to the Senate of Universiti Putra Malaysia has been accepted as

fulfillment of the requirement for the degree of Master of Science. The members of the

Supervisory Committee were as follows:

Azizah Abdul Hamid, Ph.D.


Faculty of Food Science and Technology


(Chairman)

Yaacob Che Man, Ph.D.

Professor



(Member)

Nazamid Saari, Ph.D.

Professor,



(Member)

Azizah Osman, Ph.D.

Professor,



(Member)

___________________________________

HASANAH MOHD GHAZALI, PhD

Professor and Dean

School of Graduate Studies


Date :

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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 degree at Universiti Putra Malaysia or other institutions.

____________________________


Date: 9 June 2011

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TABLE OF CONTENTS

Page

DEDICATION ii

ABSTRACT iii

ABSTRAK v

ACKNOWLEDGEMENTS vii

APPROVAL ix

DECLARATION xi

LIST OF TABLES xvii

LIST OF FIGURES xix

LIST OF ABBREVIATIONS/NOTATIONS/GLOSSARY OF TERMS xx

CHAPTER

1 GENERAL INTRODUCTION 1

2 REVIEW OF THE LITERATURE 6

2.1 Rice 6

2.1.1 Rice Production 6

2.1.2 Rice Composition 8

2.2 Rice Bran 9

2.2.1 Rice Bran Productions 10

2.2.2 Rice Bran Composition 11

2.2.3 Stabilization of Rice Bran 16

2.2.4 Health Benefit of Rice Bran 21

2.3 Rice Bran Oil (RBO) 23

2.4 Industrial Technology for Rice Bran Oil Extraction 27

2.5 Alternatives Technologies 31

2.5.1 Supercritical Fluid Extraction 31

2.5.2 Aqueous Enzymatic Oil Extraction (AEE) 36

2.5.2.1 Enzymes Used in Aqueous Enzymatic Extraction 37

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2.5.2.2 Enzymatic Treatment in Aqueous Extraction Process 39

2.5.2.3 Factors Affecting the Enzymatic Treatment and Oil

Recovery

42

2.5.3 Enzymatic Treatment in Solvent-Based and Pressing Extraction 46

2.6 Aqueous Extraction on Rice Bran 48

2.7 Aqueous Enzymatic Extraction on Rice Bran 49

2.8 Quality of Oil Extracted by Aqueous Enzymatic Extraction 54

3 CHEMICAL COMPOSITION OF RICE BRAN OBTAINED FROM

FOUR MALAYSIAN RICE (Oryza sativa L.) VARIETIES

56

3.1 Introduction 56

3.2 Materials and Methods 58

3.2.1 Source of Samples 58

3.2.2 Determination of Macronutrient 59

3.2.2.1 Proximate Analysis 59

3.2.2.2 Fatty Acid Profile 60

3.2.2.3 Amino Acid Profile 61

3.2.3 Determination of Micronutrient 62

3.2.3.1 Mineral Content 62

3.2.3.2 Tocopherol 63

3.2.3.3 Carotenoids 63

3.2.4 Statistical Analysis 64

3.3 Results and Discussions 64

3.3.1 Proximate Analysis 64

3.3.2 Fatty Acid Profile 70

3.3.3 Amino Acid Profile 72

3.3.4 Mineral 74

3.3.5 Tocopherol 76

3.3.6 Carotenoids 78

3.4 Conclusions 79

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4 AQUEOUS ENZYMATIC EXTRACTION OF SPECIALITY OIL FROM

RICE BRAN

80

4.1 Introduction 80


4.2.1 Samples 83

4.2.2 Enzymes Selection 83

4.2.2.1 Cellulase Preparation 84

4.2.2.2 Protease Preparation 84

4.2.2.3 Amylase Preparation 84

4.2.2.4 Polygalacturonase Preparation 85

4.2.2.5 Pectinase Preparation 85

4.2.3 Methods 87

4.2.3.1 Determination of Preliminary Extraction Parameter 87

4.2.3.2 Aqueous Enzymatic Extraction of Rice Bran Oil 88

4.2.3.3 Characterization of the Oil 89

4.2.3.4 Chemical Composition Rice Bran Oil 93



4.3.1 Preliminary Extraction Parameter 96

4.3.1.1 Effect of Various Enzymes 96

4.3.1.2 Effect of the Liquid to Solid Ratio 98

4.3.1.3 Effect of Aqueous Media 99

4.3.1.4 Effect of Homogenization 100

4.3.1.5 Effect of Mixing Equipment 101

4.3.1.6 Effect of Centrifugation Speed 102

4.3.2 Extractability of Oil 103

4.3.2.1 Effect of pH and Incubation Temperatures 103

4.3.2.2 Effect of Particle Size 105

4.3.2.3 Effect of Enzyme Concentration 106

4.3.2.4 Effect of Extraction Time 107

4.3.3 Quality of Aqueous Enzymatic Extraction Rice Bran Oil 108

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4.3.4 Bioactive Compounds of Aqueous Enzymatic Extraction Rice

Bran Oil

111

4.4 Conclusions 112

5 QUALITY CHARACTERISTIC AND ANTIOXIDATIVE ACTIVITIES

OF AQUEOUS ENZYMATIC EXTRACTION RICE (Oryza sativa L.)

BRAN OIL

113

5.1 Introduction 113


5.2.1 Source of Sample 115

5.2.2 Solvent Extraction (SE) 115

5.2.3 Aqueous Enzymatic Extraction of Rice Bran Oil 116

5.2.4 Determination of Quality and Chemical Composition of the Oil 116

5.2.4.1 Oil Quality 116

5.2.4.2 Chemical Composition 117

5.2.5 Determination of Antioxidant Activity of the Oil 117

5.2.5.1 Ferric Thiocyanate Test (FTC) 117

5.2.5.2 Thiobarbituric Acid Test (TBA) 118

5.2.5.3 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Radical

Scavenging Method

119



5.3.1 Oil Quality and Chemical Composition of Aqueous Enzymatic

Extraction, Solvent Extraction and Commercially Available Rice

Bran Oil

120

5.3.1.1 Oil Quality of the Rice Bran Oil 121

5.3.1.2 Chemical Composition of the Rice Bran Oil 125

5.3.1.3 Antioxidant Activity 134

5.4 Conclusions 139

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6 CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE

RESEARCH

140

6.1 General Conclusions 140

6.2 Recommendations for Future Research 144

REFERENCES 145

APPENDICES 176

BIODATA OF STUDENT 180

LIST OF PUBLICATIONS 181

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