universiti putra malaysia - psasir.upm.edu.mypsasir.upm.edu.my/id/eprint/32261/1/fbsb 2012 10r.pdfmy...
TRANSCRIPT
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
KHALILAH ABDUL KHALIL
DOCTOR OF PHILOSOPHY
UNIVERSITI PUTRA MALAYSIA
2012
© COPYRIG
HT UPM
MICROENCAPSULATION of Bifidobacterium pseudocatenulatum G4 USING
NATURAL MATRICES
By
KHALILAH ABDUL KHALIL
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
KHALILAH ABDUL KHALIL
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
KHALILAH ABDUL KHALIL
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
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Noraini binti Abdul Rahman, PhD
Senior Lecturer
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(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
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Arbakariya Ariff, PhD
Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
Mohd Yazid Abd Manap, PhD
Professor
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Member)
Rosfarizan Mohamad, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
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.
KHALILAH ABDUL KHALIL
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
5.2 Materials and Methods 102
5.3 Results and Discussion
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
6.1 Introduction 131
6.2 Materials and Methods 133
6.3 Results and Discussion
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
7.1 Introduction 191
7.2 Materials and Methods 192
7.3 Results and Discussion
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