UNIVERSITI PUTRA MALAYSIA

AIDA FIRDAUS BT MUHAMMAD NURUL AZMI

FBSB 2012 13

CHARACTERIZATION OF WATER SOLUBLE POLYSACCHARIDES FROM BAMBOO SHOOT (Gigantochloa levis Blanco Merr) AND THEIR

PREBIOTIC POTENTIAL

© COPYRIG

HT UPM

����������������� ����������������������������

��� ���� ������������������� ����������� ������

��������������������������

�

�

�

�

�

�

�

�

�

�

�

�

�

�

�

���

�

����������������� ���� � ����������� ��

�

�

�

�

�

�

�

�

�

�

�

�

� �!"!�!#$% "&&�'�& ���� �����(�)��'#�&���&#'"�!*���"+��!"&"��#&���� ����!"�*�"��

(#�("��% ��&��(�& ����,#"��% ��&!�(���& ��'�-�����(����&����(�� "��!�. ��

�

��.&�% $���/01/�

© COPYRIG

HT UPM

ii

Specially dedicated to Abahmak, my siblings, my hubby & my lovely son, wafi…

© COPYRIG

HT UPM

iii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy

CHARACTERIZATION OF WATER SOLUBLE POLYSACCHARIDES FROM BAMBOO SHOOT (Gigantochloa levis Blanco Merr) AND THEIR PREBIOTIC POTENTIAL

By

AIDA FIRDAUS BT MUHAMMAD NURUL AZMI

September 2012

Chair : Assoc. Prof. Shuhaimi bin Mustafa, PhD Faculty : Faculty of Biotechnology and Biomolecular Sciences

Health benefits of bamboo shoots are often neglected as limited study was carried

out to reveals its medicinal properties. Increasing demands in functional foods like

prebiotics have driven many researches to isolate these functional ingredients from

natural sources. Therefore this research was conducted as to explore the prebiotic

potential of bamboo shoots from Gigantochloa levis. Characterization on its

compositions, molecular weight, solubility, digestibility, intrinsic viscosity,

rheological properties, thermal stability and morphological properties were also

covered. Hot water extraction (90 0C) of water soluble polysaccharides extracted

from the shoots of Gigantochloa levis (which was named as Bamboo Shoots Crude

Polysaccharides, BSCP) was found to be 3.27 ± 0.18 % of dry basis, with molecular

weight of about 7.49 kDa, while other purified sub-fractions (F1 to F5) were

1550.96, 1471.63, 1685.78, 1691.61 and 1551.67 Da respectively.

© COPYRIG

HT UPM

iv

BSCP was composed of glucose, galactose and arabinose as the major sugar

components with the molar ratio of 10 : 5 : 4 (50.51 ± 1.16, 25.11 ± 0.31 and 19.81 ±

0.52 % molar respectively). The percentage of xylose, mannose, rhamnose and

fucose in crude BSCP were however found to be relatively low; 2.05 ± 0.03, 1.77 ±

0.25, 0.62 ± 0.53 and 0.14 ± 0.02 % molar respectively. It also posses good solubility

in water (99.81 ± 0.01 % solubility at 5 % (w/v) of BSCP) and its non-digestibility

towards artificial human gastric juices was proven to be significantly higher (> 99 %)

as to compare with the commercial fructooligosaccharides (FOS) (98 %). This might

be due to the presence of (1→3),(1→4)-β-glucan in BSCP as elucidated through

Fourier Transform Infra Red (FTIR) spectrum as well as Nuclear Magnetic Resonance

(NMR). Fingerprint of BSCP gathered through structural analysis using FTIR showed

that BSCP contains polysaccharide with traces of phenolic and uronic acids

presence.

Intrinsic viscosity of BSCP was 6.89 dl/g, with a rod-like conformation presumed in

the dilute domain through the measurement of power law model. The onset of

molecular entanglements or the critical concentration, C* for BSCP was 0.88 g/dl,

while the relative chain stiffness for BSCP was found to be 0.3086, which was about

ten times greater than pectin and alginate. This value showed a more flexible chain

of BSCP polymer, which was not aligned to the conformation of BSCP presumed

through power law model measurement. This is because rod-like polymer tends to

have a stiff chain. Atomic Force Microscopy (AFM) analysis conducted for

morphological studies of BSCP was however elucidated the conformation of BSCP to

be globular rather than rod-like polymer.

© COPYRIG

HT UPM

v

Rheological test conducted had confirmed the weak gel properties of BSCP. It was

found to be a heat-setting gel, where gel is formed upon heating of a solution.

Thermal analysis using Differential Scanning Calorimetry (DSC) also proves the

weakness of BSCP gelling properties, with a very small enthalpy value obtained

(0.03 to 0.37 mJ).

In vitro fermentation conducted using BSCP as a carbon source gave a positive

indicator as a potential prebiotic. BSCP was found to potently support the growth of

B. animalis, B. longum and L. acidophilus. On top of that, BSCP was able to suppress

the growth of Salmonella sp. pH of Trypticase Peptone Yeast (TPY) medium

supplemented with BSCP and FOS were seen to decrease as an indication of

bacterial growth. Lactic acid and SCFA produced by the probiotic strains

supplemented with BSCP were also found to be higher as compared to FOS, even

though there was no significant trend noted. Results obtained throughout this

research showed that BSCP meets most of prebiotic characteristics, which is highly

resistance to the artificial gastric juice, able to be fermented by the intestinal

microbiota like bifidobacterium and lactobacilli while suppressing the growth of

pathogens as well as producing SCFA which later could enhance the host health

indirectly.

© COPYRIG

HT UPM

vi

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Doktor Falsafah

PENCIRIAN POLISAKARIDA LARUT AIR DARIPADA REBUNG BULUH (Gigantochloa levis Blanco Merr) DAN POTENSI PREBIOTIKNYA

Oleh

AIDA FIRDAUS BT MUHAMMAD NURUL AZMI

September 2012

Pengerusi : Prof. Madya Shuhaimi bin Mustafa, PhD Fakulti : Fakulti Bioteknologi dan Sains Biomolekul

Khasiat kesihatan rebung buluh sering dipinggirkan ekoran kekurangan penyelidikan

yang dijalankan untuk membongkarkan khasiat perubatannya. Permintaan tinggi

terhadap makanan berfungsi seperti prebiotik telah mendorong ramai penyelidik

untuk mengekstrak makanan berfungsi ini daripada sumber alam. Oleh sebab itu,

penyelidikan ini telah dijalankan untuk mengkaji potensi prebiotik daripada rebung

buluh yang boleh dimakan iaitu Gigantochloa levis. Pengekstrakkan polisakarida

larut air daripada rebung Gigantochloa levis (yang dipanggil Polisakarida Kasar

Rebung Buluh, BSCP) dengan menggunakan air panas (90 0C) memberikan sebanyak

3.27 ± 0.18 % hasil dengan berat molekular sebanyak 7.49 kDa, sementara sub-

fraksi yang telah ditulenkan (F1 hingga F5) mempunyai berat molekular sebanyak

1550.96, 1471.63, 1685.78, 1691.61 and 1551.67 Da masing-masing.

© COPYRIG

HT UPM

vii

BSCP mengandungi glukosa, galaktosa dan arabinosa sebagai gula utama dengan

nisbah molar sebanyak 10 : 5 : 4 (50.51 ± 1.16, 25.11 ± 0.31 dan 19.81 ± 0.52 %

molar masing-masing). Manakala peratusan bagi xylosa, mannosa, rhamnosa dan

fukosa di dalam BSCP didapati agak rendah iaitu sebanyak; 2.05 ± 0.03, 1.77 ± 0.25,

0.62 ± 0.53 dan 0.14 ± 0.02 % molar masing-masing. Ia mempunyai kadar

keterlarutan yang tinggi di dalam air (99.81 ± 0.01 % keterlarutan pada 5 % (w/v)

BSCP) serta menunjukkan kadar ketidakhadaham yang tinggi terhadap jus gastrik

tiruan (> 99%) berbanding fruktooligosakarida (FOS) (98%). Ini mungkin disebabkan

oleh kehadiran (1→3),(1→4)-β-glucan yang ditemui melalui spektra Fourier

Fransform Infra Red (FTIR) dan juga Nuclear Magnetic Resonance (NMR). Spektra

FTIR juga menunjukkan BSCP mengandungi polisakarida beserta sedikit kandungan

phenolik dan asid uronik.

Kelikatan intrinsik bagi BSCP ialah 6.89 dl/g, dengan konformasi yang diandaikan

berbentuk rod pada tahap kelikatan paling cair menerusi pengukuran “power law

model”. Permulaan simpulan molekul atau dikenali sebagai konsentrasi kritikal, C*

bagi BSCP adalah 0.88 g/dl, dengan kekuatan rantaian relatif sebanyak 0.3086, iaitu

sepuluh kali ganda berbanding pektin dan alginate. Nilai ini menunjukkan rantaian

BSCP yang lebih fleksibel, di mana ianya tidak selari dengan konformasi BSCP yang

diandaikan menerusi pengukuran “power law model”. Ini kerana molekul berbentuk

rod cenderung untuk bersifat lebih kaku. Analisa menggunakan Atomic Force

Microscopy (AFM) untuk mengkaji keadaan morfologi BSCP walau bagaimanapun

menunjukkan konformasi BSCP berbentuk globul, berbanding bentuk polimer

berbentuk rod.

© COPYRIG

HT UPM

viii

Kajian rheologi menunjukkan bahawa BSCP mempunyai kebolehan membentuk gel

yang lemah. Ia merupakan perekat gel panas, di mana gel terbentuk apabila larutan

dipanaskan. Analisa terma menggunakan Differential Scanning Calorimetry (DSC)

juga membuktikan kelemahan ciri gel BSCP, dengan nilai entalpi yang terlalu kecil

(0.03 hingga 0.37 mJ).

Fermentasi secara in vitro menggunakan BSCP sebagai sumber karbon menunjukkan

potensi yang positif sebagai prebiotik. BSCP didapati mampu untuk menyokong

pertumbuhan B. animalis, B. longum dan L. acidophilus. Disamping itu juga, BSCP

mampu untuk membendung pertumbuhan Salmonella sp . pH bagi media Trypticase

Peptone Yeast (TPY) yang mengandungi BSCP didapati menurun sebagai tanda

pertumbuhan bakteria. Laktik asid dan asid lemak rantaian pendek (SCFA) yang

dihasilkan oleh bakteria yang diberi medium mengandungi BSCP menunjukkan

kuantiti yang tinggi berbanding FOS, walaupun tiada corak yang siknifikan

diperhatikan.

© COPYRIG

HT UPM

ix

ACKNOWLEDGEMENT

In the name of Allah, The Most Gracious, The Most Merciful…

After years of struggling and hardworking, I couldn’t say more than

Alhamdulillah. Praises to Allah SWT for His blessing, love and endurance He gave me

all the way through my PhD journey. I would also like to express greatest gratitude

to all my supervisory committee; Assoc. Prof. Dr. Shuhaimi Bin Mustafa, Prof. Dr.

Mohd. Yazid bin Abd. Manap, Mr. Dzulkifly bin Md. Hashim as well as Prof. Dr. Amin

bin Ismail for their kind help, assistance and guidance throughout my entire study.

May Allah bless you!

My appreciation also goes to all kind hearted people surrounds me, who has

helped me a lot during my candidature. They are: i) researchers from Institute of

Food Research Institute (IFR), Norwich, United Kingdom; Prof. V.J. Morris, Andrew

Kirby, Patrick Gunning, Dr. Nicholas Wellner, Dr. Ian Colquhoun, Mr. Samuel Collin,

Dr. Mary Parker as well as Dr. Christina, ii) Dr. Anis Shobirin Meor Hussein and her

family, Noorzela, Hakim, Noorarmylisas, Norlaili Che Rose as well as Maslina

Mohamad and her family for their kindness during my stay in UK, iii) UPM Science

Officers; Mr. Halim, Mr. Azman, Mr. Amran, Ms. Norliza, Ms. Mafuzah, Ms. Ros,

Mr. Hamizan and Mr. Faizal, iv) Cikgu Manas and his wife, Pn. Zaharaton as well as

his family members for being very supportive and helpful. His love and passion

towards bamboo industry has really inspires me! v) my lovely and supportive

labmates; Noorzaliza, Khalilah, Yamin, Sadeq, Mojdeh, Salma, Dr. Mehrnoush,

© COPYRIG

HT UPM

x

Kabeir, Aisyah, Rafidah, Nurul Aqilah, Noorhidayah, Afinah, Hajar, Lian, Suresh,

Safuan, Qadyr, Leila, Bita and to name a few and not forgotten vi) my best buddies;

Nurulaida, Rohaieza, Shazlin, Hanim, Norazrina and Siti Khairani. All of you are the

greatest gift from Allah! Thank you for being there when Im in need. May Allah

bless your helping hands, motivational thoughts and spirit…

To the most and VERY IMPORTANT PERSON in my life; Muhammad Nurul

Azmi bin Ahmad Mukhtar and Nur’Aini bt Yahya, YOU ARE MY EVERYTHING. You

brought me to this world with your true love and raised me up with your endless

love. I LOVE YOU more than you ever thought of. Thank you for everything – your

love, your passion, your advice, your thought and your guide for me to live this life. I

may not be able to redeem your kindness, but I may promise you A THING – that I

will always do my best and make you proud of me here and thereafter! InsyaAllah...

May Allah grant His Jannah to both of you.

The same goes to my beloved brothers and sisters; Adib Fadli and his wife

and son, Athirah Najwa, Aiman Najib and Aina Jihan for being so supportive and

helpful. May this become your future source of inspiration to soar higher. Lastly, I

would like to thank to my beloved husband, Mohd. Khairil Hasni bin Mohd. Hayat

for his understanding, love, courage and support since the first time we met. We

have made it this far and may Allah give us His blessing to be together till Jannah.

Last but not least, to my little angle, Muhammad Wafi Darwisy bin Mohd. Khairil

Hasni – mama loves you so much! Thanks for being such a great motivator for

mama to win this battle to the end… Alhamdulillah!

© COPYRIG

HT UPM

xi

I certify that an Examination Committee has met on 03 SEPTEMBER 2012 to conduct the final examination of AIDA FIRDAUS BT MUHAMMAD NURUL AZMI on her DOCTOR OF PHILOSOPHY thesis entitled "Characterization of Water Soluble Polysaccharides from Edible Bamboo Shoot (Gigantochloa levis) and Its Prebiotic Potential” in accordance with Universiti Putra Malaysia (Higher Degree) Act 1980 and Universiti Putra Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the student be awarded the Doctor of Philosophy. Members of the Examination Committee were as follows: Assoc. Prof. Dr. Shuhaimi bin Mustafa, PhD Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Dr. Syahida Ahmad, PhD Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Assoc. Prof. Dr. Janna Ong Abdullah, PhD Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Dr. Marilena Marino, PhD Dipartimento di Scienze degli Alimenti, Università degli Studi di Udine, Udine, Italy (External Examiner) ________________________ SEOW HENG FONG, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

© COPYRIG

HT UPM

xii

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows: Shuhaimi bin Mustafa, PhD Associate Professor Faculty of Biotecnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Mohd. Yazid bin Abd. Manap, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Member) Amin bin Ismail, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member)

_________________________

BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date:

© COPYRIG

HT UPM

xiii

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 concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution.

______________________________________

AIDA FIRDAUS BT MUHAMMAD NURUL AZMI

Date:______________

© COPYRIG

HT UPM

xiv

TABLE OF CONTENTS

DEDICATION ii

ABSTRACT iii

ABSTRAK vi

ACKNOWLEDGEMENTS ix

APPROVAL xi

DECLARATION xiii

LIST OF TABLES xviii

LIST OF FIGURES xx

LIST OF ABBREVIATIONS xxii

CHAPTER

1 INTRODUCTION 01

2 LITERATURE REVIEW 2.1 Introduction 06 2.2 Nutritional Composition of Bamboo Shoots 11 2.3 Food Carbohydrate 14 2.4 Current trends on Functional Foods 15 2.5 A Concept of Prebiotic 17 2.6 Characteristics of a Prebiotic 20 2.7 Benefits of Prebiotics 23 2.8 Isolation, Purification and Identification of Polysaccharides 25 2.9 Chain Conformational Analysis of Polysaccharides in Solution 28 2.10 Rheological Measurement of Polysaccharides 30 2.11 Thermal Analysis (TA) 31 2.12 Fourier Transform Infra-Red (FTIR) 32 2.13 Atomic Force Microscopy (AFM) 33

© COPYRIG

HT UPM

xv

3 PROXIMATE COMPOSITION OF BAMBOO SHOOTS 3.1 Introduction 35 3.2 Materials and Methods 37

3.2.1 Bamboo Shoots 37 3.2.2 Proximate Analysis of Bamboo Shoots 38

3.2.2.1 Moisture Content 39 3.2.2.2 Crude Protein Content 39 3.2.2.3 Crude Fat Content 39 3.2.2.4 Ash Content 40 3.2.2.5 Carbohydrate Content 40

3.2.3 Statistical Analysis 40

3.3 Results and Discussions 41 3.3.1 Moisture Contents 41 3.3.2 Protein Content 42 3.3.3 Crude Fat Content 43 3.3.4 Ash Content 43 3.3.5 Carbohydrate Content 45

3.4 Conclusions 46 4 ISOLATION, PURIFICATION AND IDENTIFICATION OF WATER

SOLUBLE POLYSACCHARIDES FROM BAMBOO SHOOT (Gigantochloa levis)

4.1 Introduction 47 4.2 Materials and Methods 49

4.2.1 Isolation of Crude Polysaccharides 49 4.2.1.1 Preparation of Dialysis Tubing 50

4.2.2 Purification of Crude Polysaccharides 51 4.2.2.1 Phenol-sulphuric Acid Method 52

4.2.3 Sugar Compositions Determination 53 4.2.4 Molecular Weight Estimation of Crude Polysaccharides 55 4.2.5 Solubility of Crude Polysaccharides 55 4.2.6 Digestibility of Crude Polysaccharides 56

4.2.6.1 DNS Method 57 4.2.7 Identification of Crude Polysaccharides 58

4.2.7.1 Fourier Transform Infra Red (FTIR) 58 4.2.8 Statistical Analysis 59

© COPYRIG

HT UPM

xvi

4.3 Results and Discussion 59 4.3.1 Isolation of Crude Polysaccharides 59 4.3.2 Purification and Molecular Weight of Crude 61 Polysaccharides and Its Fractions 4.3.3 Sugar Compositions 65 4.3.4 Solubility of Crude Polysaccharides 66 4.3.5 Digestibility of Crude Polysaccharides 67 4.3.6 Identification of Crude Polysaccharides 71

4.3.6.1 Fourier Transform Infra Red (FTIR) 71

4.4 Conclusions 76

5 STRUCTURE-FUNCTION RELATIONSHIP OF BAMBOO SHOOT CRUDE POLYSACCHARIDES (BSCP)

5.1 Introduction 78 5.2 Materials and Methods 81

5.2.1 Chain Conformational Analysis of Polysaccharides in Solution 81 5.2.1.1 Intrinsic Viscosity 81 5.2.1.2 Determination of the Molecular Conformation and 86 Polymer Interaction 5.2.1.3 Salt Tolerance (S) and Relative Stiffness Parameter (B) 86

5.2.2 Rheological Measurement of Polysaccharides 88 5.2.3 Differential Scanning Calorimetry (DSC) 89 5.2.4 Atomic Force Microscopy (AFM) 89 5.2.5 Statistical Analysis 90

5.3 Results and Discussions 90 5.3.1 Intrinsic Viscosity 90 5.3.2 Determination of the Molecular Conformation and Polymer 97 Interaction 5.3.3 Salt Tolerance (S) and Relative Chain Stiffness Parameter (B) 100 5.3.4 Rheological Measurement of Polysaccharides 104

5.3.4.1 Dynamic Strain Sweep 104 5.3.4.2 Viscoelastic properties 105 5.3.4.3 Steady Shear Viscosity 110 5.3.4.5 Temperature Dependence 113

5.3.5 Differential Scanning Calorimetry (DSC) 118 5.3.6 Atomic Force Microscopy (AFM) 121

5.4 Conclusions 126

© COPYRIG

HT UPM

xvii

6 PREBIOTIC POTENTIAL OF BAMBOO SHOOT CRUDE POLYSACCHARIDES (BSCP) ON SELECTED GUT MICROBIOTA STRAINS

6.1 Introduction 129

6.2 Materials and Methods 132

6.2.1 Maintenance of Bacterial Strains 132 6.2.2 Bacterial Identification and Confirmation 132 6.2.3 Medium and Fermentation Condition 134 6.2.4 Determination of Optical Density (OD) 135 6.2.5 Enumeration of Viable Cell 135 6.2.6 Determination of pH 136 6.2.7 Determination of Lactic Acid and Short Chain Fatty Acid (SCFA) 136 6.2.8 Statistical Analysis 137

6.3 Results and Discussions 137 6.3.1 Optical Density (OD) and Total Viable Count (TVC) 137 6.3.2 pH and Short Chain Fatty Acids Production (SCFA) 148

6.4 Conclusions 157 7 GENERAL CONCLUSIONS AND RECOMMENDATIONS 159 8 REFERENCES 164

9 APPENDICES 181

10 BIODATA OF STUDENT 237

11 LIST OF PUBLICATIONS 238