UNIVERSITI PUTRA MALAYSIA
HADIZA ALTINE ADAMU
IB 2012 15
BIOTRANSFORMATION OF FERULIC ACID, ANTIOXIDANT ACTIVITY, AND CHOLESTEROL GENE REGULATION OF RICE BRAN BYPRODUCTS
© COPYRIG
HT UPM
BIOTRANSFORMATION OF FERULIC ACID, ANTIOXIDANT ACTIVITY,
AND CHOLESTEROL GENE REGULATION OF RICE BRAN
BYPRODUCTS
HADIZA ALTINE ADAMU
MASTER OF SCIENCE
UNIVERSITI PUTRA MALAYSIA
December 2012
© COPYRIG
HT UPM
BIOTRANSFORMATION OF FERULIC ACID, ANTIOXIDANT ACTIVITY,
AND CHOLESTEROL GENE REGULATION OF RICE BRAN
BYPRODUCTS
By
HADIZA ALTINE ADAMU
Thesis Submitted to the School of Graduate Studies, Universiti Putra
Malaysia, in Fulfilment of the Requirements for the Degree of Master Science
December 2012
© COPYRIG
HT UPM
iii
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment
of the requirement for the degree of Master of Science
BIOTRANSFORMATION OF FERULIC ACID, ANTIOXIDANT ACTIVITY,
AND CHOLESTEROL GENE REGULATION OF RICE BRAN
BYPRODUCTS
By
HADIZA ALTINE ADAMU
December 2012
Chairman : Professor Maznah Ismail, PhD
Faculty : Institute of Bioscience
An astronomical proportion of lignocellulosic waste such as husks, bran, bagasse,
cobs, lint and fibre are produced via paper-pulp, timber, forestry and agricultural
industries. The said “waste” can be transformed into numerous value added products
such as biofuels, chemicals, cheap energy sources for fermentation, improved animal
feed and human nutrients. In this study, the potential of Lactobacillus farciminis
29644, Lactobacillus reuteri 23272 and Lactobacillus fermentum 11976 to
biotransform ferulic acid via submerged fermentation and solid state fermentation of
rice bran to other phenolic compounds, their antioxidant activities and ability to
regulate the genes involved in cholesterol metabolism were investigated. Ferulic acid
is an important bioactive constituent of agro-industrial wastes such as brans, husks,
cobs, bagasse, fiber and lint. Biotransformation started after 5 h incubation of L.
farciminis 29644 with ferulic acid in Man Rogosa and Sharpe (MRS) broth at 37 °C
under 5% CO2 and production rate was at its peak after 48 h. The impact of initial
concentrations of ferulic acid and bacteria on the production of biotransformed
products in particular 4-vinyl-guaiacol (4VG) was studied. Biotransformation via
© COPYRIG
HT UPM
iv
solid state fermentation (SSF) using rice bran (RB) as substrate was equally
determined over a 24 h period. Identification and quantification of biotransformed
products was by high performance liquid chromatography (HPLC). Subsequently the
antioxidant capacity of the biotransformed product from submerged and SSF along
with the ability of the byproduct from SSF of rice bran to regulate the genes involved
in cholesterol metabolism was investigated. The results indicated that the production
of 4VG via submerged fermentation was significantly affected by the initial
concentration of ferulic acid, and empirically 15, 25, 35 and 50 mg/l of ferulic acid
yielded 3.34, 6.84, 8.93 and 10.26 mg/l of 4VG, respectively. Under SSF, the
biotransformed products after 24 h were considered the most significant. The
production of 4VG and vanillin were at their highest after 1h at 29.0 and 7.1 mg/l
respectively while ferulic acid and vanillic acid were at 17h at 19.6 and 9.6 mg/l
respectively. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2´-azinobis (3-ethyl-
benzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity of the
byproduct from submerged fermentation showed that the higher the ferulic acid
concentration the better the activity as was observed with 25, 35 and 50 mg/l of
ferulic acid. The same trend was equally observed for the byproduct from SSF. As
for the ability of the SSF byproduct to regulate genes involved in cholesterol
metabolism, 5µg/ml of byproduct up regulated LDLR, 5 and 20µg/ml of byproduct
down regulated ApoB100, 20µg/ml of byproduct down regulated HMG-CoAR, and
40µg/ml of byproduct up regulated ApoA1. The findings are a milestone towards
economical high yielding means of biotransforming some common agro-industrial
wastes to a value added product such as nutraceuticals.
© COPYRIG
HT UPM
v
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Master Sains
BIOTRANSFORMASI ASID FERULIC, AKTIVITI ANTIOKSIDAN, DAN
KAWALATUR GEN KOLESTEROL OLEH HASIL SAMPINGAN
Oleh
HADIZA ALTINE ADAMU
Disember 2012
Pengerusi : Profesor Maznah Ismail, PhD
Fakulti : Institut Biosains
Kebanyakan sisa lignoselulosik seperti sekam, dedak, hampas tebu, tongkol, lin dan
serat adalah hasil daripada industri pulpa kertas, pembalakan, perhutanan dan
pertanian. “Sisa” tersebut boleh ditransformasikan kepada bahan api bio, bahan
kimia, sumber tenaga murah bagi proses penapaian, makanan haiwan dan manusia
serta pelbagai produk lain yang mempunyai nilai tambah. Dalam kajian ini, potensi
Lactobacillus farciminis ATCC 29644 Lactobacillus reuteri 23272 and Lactobacillus
fermentum 11976 bagi proses biotransformasi asid ferulik melalui penapaian
tenggelam dan penapaian dedak beras dalam keadaan pepejal kepada sebatian fenol
yang lain, aktiviti antioksidan serta keupayaan untuk mengawalatur gen yang terlibat
dalam metabolisme kolesterol telah diselidik. Asid ferulik adalah sebatian bioaktif
utama dalam sisa buangan industri pertanian seperti dedak, sekam, tongkol, hampas
tebu, serat dan lin. Proses biotransformasi asid ferulik bermula selepas lima jam
inkubasi L. farciminis dengan acid ferulik dalam media „Man Rogosa and Sharpe‟
(MRS) pada 37 °C dan 5% CO2. Kadar penghasilan mencapai kemuncaknya selepas
48 jam. Kesan konsentrasi awal asid ferulik dan bakteria dalam pengeluaran produk
biotransformasi terutamanya 4-vinyl-guaiacol (4VG) turut dikaji. Proses
© COPYRIG
HT UPM
vi
biotransformasi melalui penapaian dalam keadaan pepejal (SSF) dengan
menggunakan dedak beras (RB) sebagai substrat telah diselidik sepanjang tempoh 24
jam. Proses identifikasi dan kuantifikasi produk biotransformasi telah dijalankan
dengan menggunakan kromatografi cecair berprestasi tinggi (HPLC). Sementara itu,
kapasiti antioksidan serta keupayaan produk biotransformasi penapaian tenggelam
dan produk sampingan SSF dalam mengawalatur gen yang terlibat dalam
metabolisme kolesterol juga dikaji. Hasil kajian menunjukkan produksi 4VG melalui
penapaian tenggelam dipengaruhi dengan signifikan oleh konsentrasi awal asid
ferulik, di mana 15, 25, 35 dan 50 mg/l asid ferulik telah menghasilkan 3.34, 6.84,
8.93 dan 10.26 mg/l 4VG masing-masing. Di bawah keadaan SSF, produk
biotransformasi pada jam ke-24 adalah yang paling signifikan. Produksi 4VG dan
vanillin adalah paling tinggi selepas 1 jam, mencapai 29.0 dan 7.1 mg/l masing-
masing, sementara asid ferulik dan asid vanilik mencapai tahap maksimum selepas
17 jam, dengan produksi sebanyak 19.6 dan 9.6 mg/l masing-masing. Aktiviti
memerangkap radikal 2,2-diphenyl-1-picrylhydrazyl (DPPH) dan 2,2´-azinobis (3-
ethyl-benzothiazoline-6-sulphonic acid) (ABTS) oleh produk sampingan penapaian
tenggelam menunjukkan aktiviti yang lebih tinggi dengan konsentrasi asid ferulik
yang lebih pekat, seperti yang diperhatikan dengan 25, 35 dan 50 mg/l asid ferulik.
Gaya yang sama turut diperhatikan dengan produk sampingan SSF. Produk
sampingan SSF turut berupaya mengawalatur gen yang terlibat dalam metabolisme
kolesterol. 5µg/ml produk sampingan meningkatkan regulasi LDLR, 5 and 20µg/ml
produk sampingan mengurangkan regulasi ApoB100, 20µg/ml produk sampingan
mengurangkan regulasi HMG-CoAR, dan 40µg/ml produk sampingan meningkatkan
regulasi ApoA1. Penemuan dalam kajian ini membantu mewujudkan cara yang
© COPYRIG
HT UPM
vii
berhasil tinggi dan berekonomikal dalam proses biotransformasi sisa buangan
industri pertanian kepada produk bernilai tambah seperti nutraseutikal.
© COPYRIG
HT UPM
viii
ACKNOWLEDGEMENTS
Thanks be to Almighty Allah, for seeing me through this work. I acknowledge with
great appreciation, my able and dynamic supervisor, Prof. Dr. Maznah Ismail for her
constructive criticism, meticulous guidance and patience during the course of this
project. I am greatly indebted to my family especially my parents Dr. and Mrs.
Adamu for their love, financial and parental encouragement and to my dear brothers
Ibrahim and Abdullahi. My appreciation also goes to the staff of the laboratory, my
colleagues and friends for their support and assistance.
© COPYRIG
HT UPM
ix
I certify that an Examination Committee has met on date of viva voce to conduct the
final examination of Hadiza Altine Adamu on her Master of Science thesis entitled
“Biotransformation of ferulic acid, antioxidant activity and cholesterol gene
regulation of rice bran byproducts” is in accordance with the Universiti Pertanian
Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher
Degree) Regulation 1981. The Committee recommends that the student be awarded
the Master of Science.
Members of the Examination Committee were as follows:
Md. Zuki Abu Bakar, PhD
Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Chairman)
Noorjahan Banu Mohamed Alitheen, PhD
Assoc. Professor
Faculty of Biotechnology and Biomolecular sciences
Universiti Putra Malaysia
(Internal Examiner)
Norhaizan Mohd. Esa, PhD
Assoc. Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Internal Examiner)
Musalmah Mazlan, PhD
Professor
Universiti Kebangsaan Malaysia
(External Examiner)
________
SEOW HENG FONG, PhD
Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
© COPYRIG
HT UPM
x
This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Master of Science. The
members of the Supervisory Committee were as follows:
Maznah Ismail, PhD
Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Chairman)
Dzulkefly Kuang Abdullah, PhD
Professor
Faculty of Science
Universiti Putra Malaysia
(Member)
____________________
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
\
© COPYRIG
HT UPM
xi
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 currently submitted for any other degree at Universiti Putra Malaysia or at any
other institution.
___________________
HADIZA A. ADAMU
Date: 18 December 2012
© COPYRIG
HT UPM
xii
TABLE OF CONTENTS
Page
ABSTRACT iii
ABSTRAK v
ACKNOWLEDGEMENTS viii
APPROVAL ix
DECLARATION xi
LIST OF TABLES xiv
LIST OF FIGURES xv
LIST OF EQUATIONS xviii
LIST OF ABBREVIATIONS xix
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 5
2.1 Biotransformation 5
2.1.1 Ferulic acid 6
2.1.2 Vanillin 7
2.1.3 4-Vinyl guaiacol 9
2.1.4 Solid state and submerged fermentation 10
2.2 Bioactive compounds 11
2.3 Rice bran 13
2.3.1 Bioactive compounds in rice bran 16
2.3.2 Health promoting activity of rice bran 17
2.4 Extraction 20
2.4.1 High performance liquid chromatography (HPLC) 21
2.5 Free radicals and reactive species 23
2.5.1 Origin and terms 23
2.5.2 Beneficial effects 25
2.5.3 Damaging effects 25
2.6 Antioxidants 26
2.6.1 Complex nature of antioxidants 26
2.6.2 Antioxidant and its mechanism 27
2.6.3 Phenolic and polyphenolic compounds 29
2.7 Antioxidant activity assay 31
2.7.1 Free radical scavenging activity 31
(a) 2,2-diphenyl-1-picrylhydrazyl (DPPH) 32
(b) 2,2´-azinobis (3-ethyl-benzothiazoline-6-
sulphonic acid) (ABTS)
33
2.8 Cholesterol 34
2.8.1 Regulation of cholesterol homeostasis 36
36
3 MATERIALS AND METHODS 39
3.1 Chemicals 39
3.2 Microorganism 40
© COPYRIG
HT UPM
xiii
3.2.1 Man, Rogosa and Sharpe (MRS) agar and broth
preparation
40
3.2.2 Screening of microorganism 42
3.2.3 Colony count, inoculum preparation and
biotransformation
42
3.2.4 Analysis of culture supernatant 43
(a) Identification of 4VG by gas chromatography–
mass spectrometry (GC-MS)
43
(b) Quantification of 4VG by HPLC 44
3.2.5 Experimental design 44
(a) Initial bacterial concentration 45
(b) Initial substrate concentration 45
3.3 Solid state fermentation of rice bran 45
3.3.1 Duration of incubation 46
3.3.2 Culture conditions 46
3.3.3 Extract preparation 46
3.3.4 Thin layer chromatography 47
3.4 Antioxidant activity assays 47
3.4.1 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical
scavenging activity
47
(a) Preparation of solutions 47
(b) Procedure 48
3.4. 2 2, 2‟-azino-bis (3-ethylbenzthiazoline-6-sulfonic)
acid radical cation scavenging (ABTS+·
) assay
48
(a) Preparation of solutions 48
(b) Procedure 49
3.5 Media preparation 49
3.5.1 Culture of HepG2 cells 49
3.5.2 Measurement of cell viability 50
3.5.3 Treatment 51
3.6 Gene expression studies 51
3.6.1 RNA extraction 51
3.6.2 cDNA synthesis 52
3.6.3 Quantitative real time PCR 52
3.7 Statistical analysis 55
4 RESULTS AND DISCUSSION 56
4.1 Screening of microorganisms for biotransformation 56
4.1.1 pH 56
4.1.2 O2 availability and duration of incubation 57
4.2 Optimization of biotransformation of ferulic acid by
selected lactic acid bacteria and antioxidant activity of
biotransformed product
59
4.2.1 Biotransformation process in submerged
fermentation
59
4.2.2 Initial ferulic acid concentration 63
© COPYRIG
HT UPM
xiv
4.2.3 Initial bacterial concentration 64
4.3 Antioxidant activity assay of byproducts from submerged
fermentation
71
4.3.1 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical
scavenging activity
71
4.3.2 2, 2‟-azino-bis (3-ethylbenzthiazoline-6-sulfonic)
acid radical cation scavenging (ABTS+·
) assay
75
4.4 Solid state fermentation of stabilized rice bran with
lactobacillus farciminis
77
4.4.1 Viable cell count of Lactobacillus farciminis
cultured in rice bran
77
4.4.2 Extraction of biotransformed product 79
4.4.3 Biotransformation process in solid state
fermentation
79
4.5 Antioxidant activity assay of byproducts from solid state
fermentation
84
4.5.1 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical
scavenging activity
84
4.5.2 2, 2‟-azino-bis (3-ethylbenzthiazoline-6-sulfonic)
acid radical cation scavenging (ABTS+·
) assay
86
4.6 Regulation of genes involved in cholesterol metabolism by
biotransformed product from rice bran in HepG2 cells
89
4.6.1 Cell viability assay 89
4.6.2 Regulation of LDLR gene by SSF RB extract 90
4.6.3Regulation of HMG-CoAR gene by SSF RB extract 92
4.6.4 Regulation of ApoB 100 gene by SSF RB extract 93
4.6.5 Regulation of Apo A1 gene by SSF RB extract 94
5 CONCLUSION 99
5.1 Future work
100
REFERENCES 101
APPENDICES 120
BIODATA OF STUDENT 124
LIST OF PUBLICATIONS AND PROCEEDINGS 125