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UNIVERSITI PUTRA MALAYSIA
OPTIMIZATION OF MEDIUM FORMULATION FOR FOLATE BIOSYNTHESIS BY Lactobacillus plantarum I-UL4 USING RESPONSE
SURFACE METHODOLOGY
NORFARINA BT MUHAMAD NOR
FBSB 2011 33
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OPTIMIZATION OF MEDIUM FORMULATION FOR FOLATE
BIOSYNTHESIS BY Lactobacillus plantarum I-UL4 USING RESPONSE
SURFACE METHODOLOGY
By
NORFARINA BT MUHAMAD NOR
Thesis submitted to the School of Graduate Studies, Universiti Putra Malaysia, in
Fulfillment of the Requirements for the Degree of Master Science
May 2011
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in the fulfillment
of the requirement of the Degree of Master Science
OPTIMIZATION OF MEDIUM FORMULATION FOR FOLATE
BIOSYNTHESIS BY Lactobacillus plantarum I-UL4 USING RESPONSE
SURFACE METHODOLOGY
By
NORFARINA BT MUHAMAD NOR
May 2011
Chairman : Assoc. Prof. Rosfarizan Mohamad, PhD
Faculty : Biotechnology and Biomolecular Sciences
Folate plays an important role in human life as one of the most important components
for the synthesis of purine, guanine, adenine, pyrimidine and thymine. In this study,
lactic acid bacteria (Lactococcus lactis NZ 9000 and MG 1363, Lactobacillus plantarum
I-UL4, and Lactobacillus johnsonii DSM 20553) were investigated for the biosynthesis
of folate. The total folate concentrations in lactic acid bacteria were determined by
microbiological assay as a reliable method to evaluate the folate level and Lactobacillus
rhamnosus (Lactobacillus casei, ATCC 7469) was used to analyze total folate and
responds to most native folates. From this study, all lactic acid bacteria able to
synthesized folate and Lb. plantarum I-UL4 exhibited as the best folate biosynthesis and
used throughout investigation on the medium optimization.
Five components of medium composition (carbon source, lactose, nitrogen source, meat
extract and p-aminobenzoic acid (PABA)) were optimized conventionally and the
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cultivation process was conducted in shake-flask experiment. From the investigation,
lactose, meat extract and PABA at concentration 20 g/L, 15 g/L and 10 µM respectively
gave significant effect towards growth of Lb. plantarum I-UL4 and folate biosynthesis.
Thus, these three concentrations were subsequently used for further medium
optimization study based on response surface methodology (RSM).
Through RSM approach, twenty experiments of three factors in response to folate
biosynthesis were carried out. Estimated optimize conditions of the factors for the
growth of Lb. plantarum I-UL4 and folate biosynthesis as suggested by RSM are as
follows: lactose, 20 g/L; meat extract, 16.57 g/L and PABA concentration, 10 µM. The
optimized medium composition was then further applied in the cultivation process using
2 L stirred tank bioreactor. Finally, about 61.89 µg/L of folate was synthesized by Lb.
plantarum I-UL4 using the bioreactor, which was slightly higher than in shake-flask
experiment (59.88 µg/L) using the optimized medium composition. In a conclusion, a
better understanding of the relationship among the factors was obtained by RSM, which
was used as a statistical tool to improve the folate biosynthesis of Lb. plantarum I-UL4
in small and large scale cultivation.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Master Sains
PENGOPTIMUMAN FORMULASI MEDIA UNTUK BIOSINTESIS FOLAT
OLEH Lactobacillus plantarum I-UL4 MENGGUNAKAN KAEDAH
RANGSANGAN PERMUKAAN
Oleh
NORFARINA BT MUHAMAD NOR
Mei 2011
Pengerusi : Profesor Madya Rosfarizan Mohamad, PhD
Fakulti : Bioteknologi dan Sains Biomolekul
Folat memainkan peranan yang penting dalam kehidupan manusia dimana ianya
digunakan sebagai satu komponen untuk sintesis purin, guanin, adenin, piarimidin dan
tiamin. Dalam kajian ini, bakteria asid laktik (Lactococcus lactis NZ 9000 and MG
1363, Lactobacillus plantarum I-UL4, dan Lactobacillus johnsonii DSM 20553) telah di
uji untuk biosintesis folat. Jumlah kepekatan folat dalam bakteria asid laktik ditentukan
dengan kaedah mikrobiologi iaitu kaedah yang tepat untuk menilai tahap folat dan
Lactobacillus rhamnosus (Lactobacillus casei, ATCC 7469) digunakan untuk
menganalisis jumlah folat dan semua folat natif. Dalam kajian ini, semua bakteria asid
laktik boleh mensintesiskan folat dan Lb. plantarum I-UL4 telah menunjukkan
keupayaan untuk biosintesis folat yang terbaik dan digunakan dalam seluruh kajian
pengoptimuman medium.
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Lima komponen komposisi media (sumber karbon, laktosa, sumber nitrogen, ekstrak
daging dan asid p-aminobenzoik) telah di optimumkan secara konvensional dan proses
pertumbuhan ini dijalankan di dalam kelalang kon. Daripada kajian, laktosa, ekstrak
daging dan PABA pada kepekatan 20 g/L, 15 g/L dan 10 µM masing-masing
memberikan kesan pada pertumbuhan Lb. plantarum I-UL4 dan folat biosintesisnya.
Demikian juga tiga kepekatan ini digunakan untuk kajian seterusnya dalam
pengoptimuman medium berasaskan kaedah rangsangan permukaan (RSM).
Melalui kaedah rangsangan permukaan, dua puluh eksperimen untuk tiga faktor telah
dijalankan. Anggaran kondisi faktor yang optimum untuk pertumbuhan Lb. plantarum I-
UL4 dan penghasilan folat seperti yang disyorkan oleh RSM adalah seperti berikut:
laktosa, 20 g/L; ekstrak daging, 16.57 g/L dan kepekatan PABA, 10 µM masing-masing.
Medium yang optimum pada proses pertumbuhan seterusnya telah dikaji menggunakan
bioreaktor 2 L yang berpengaduk. Akhir sekali, biosintesis folat oleh Lb. plantarum I-
UL4 menghasilkan folat sebanyak 61.89 µg/L. Dengan menggunakan medium yang
optimum yang sama seperti di dalam bioreaktor, folat di dalam kelalang kon dihasilkan
sebanyak 59.88 µg/L. Sebagai kesimpulan, kefahaman yang mendalam mengenai
hubungan antara faktor-faktor tersebut telah diperolehi dari RSM, dimana ianya
digunakan sebagai alat statistik untuk memperbaiki biosintesis folat oleh Lb. plantarum
I-UL4 dalam skala yang kecil mahupun yang besar.
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ACKNOWLEDGEMENT
All praise to ALLAH S.W.T who has guide my safety, give me a great health that I
could not adequately thank for. The strength given by ALLAH has provided me to finish
my study. Alhamdulillah.
I would like to express a great gratitude to my supervisor, Assoc. Prof. Dr. Rosfarizan
Mohamad, who have always had enormous patience and helped me in every way
possible. Thanks for the great help. My gratitude also goes to my co-supervisors, Prof
Dr. Raha Abdul Rahim and Assoc. Prof. Dr. Foo Hooi Ling for their invaluable
guidance, constant encouragement and constructive suggestions throughout this study.
Thanks also extended to my amazing friends at the Department Bioprocess Technology,
who are directly or indirectly supporting and helping.
I could not finish these lines without extending special thanks to my husband, Khairil
Amri b. Anuwar for his continuous support and encouragement during the realization of
this work. He really deserves all the love and trust that I have placed in him. To my baby
Nurfaliha Qaisara, thanks for your smile. Your smiles always brighten my day life.
Finally, I would like to mention the invaluable help, enormous support, endless loves,
cares and patience to my family members. My higher gratitude also expressed to my
beloved father, Mr. Muhamad Nor bin Sahar and my beloved mother, Madam Amanah
bt Sajak for really understand with my study. All prayers from both of you have brought
success to my life. Thank you for all.
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I certify that an Examination Committee has met on 24/05/2011 to conduct the final
examination of Norfarina bt Muhamad Nor on her thesis entitled “Optimization Of
Medium Formulation for Folate Biosynthesis by Lactobacillus Plantarum I-UL4 Using
Response Surface Methodology” in accordance with Universities and University
Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106]
15 March 1998. The Committee recommends that the student be awarded the Master of
Science Industrial Biotechnology.
Members of the Examination Committee were as follows:
Nor’Aini binti Abdul Rahman, PhD
Department of Biochemistry
Fakulti of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Shuhaimi bin Mustafa, PhD
Associate Professor
Institute of Halal Products Research
Universiti Putra Malaysia
(Internal Examiner)
Umi Kalsom binti Md Shah, PhD
Associate Professor
Department of Bioprocess Technology
Fakulti of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Jamaliah binti Md. Jahim, PhD
Associate Professor
Department of Process Engineering and Chemistry
Universiti Kebangsaan Malaysia
(External Examiner)
_______________________________
SHAMSUDDIN SULAIMAN, PhD
Professor and 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 and has been
accepted as fulfillment of the requirement for the degree of Master of Science. The
members of the Supervisory Committee were as follows
Rosfarizan Mohamad, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Raha Abdul Rahim, PhD Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
Foo Hooi Ling, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
___ ______________________
HASANAH MOHD GHAZALI, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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DECLARATION
I hereby declare that the thesis is based on my original work except for quotations and
citation, which have been duly acknowledged. I also declare that it has not been
previously or concurrently submitted for any other at Universiti Putra Malaysia or other
institutions.
_______________________________
NORFARINA BT MUHAMAD NOR
Date: 24th
May 2011
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TABLE OF CONTENTS
Page
ABSTRACT ii
ABSTRAK iv
ACKNOWLEDGEMENTS vi
APPROVAL SHEETS viii
DECLARATION FORM ix
LIST OF TABLES xiii
LIST OF FIGURES xv
LIST OF ABBREVIATIONS xvii
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW
2.1 Microorganism 5
2.1.1 Lactic Acid Bacteria 5
2.1.2 Lactobacillus plantarum 6
2.2 Properties of Folate 8
2.3 Folate Biochemistry 10
2.3.1 Chemical Structure 10
2.3.2 Folate Biosynthesis 13
2.4 Sources and Application of Folate 15
2.4.1 Sources of Folate 15
2.4.2 Application of Folate 19
2.5 Measurements of Folate Level 21
2.6 Cultivation Techniques 22
2.6.1 Batch Culture 22
2.7 Medium Composition 26
2.7.1 Carbon Sources 28
2.7.2 Nitrogen Sources 29
2.7.3 Precursor 30
2.8 Response Surface Methodology 32
2.9 Concluding Remarks 35
3 GENERAL MATERIALS AND METHODS
3.1 Bacterial Strains 37
3.2 Inoculum Preparation 38
3.3 Medium 38
3.4 Batch Cultivation 40
3.4.1 Shake-flask 40
3.4.2 Stirred Tank Bioreactor 41
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3.5 General Experimental Plan 44
3.6 Analytical Methods 46
3.6.1 Determination of Folate Concentration 46
3.6.2 Cell and substrate Concentration 47
4 SELECTION OF THE BEST FOLATE PRODUCER FROM LACTIC
ACID BACTERIA
4.1 Introduction 49
4.2 Materials and Methods 50
4.2.1 Inoculum Preparation 50
4.2.2 Medium 50
4.2.3 Assays 51
4.3 Results and Discussion 51
4.3.1 Folate Biosynthesis by LAB 53
4.3.2 Comparison of folate Biosynthesis by LAB 56
4.4 Conclusion 60
5 EFFECT OF MEDIUM FORMULATION ON FOLATE
BIOSYNTHESIS BY Lactobacillus plantarum I-UL4
5.1 Introduction 61
5.2 Materials and Methods 62
5.2.1 Inoculum Preparation 62
5.2.2 Medium 62
5.2.3 Batch Cultivation 63
5.2.4 Assays 63
5.3 Results and Discussion 64
5.3.1 Carbon Source 64
5.3.2 Lactose 67
5.3.3 Nitrogen Source 71
5.3.4 Meat Extract 74
5.3.5 p-Aminobenzoic Acid 78
5.4 Conclusion 82
6 IMPROVEMENT OF FOLATE BIOSYNTHESIS BY Lactobacillus
plantarum I-UL4 USING RESPONSE SURFACE METHODOLOGY
6.1 Introduction 83
6.2 Materials and Methods 84
6.2.1 Inoculum Preparation 84
6.2.2 Medium 84
6.2.3 Experimental Design for RSM 84
6.2.4 2 L Stirred Tank Bioreactor 86
6.3 Results and Discussion 86
6.3.1 Central Composite Design 86
6.3.2 2 L Stirred Tank Bioreactor 96
6.4 Conclusion 99
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7 GENERAL DISCUSSION, CONCLUSIONS AND SUGGESTIONS
FOR FURTHER WORK 100
REFERENCES 105
APPENDICES 116
BIODATA OF THE STUDENT 121
LIST OF PUBLICATION 122