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

Transcription

1 UNIVERSITI PUTRA MALAYSIA BIOTRANSFORMATION OF FERULIC ACID, ANTIOXIDANT ACTIVITY, AND CHOLESTEROL GENE REGULATION OF RICE BRAN BYPRODUCTS HADIZA ALTINE ADAMU IB

2 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

3 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

4 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 and Lactobacillus fermentum 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 with ferulic acid in Man Rogosa and Sharpe (MRS) broth at 37 C under 5% CO 2 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 iii

5 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 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-ethylbenzothiazoline-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. iv

6 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 Lactobacillus reuteri and Lactobacillus fermentum 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% CO 2. 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 v

7 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 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 masingmasing, 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 vi

8 berhasil tinggi dan berekonomikal dalam proses biotransformasi sisa buangan industri pertanian kepada produk bernilai tambah seperti nutraseutikal. vii

9 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. viii

10 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 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: ix

11 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: x

12 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 xi

13 TABLE OF CONTENTS ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF EQUATIONS LIST OF ABBREVIATIONS CHAPTER 1 INTRODUCTION 1 Page iii v viii ix xi xiv xv xviii xix 2 LITERATURE REVIEW Biotransformation Ferulic acid Vanillin Vinyl guaiacol Solid state and submerged fermentation Bioactive compounds Rice bran Bioactive compounds in rice bran Health promoting activity of rice bran Extraction High performance liquid chromatography (HPLC) Free radicals and reactive species Origin and terms Beneficial effects Damaging effects Antioxidants Complex nature of antioxidants Antioxidant and its mechanism Phenolic and polyphenolic compounds Antioxidant activity assay Free radical scavenging activity 31 (a) 2,2-diphenyl-1-picrylhydrazyl (DPPH) 32 (b) 2,2 -azinobis (3-ethyl-benzothiazoline-6-33 sulphonic acid) (ABTS) 2.8 Cholesterol Regulation of cholesterol homeostasis MATERIALS AND METHODS Chemicals Microorganism 40 xii

14 3.2.1 Man, Rogosa and Sharpe (MRS) agar and broth 40 preparation Screening of microorganism Colony count, inoculum preparation and biotransformation Analysis of culture supernatant 43 (a) Identification of 4VG by gas chromatography 43 mass spectrometry (GC-MS) (b) Quantification of 4VG by HPLC Experimental design 44 (a) Initial bacterial concentration 45 (b) Initial substrate concentration Solid state fermentation of rice bran Duration of incubation Culture conditions Extract preparation Thin layer chromatography Antioxidant activity assays , 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity 47 (a) Preparation of solutions 47 (b) Procedure , 2 -azino-bis (3-ethylbenzthiazoline-6-sulfonic) 48 acid radical cation scavenging (ABTS + ) assay (a) Preparation of solutions 48 (b) Procedure Media preparation Culture of HepG2 cells Measurement of cell viability Treatment Gene expression studies RNA extraction cdna synthesis Quantitative real time PCR Statistical analysis 55 4 RESULTS AND DISCUSSION Screening of microorganisms for biotransformation ph O 2 availability and duration of incubation Optimization of biotransformation of ferulic acid by selected lactic acid bacteria and antioxidant activity of biotransformed product Biotransformation process in submerged 59 fermentation Initial ferulic acid concentration 63 xiii

15 4.2.3 Initial bacterial concentration Antioxidant activity assay of byproducts from submerged 71 fermentation , 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity , 2 -azino-bis (3-ethylbenzthiazoline-6-sulfonic) 75 acid radical cation scavenging (ABTS + ) assay 4.4 Solid state fermentation of stabilized rice bran with 77 lactobacillus farciminis Viable cell count of Lactobacillus farciminis 77 cultured in rice bran Extraction of biotransformed product Biotransformation process in solid state 79 fermentation 4.5 Antioxidant activity assay of byproducts from solid state 84 fermentation , 2-diphenyl-1-picrylhydrazyl (DPPH) radical 84 scavenging activity , 2 -azino-bis (3-ethylbenzthiazoline-6-sulfonic) acid radical cation scavenging (ABTS + ) 86 assay 4.6 Regulation of genes involved in cholesterol metabolism by 89 biotransformed product from rice bran in HepG2 cells Cell viability assay Regulation of LDLR gene by SSF RB extract Regulation of HMG-CoAR gene by SSF RB extract Regulation of ApoB 100 gene by SSF RB extract Regulation of Apo A1 gene by SSF RB extract 94 5 CONCLUSION Future work 100 REFERENCES 101 APPENDICES 120 BIODATA OF STUDENT 124 LIST OF PUBLICATIONS AND PROCEEDINGS 125 xiv