universiti putra malaysia khalilah abdul khalil fbsb

UNIVERSITI PUTRA MALAYSIA

KHALILAH ABDUL KHALIL

FBSB 2012 10

MICROENCAPSULATION of Bifidobacterium pseudocatenulatum G4 USING NATURAL MATRICES

© COPYRIG

HT UPM

MICROENCAPSULATION of Bifidobacterium pseudocatenulatum G4 USING

NATURAL MATRICES


DOCTOR OF PHILOSOPHY

UNIVERSITI PUTRA MALAYSIA

2012

© COPYRIG

HT UPM

MICROENCAPSULATION of Bifidobacterium pseudocatenulatum G4 USING

NATURAL MATRICES

By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfillment of the Requirements for the Degree of Doctor of Philosophy

July 2012

© COPYRIG

HT UPM

i

Dedicated to…..

My beloved mother, Rahmah, my dearest husband, Awis Qurni, and my adorable

children, Shahira, Ammar, Ammir and Amsyar. As well goes to teachers,

researchers, scientists and peoples who contribute the knowledge in this field.

© COPYRIG

HT UPM

ii

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

fulfillment of the requirement for the degree of Doctor of Philosophy

MICROENCAPSULATION of Bifidobacterium pseudocatenulatum G4

USING NATURAL MATRICES

By


July 2012

Chairman: Associate Professor Shuhaimi Mustafa, PhD

Faculty : Biotechnology and Biomolecular Sciences

Probiotic cultures, nowadays, are widely used in food products for health

enhancement. Low survivability of probiotic cultures in acidic environment such

as in the stomach region has limited their potential benefits. The possibility of

using encapsulation method to improve the survivability of probiotic bacterium,

Bifidobacterium pseudocatenulatum G4 (G4), during passage through the

gastrointestinal tract was investigated in this study. Bovine and fish gelatin with

the combination of genipin (a plant extract) and sodium alginate were used as

encapsulating matrices. The study was accomplished through the following

approaches: 1) formulation of medium based on skim milk and yeast extract for

development of active inoculum for G4 cultivation; 2) optimization of inoculum

medium prior to subsequent fermentation, 3) optimization of the encapsulation

matrices for improvement of encapsulation yield (%) and beads strength (g)

before and after being exposed to simulated gastric (SGF) and intestinal fluids

(SIF), and 4) determination of cell release activities based on swelling rate (%),

© COPYRIG

HT UPM

iii

release activity (OD), survival assay (cfumL-1

) and beads morphology using scan

electron microscope (SEM) during exposures to SGF and SIF.

The use of 2 and 4% (w/v) skim milk as inoculum medium has elevated only 1

log cfumL-1

after 24 h of cultivation. Skim milk concentration ranging from 6 to

10% (w/v) greatly enhanced cell growth with more efficient carbon and free

amino nitrogen usage as well as higher production of -galactosidase. Through

statistical modeling based on the Face Centered Central Composite Design

(FCCD), the optimum concentration of combined skim milk and yeast extract was

determined as 7.02 and 1.73% (w/v), respectively. A validation experiment

proved that the predicted and experimented values were not significantly different

(p > 0.05). Substantial improvement in biomass production (11.72 cfumL-1

) was

achieved in cultivation with optimized medium in 2-L stirred tank bioreactor for

18 h, and this biomass production was not statistically different (p > 0.05) as

compared to the cultivation using commercial inoculum medium.

FCCD was also employed for the optimization of encapsulating matrices. The

optimum concentration for bovine gelatin-genipin-alginate was predicted at

11.21% (w/v), 13.96 mM and 2.60% (w/v), respectively. While, in the case of fish

gelatin-genipin-alginate, combined matrices at 12.57% (w/v), 19.12 mM and 5%

(w/v) was predicted to generate optimum responses. Upon verification,

experimental data of bovine gelatin-genipin-alginate and fish gelatin-genipin-

alginate remained close value to the predicted data with low error for all the

responses. As compared to porcine-genipin-alginate encapsulating matrices, the

optimized bovine and fish gelatin-genipin-alginate have both demonstrated lower

strength (p < 0.05) after SIF exposure.

© COPYRIG

HT UPM

iv

The performances of the optimized bovine gelatin-genipin-alginate and fish

gelatin-genipin-alginate in protecting G4 and other probiotics were also

determined. Eight groups of encapsulating matrices were evaluated: 1) optimized

bovine gelatin-genipin-alginate, 2) optimized fish gelatin-genipin-alginate, 3)

porcine gelatin-genipin-alginate, 4) optimized bovine gelatin-alginate, 5)

optimized fish gelatin-alginate, 6) porcine gelatin-alginate, 7) alginate alone, 8)

free cell (unencapsulate). Low encapsulation yield was observed in groups 2 and

5, respectively. Meanwhile group 1 showed highest in encapsulation yield. Slow

swelling rate during the SGF exposure was shown by group 1, 3, 4 and 6 while

groups 5 was demonstrated progressive swelling and slightly erode at 120 min of

exposure. The releases of cells occur when the beads disintegrate and these were

observed through the cells release activity analysis. All groups were presented

positive performance in releasing cells into the intestinal region with higher

optical density and lower survivability of entrapped cells obtained under SIF

exposure except for groups 3 and 6, respectively. Factors like gelatin source,

bloom strength and the presence of alginate played important roles in stabilizing

the chemical cross link of the gelatin especially in acidic environment.

Encapsulations of both bovine and fish gelatin with genipin and alginate

combinations have successfully improved the survival and cells release and this

approach could potentially be useful in replacing porcine gelatin for the delivery

of probiotic culture to the target area in the intestinal region.

© COPYRIG

HT UPM

v

Abstrak tesis yang dikemukakan Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

MIKROENKAPSULASI Bifidobacterium pseudocatenulatum G4

MENGGUNAKAN MATRIKS ASLI

Oleh


Julai 2012

Pengerusi: Profesor Madya Shuhaimi Mustafa, PhD

Fakulti : Bioteknologi and Sains Biomolekul

Pada masa sekarang, kultur probiotik digunakan secara meluas di dalam produk

makanan untuk tujuan kesihatan. Akan tetapi, tahap kehidupan probiotik kultur

ini adalah rendah di dalam persekitaran berasid seperti di dalam perut. Oleh itu,

kaedah enkapsulasi dikaji untuk melihat potensi bagi memperbaiki tahap

kehidupan probiotik, Bifidobacterium pseudocatenulatum G4, semasa melalui

sistem pencernaan. Kombinasi bahan enkapsulasi seperti gelatin lembu dan ikan

bersama genipin (ekstrak tumbuhan) dan sodium alginat telah digunakan. Kajian

in melibatkan beberapa peringkat seperti: 1) meningkatkan pengaktifan inokula

G4 dengan mencari formulasi susu skim and ekstrak yis yang sesuai sebagai

persediaan media untuk inokula, 2) mengoptimasikan media tersebut bagi

meningkatkan populasi G4 sebelum proses penapaian yang selanjutnya, 3)

mengoptimasikan kombinasi bahan enkapsulasi yang digunakan berdasarkan

kadar enkapsulasi sel (%) dan kekuatan kapsul yang dihasilkan (g) sebelum dan

selepas pendedahan kepada bendalir gastrik (SGF) dan pencernaan (SIF), 4)

mengkaji aktiviti pelepasan sel probiotik ketika di dalam SGF dan SIF

berdasarkan kadar pembengkakan kapsul (%), analisa pelepasan sel (OD), kadar

© COPYRIG

HT UPM

vi

tahap kehidupan sel di dalam kapsul (cfumL-1

) dan perubahan bentuk kapsul

dengan menggunakan mikroskop imbasan elektron (SEM).

Penggunaan 2 dan 4% (w/v) susu skim sebagai media pengaktifan inokula hanya

memberi peningkatan sel sebanyak 1 log10cfumL-1

selepas 24 jam kultivasi.

Sebaliknya, kepekatan susu skim dari 6 hingga 10% (w/v), memberi penambahan

bilangan sel yang besar dengan penggunaan karbon dan amino nitrogen yang

cekap serta penghasilan -galaktosidase yang tinggi. Model statistik berdasarkan

“Face Centered Central Composite Design” (FCCD) memberi titik optima

kepekatan bagi susu skim dan ekstrak yis iaitu 7.02 dan 1.73% (w/v). Validasi

eksperimen membuktikan yang nilai ramalan dan eksperimen yang dijalankan

tiada perbezaan ketara (p > 0.05). Peningkatan yang tinggi dalam penghasilan sel

(11.72 cfumL-1

) tercapai di dalam bioreaktor 2-L selepas 18 jam kultivasi dan

peningkatan ini tiada perbezaan statistik (p > 0.05) dengan sel yang diaktifkan

menggunakan media komersial.

FCCD juga digunakan untuk mengoptimasikan bahan enkapsulasi. Titik optima

kepekatan untuk gelatin lembu-genipin-alginat adalah pada 11.21% (w/v), 13.96

mM dan 12.57% (w/v). Manakala, gelatin ikan-genipin-alginat ialah pada 12.57%

(w/v), 19.12 mM dan 5% (w/v) untuk memberi kesan yang optima. Berdasarkan

verifikasi, nilai eksperimen bagi kapsul yang dioptimakan tidak menunjukkan

perbezaan besar dengan nilai ramalan. Perbandingan dibuat dengan kapsul dari

gelatin khinzir-genipin-alginat dan menunjukkan kapsul gelatin lembu-genipin-

alginat dan gelatin ikan-genipin-alginat yang dioptimakan memberi kesan

kekuatan yang rendah berbanding dengan kapsul gelatin khinzir-genipin-alginat

apabila didedahkan kepada SIF.

© COPYRIG

HT UPM

vii

Tahap perlindungan bagi G4 dan probiotik lain dengan menggunakan bahan

enkapsulasi yang dioptimakan telah dikaji. Lapan formulasi bahan enkapsulasi

yang dioptimakan melibatkan: 1) gelatin lembu-genipin-alginat, 2) gelatin ikan-

genipin-alginat, 3) gelatin khinzir-genipin-alginat, 4) gelatin lembu-alginat, 5)

gelatin ikan-alginat, 6) gelatin khinzir-alginat, 7) alginat sahaja, 8) sel bebas

(tidak dienkapsulasikan). Kadar enkapsulasi sel yang rendah telah diperolehi dari

kumpulan 2 dan 5. Manakala, kumpulan 1 menunjukkan kadar enkapsulasi sel

yang paling tinggi. Kadar pembengkakan yang rendah telah dilihat dari kumpulan

1, 3, 4 dan 6, manakala kumpulan 5 menunjukkan pembengkakan yang cepat

setelah 120 minit di dalam SGF. Pembebasan sel berlaku apabila kapsul mula

pecah dan ini boleh dilihat menerusi analisa aktiviti pembebasan sel. Kesemua

kumpulan kecuali kumpulan 3 dan 6, menunjukkan kesan positif di dalam

membebaskan sel ketika dalam SIF dengan menunjukkan nilai ketumpatan optik

(OD) yang tinggi dan tahap kehidupan sel tertinggal di dalam kapsul adalah

rendah. Faktor seperti jenis gelatin, kekuatan “bloom” dan penggunaan alginat

memainkan peranan penting dalam penstabilan gelatin struktur kimianya terutama

ketika di dalam persekitaran yang berasid. Enkapsulasi menggunakan gelatin

lembu dan ikan dapat memperbaiki tahap kehidupan dan pembebasan sel serta

berpotensi untuk mengantikan penggunaan kapsul dari gelatin khinzir untuk

penghantaran probiotik kultur ke tempat sasaran di dalam usus.

© COPYRIG

HT UPM

viii

AKNOWLEDGEMENT

First and foremost, GOD ALMIGHTY for give strength and courage to face the

challenges I encountered throughout this course.

I would like to give my sincere thanks to my supervisor and all co-supervisors for

their advice, support and guidance, will always be highly appreciated.

My friends especially from lab 202, Biotech 2 for always accompanied me during

overnight experiments.

Universiti Teknologi MARA (UiTM) and Malaysia Ministry of High Education

for financial support.

Last but not least, my dearest husband (Awis Qurni), my beloved mom (Rahmah)

and lovely kids (Shahira, Ammar, Amir and Amsyar) deserve thanks for their

inspiration, encouragement, kindness, understanding and patience throughout my

whole studies.

© COPYRIG

HT UPM

ix

APPROVAL I certify that a Thesis Examination Committee has met on 5

th July 2012 to

conduct the final examination of Khalilah Abdul Khalil on her thesis entitled

“Enhanced survivability of Bifidobacterium pseudocatenulatum G4 by

encapsulation using gelatin-genipin-alginate matrices” in accordance with the

Universities and University Colleges Act 1971 and the Constitution of the Putra

Malaysia [ P.U.(A) 106] 15 March 1998. The Committee recommends that the

student be awarded the degree of Doctor Philosophy.

Member of the Examination Committee were as follows:

Muhajir bin Hamid, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Umi Kalsom binti Md Shah, PhD

Associate Professor



(Internal Examiner)

Noraini binti Abdul Rahman, PhD

Senior Lecturer



(Internal Examiner)

Satya Prakash, PhD

Professor

Biomedical Engineering, Faculty of Medicine

McGill University

Quebec, Canada

(External Examiner)

BUJANG KIM HUAT,PhD Professor and Deputy Dean

School of Graduate Studies

Universti Putra Malaysia

Date:

© COPYRIG

HT UPM

x

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Doctor of Philosophy.

The members of the Supervisory Committee were as follows:

Shuhaimi Mustafa, PhD

Associate Professor



(Chairman)

Arbakariya Ariff, PhD

Professor



(Member)

Mohd Yazid Abd Manap, PhD

Professor

Faculty of Food Science and Technology


(Member)

Rosfarizan Mohamad, PhD

Associate Professor



(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

© COPYRIG

HT UPM

xi

DECLARATION

I declare that the thesis is based on my original work except for quotations and

citations, which have been duly acknowledged. I also declare that is has not been

previously and is not concurrently, submitted for any other degree at Universti

Putra Malaysia or at any other institution.


Date: 5 July 2012

© COPYRIG

HT UPM

xii

TABLES OF CONTENTS

Page

DEDICATION i

ABSTRACT ii

ABSTRAK v

AKNOWLEDGEMENTS viii

APPROVAL ix

DECLARATION xi

LIST OF TABLES xvii-xx

LIST OF FIGURES xxi-xxiv

LIST OF PLATES xxv

LIST OF ABBREVIATIONS xxvii-xxix

CHAPTER

Pages

1 INTRODUCTION

1

2 LITERATURE REVIEW

2.1 Intestinal Microflora 7

2.2 Probiotic 9

2.2.1 Suitable Dosage of Probiotics for Health Benefit

Effects

2.2.2 Antagonistic and Other Effects of Probiotic

2.2.3 Probiotic Criteria

2.2.4 Probiotic Applications

2.2.5 Probiotic Challenges

2.2.6 Bifidobacterium pseudocatenulatum G4 as a

Potential Probiotic

10

11

12

12

13

15

2.3 Milk as Probiotic Cultivation Media 16

2.4 Technologies in Improving Probiotic Shelf Life 19

2.5 Cells Encapsulation

2.5.1 Encapsulation Methods

2.5.1.1 Extrusion

2.5.1.2 Emulsification

2.5.1.3 Atomization

2.5.2 Encapsulation Approach for Enhancing Probiotic

Viability

2.5.2.1 Starter Cultures Preparation

2.5.2.2 Fermentation

2.5.2.3 Food Matrix

2.5.2.4 Various Encapsulating Matrices Used for

Cells Protection During Gastrointestinal

Tract Passage

20

20

22

23

24

27

27

27

28

31

© COPYRIG

HT UPM

xiii

2.5.3 Encapsulation Matrices

2.5.3.1 Alginate

2.5.3.2 Gelatin

2.5.3.3 Genipin

33

34

37

3 METHODOLOGY

3.1 Bifidobacterium Strains

3.2 Inoculum Preparation

3.3 Macroscopic and Microscopic Confirmation

3.4 Further Confirmation Using 16s rRNA-Gene-Targeted

Primers

3.4.1 Bacterial Strains

3.4.2 DNA Extraction

3.4.3 Genus-Specific PCR

3.4.4 Species-Species PCR

3.4.5 Gel Electrophoresis

3.5 Medium Preparation in Schott bottle

3.6 Fermentation in 2-L Stirred Tank Bioreactor

3.6.1 Set up and Geometry of 2-L Stirred Tank Bioreactor

3.6.2 Fermentation Preparation

3.7 B. pseudocatenulatum G4 Preparation for Encapsulation

3.7.1 Cultivation

3.8 Encapsulation of B. pseudocatenulatum G4

3.9 Preparation of Simulated Gastric Fluid (SGF)

3.10 Preparation of Simulated Intestinal Fluid (SIF)

3.11 Analytical Techniques

3.11.1 Microbiological Analysis

3.11.2 Growth Studies

3.11.3 Organic Acids analysis

3.11.4 Sugar Analysis

3.11.5 Free Amino Nitrogen Analysis

3.11.6 β-galactosidase Analysis

3.11.7 Enumeration of Encapsulated Cells

3.11.8 Beads Strength Determination

3.11.9 Beads Stability in SGF and SIF

3.11.10 Encapsulated Cells Released in SGF and SIF

3.11.11 Survival Assay of Encapsulated Cells in SGF and

SIF

3.11.12 Beads Surface Morphology Observation Using

Scanning Electron Microscope (SEM)

3.12 Statistical Analysis

3.13 Experimental Flow Chart

38

38

39

39

39

40

41

42

43

44

45

45

46

46

49

49

50

50

51

51

52

53

54

55

56

57

57

58

59

60

© COPYRIG

HT UPM

xiv

4 GROWTH CHARACTERISTICS OF B. pseudocatenulatum G4 IN

MILK BASED INOCULUM MEDIUM PRIOR TO FURTHER

FERMENTATION

4.1 Introduction 61

4.2 Materials and Methods 63

4.3 Results and Discussion

4.3.1 Morphology Observation

4.3.2 Confirmation of B. pseudocatenulatum G4 Using

16S rRNA-Gene-Targeted Primers

4.3.3 Cultivation of B. pseudocatenulatum G4 Using

Standing Culture

4.3.3.1 Growth and pH Changes

4.3.3.2 Organic Acids Production

4.3.3.3 Sugar Metabolism

4.3.3.4 Determination of β-galactosidase

Production

4.3.3.5 Free Amino Nitrogen Analysis

4.3.3.6 Growth Performance of B.

pseudocatenulatum G4 in 2-L Stirred

Tank Bioreactor

4.3.3.7 β-galactosidase Production

65

67

69

73

77

81

86

89

94

4.4 Conclusions

99

5 OPTIMIZATION OF SKIM MILK BASED INOCULUM

MEDIUM FOR Bifidobacterium pseudocatenulatum G4

CULTIVATION PRIOR TO FERMENTATION USING

RESPONSE SURFACE METHODOLOGY

5.1 Introduction 100



5.3.1 Initial Screening of Significant Medium

Components and Steepest Ascent

5.3.2. Optimization of Medium Components

5.3.2.1 Biomass Production Response (y1)

5.3.2.2 β-galactosidase Production Response (y2)

5.3.2.3 Lactose Residue Response (y3)

5.3.2.4 FAN Residue Response (y4)

5.3.3 Validation of Optimized Medium.

5.3.4. Performance of G4 from Optimized Medium During

Fermentation in 2-L Stirred Tank Bioreactor

106

111

116

120

122

126

127

5.4 Conclusions

129

© COPYRIG

HT UPM

xv

6 OPTIMIZATION OF ENCAPSULATING MATRICES FOR

Bifidobacterium pseudocatenulatum G4 USING BOVINE/

FISH GELATIN WITH GENIPIN-SODIUM ALGINATE

COMBINATIONS BASED ON RESPONSE SURFACE

METHODOLOGY




6.3.1 Bovine Gelatin, Genipin and Sodium Alginate

6.3.1.1 Screening for Encapsulation Matrices

6.3.1.2 Optimization Using Face-Centered Full

Factorial Design (FCCD)

6.3.1.3 Verification

6.3.2 Fish gelatin, Genipin and Sodium Alginate.

6.3.2.1 Screening for the Encapsulation Matrices

6.3.2.2 Optimizing Using Face-Centered Full

Factorial Design (FCCD)

6.4.2.3 Verification

6.3.3 Comparison Performances of Both Optimized

Encapsulation Matrices

6.3.3.1 Beads Strength (g)

6.3.3.2. Encapsulation Yield (%)

137

144

162

163

167

185

187

188

6.4 Conclusions

189

7 CELLS RELEASE FROM OPTIMIZED BOVINE AND FISH

GELATIN WITH THE COMBINATION OF ALGINATE-

GENIPIN MATRICES




7.3.1 Encapsulation Yield of Probiotic Cells

7.3.2 Beads Stability

7.3.2.1 Simulated Gastric Fluid (SGF) Exposure

7.3.2.2 Simulated Intestinal Fluid (SIF) Exposure

7.3.3 In vitro Release Studies of Probiotic from the Beads

7.3.4 Survival of Entrapped Cells During Sequential

Incubation in SGF and SIF

7.3.4.1 Survival of Entrapped Cells in SGF

7.3.4.2 Survival of Entrapped Cells in SIF

7.3.5 SEM Observation

194

198

203

206

210

214

217

7.4 Conclusions

224

© COPYRIG

HT UPM

xvi

8 GENERAL DICUSSIONS, CONCLUSIONS AND

RECOMMENDATIONS

226

REFERENCES

APPENDICES

BIODATA OF STUDENT

PUBLICATIONS

universiti putra malaysia khalilah abdul khalil fbsb

Documents