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UNIVERSITI PUTRA MALAYSIA

HADIZA ALTINE ADAMU

IB 2012 15

BIOTRANSFORMATION OF FERULIC ACID, ANTIOXIDANT ACTIVITY, AND CHOLESTEROL GENE REGULATION OF RICE BRAN BYPRODUCTS

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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

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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

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

of the requirement for the degree of Master of Science



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

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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.

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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

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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

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berhasil tinggi dan berekonomikal dalam proses biotransformasi sisa buangan

industri pertanian kepada produk bernilai tambah seperti nutraseutikal.

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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.

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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


(Internal Examiner)

Norhaizan Mohd. Esa, PhD

Assoc. Professor

Faculty of Medicine and Health Sciences


(Internal Examiner)

Musalmah Mazlan, PhD

Professor

Universiti Kebangsaan Malaysia

(External Examiner)

________

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies


Date:

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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


(Chairman)

Dzulkefly Kuang Abdullah, PhD

Professor

Faculty of Science


(Member)

____________________

BUJANG BIN KIM HUAT, 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 currently submitted for any other degree at Universiti Putra Malaysia or at any

other institution.

___________________

HADIZA A. ADAMU

Date: 18 December 2012

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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

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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

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4.2.3 Initial bacterial concentration 64

4.3 Antioxidant activity assay of byproducts from submerged

fermentation

71


scavenging activity

71

4.3.2 2, 2‟-azino-bis (3-ethylbenzthiazoline-6-sulfonic)


) 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


scavenging activity

84

4.5.2 2, 2‟-azino-bis (3-ethylbenzthiazoline-6-sulfonic)


) 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

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