optimization of medium formulation for folate … fileoptimized medium composition was then further...

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


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


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


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


(Internal Examiner)

Umi Kalsom binti Md Shah, PhD

Associate Professor

Department of Bioprocess Technology

Fakulti of Biotechnology and Biomolecular Sciences


(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


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


(Chairman)

Raha Abdul Rahim, PhD Professor



(Member)

Foo Hooi Ling, PhD

Associate Professor



(Member)

___ ______________________

HASANAH MOHD GHAZALI, PhD

Professor and Dean

School of Graduate Studies


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.

_______________________________


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



5.2.2 Medium 62

5.2.3 Batch Cultivation 63

5.2.4 Assays 63


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



6.2.2 Medium 84

6.2.3 Experimental Design for RSM 84

6.2.4 2 L Stirred Tank Bioreactor 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

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