UNIVERSITI PUTRA MALAYSIA PROBIOTIC …psasir.upm.edu.my/id/eprint/9834/1/FBSB_2010_1_A.pdfinhibited the Pediococcus acidilactici 4-46, Enterococcus faecalis N-103, Enterococcus faecium

UNIVERSITI PUTRA MALAYSIA

PROBIOTIC CHARACTERISTICS OF BACTERIOCINOGENIC LACTOBACILLUS PLANTARUM STRAINS ISOLATED FROM

MALAYSIAN FOODS

LIM YIN SZE FBSB 2010 1

PROBIOTIC CHARACTERISTICS OF BACTERIOCINOGENIC

LACTOBACILLUS PLANTARUM STRAINS ISOLATED FROM

MALAYSIAN FOODS

By

LIM YIN SZE

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

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

February 2010

ii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia

in fulfilment of the requirement for the degree of Doctor of Philosophy

PROBIOTIC CHARACTERISTICS OF BACTERIOCINOGENIC

LACTOBACILLUS PLANTARUM STRAINS ISOLATED FROM

MALAYSIAN FOODS

By

LIM YIN SZE

February 2010

Chair : Foo Hooi Ling, PhD

Faculty : Biotechnology and Biomolecular Sciences

The usage of antimicrobial drugs, such as antibiotics, as growth enhancer in

livestock farming is prohibited in many countries. In recent years, antimicrobial

resistance associated with the use of antimicrobial drugs in animals has triggered

vast public attention. Thus, recent approach to enhance the growth performance of

livestock is through supplementation of probiotic and digestive enzymes. Probiotic

are live microorganisms, when consumed in adequate amounts will confer health

benefit on the host.

Generally recognised as safe status of lactic acid bacteria (LAB) and the increasing

demand of probiotic in food and feed industry have driven current attempt to

determine the probiotic potential of six bacteriocinogenic Lactobacillus plantarum

strains (UL4, TL1, RS5, RI11, RG11 and RG14) isolated from Malaysian foods,

through in vitro probiotic characterisation study and in vivo animal trials.

iii

In the in vitro study, the antimicrobial potential of Lb. plantarum strains and

bacteriocins was carried out, followed by the study to assess the ability of these

strains to overcome simulated gastric and small intestinal tracts, deconjugate bile

salts (sodium taurocholate, sodium glycocholate, sodium taurodeoxycholate and

sodium glycodeoxycholate), assimilate cholesterol and adhere to intestinal epithelial

Caco-2 cell line. In vivo animal trials were then carried out to evaluate the

effectiveness of Lb. plantarum strains and bacteriocins produced on growth

performance, intestinal microflora and histomorphological changes, and alteration of

short chain fatty acids and serum cholesterol level of rats.

In the in vitro study, Lb. plantarum strains and their bacteriocins had exhibited

strong bacteriocidal and bacteriostatic antimicrobial action against Gram-positive

and –negative bacteria. Lb. plantarum strains had strongly inhibited the growth of

Bacillus cereus B43 and Listeria monocytogenes L55, whereas bacteriocins had

inhibited the Pediococcus acidilactici 4-46, Enterococcus faecalis N-103,

Enterococcus faecium ATCC 19434, Listeria monocytogenes L55, Bacillus cereus

B43, Streptococcus pneumoniae S972 and Staphylococcus aureus S942. Lb.

plantarum strains were highly tolerance to the exposure of simulated gastric and

small intestinal juices. They were able to deconjugate bile salts and assimilated 56.7

– 100.0 % of cholesterol. Lb. plantarum strains adhered strongly to the Caco-2

epithelial cell line and reduced the adherence of L. monocytogenes L55 substantially.

In in vivo animal trial, rats were force-fed with six Lb. plantarum strains (108

CFU/mL daily) for 28 days. The high LAB population found in faecal and intestinal

samples indicated that the fed Lb. plantarum strains could survive and colonise in

iv

gastrointestinal tract. The small intestinal villous height and crypt depth were

significantly (P < 0.05) greater than Control group. Furthermore, Lb. plantarum

RI11 and RG11 had significantly reduced the serum cholesterol level of rats by 24.4

% (P < 0.05) and 19.3 % (P < 0.01), respectively.

In the subsequent animal trial, rats were force-fed with six bacteriocins (800 AU/mL

daily) produced by Lb. plantarum strains for 28 days. Bacteriocins had reduced the

colonisation of Enterobacteriaceae in the intestine, while maintaining the intestinal

LAB population. All bacteriocins had significantly increased (P < 0.05) the crypt

depth of small intestine and caecum. Bacteriocins RS5, RI11 and RG11 had

significantly reduced the serum cholesterol level of rats by 21.9 % (P < 0.05), 15.6

% (P < 0.05) and 15.2 % (P < 0.01) respectively. Current study showed that the

studied Lb. plantarum strains and bacteriocins produced had demonstrated certain

extent of probiotic effects in in vitro and in vivo. These probiotic effects are specific

or limited to the Lb. plantarum strains or bacteriocins tested.

v

Abstrak ini dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi

keperluan untuk ijazah Doktor Falsafah

PENCIRIAN PROBIOTIK BAGI STRAIN-STRAIN BAKTERIOSINOGENIK

LAKTOBASILUS PLANTARUM YANG DIASING DARIPADA MAKANAN

MALAYSIA

Oleh

LIM YIN SZE

Februari 2010

Pengerusi : Foo Hooi Ling, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Penggunaan ubat antimikrob seperti antibiotik sebagai penggalak pertumbuhan

dalam bidang ternakan telah dilarangkan oleh kebanyakan negara. Kebelakangan ini,

kewujudan resistant antimikrob berikutan dengan pengunaan ubat antimukrob dalam

binatang ternakan telah menarik perhatian umum. Oleh itu, pendekatan terkini untuk

meningkatkan prestasi pertumbuhan ternakan adalah dengan penggunaan probiotik

dan enzim-enzim penghadaman. Probiotik adalah mikroorganisma hidup yang

apabila dimakan dalam jumlah secukupnya, dapat memanfaatkan kesihatan

penggunanya.

Status „dikenali sebagai selamat secara umumnya‟ untuk bakteria asid laktik (LAB)

dan peningkatan permintaan untuk probiotik dalam industri makanan dan makanan

ternakan telah mendorong pelaksanaan kajian ini untuk menentukan potensi

probiotic bagi enam strain-strain bakteriosinogenik Laktobasilus plantarum (UL4,

vi

TL1, RS5, RI11, RG11 dan RG14) yang diasingkan daripada makanan Malaysia,

melalui kajian pencirian probiotik secara in vitro dan kajian in vivo haiwan.

Dalam kajian in vitro, potensi antimikrob bagi enam strain Lb. plantarum dan

bakteriosin yang dihasilkan telah dijalankan, diikuti dengan kajian untuk menilai

keupayaan strain ini untuk mengatasi keadaan simulasi saluran gastrik dan usus

kecil, dekonjugasi garam hempedu (sodium taurokolat, sodium glikokolat, sodium

taurodeoksikolat dan sodium glikodeoksikolat), asimilasi kolesterol dan pelekatan ke

Caco-2 sel epithelium usus. Kajian in vivo haiwan seterusnya dilaksanakan untuk

menilai keberkesanan strain Lb. plantarum dan bakteriosin yang dihasilkan ke atas

prestasi pertumbuhan, mikroflora usus dan perubahan-perubahan histomofologikal,

perubahan rantaian pendek asid lemak dan paras kolesterol serum tikus.

Dalam kajian in vitro, strain Lb. plantarum dan bakteriosinnya telah mempamerkan

tindakan antimikrob bakteriosidal dan bakteriostatik yang kuat terhadap patogen-

patogen. Strain Lb. plantarum telah menyinkirkan pertumbuhan Basilus cereus B43

dan Listeria monocytogenes L55, manakala bakteriosinnya telah merencatkan

bacteria Pediococcus acidilactici 4-46, Enterococcus faecalis N-103, Enterococcus

faecium ATCC 19434 Listeria monocytogenes L55, Bacillus cereus B43,

Streptococcus pneumoniae S972 dan Staphylococcus aureus S942. Strain Lb.

plantarum telah mempamerkan tolerasi yang tinggi dalam simulasi saluran gastrik

dan usus kecil. Mereka ini boleh mendekonjugasikan garam hempedu dan

mengasimilasikan 56.7 – 100.0 % kolesterol. Strain Lb. plantarum juga melekat

dengan kuat pada Caco-2 sel epithelium usus, dan telah mengurangkan pelekatan L.

monocytogenes L55 dengan nyatanya.

vii

Dalam kajian in vivo haiwan, tikus telah diberi enam strain Lb. plantarum (108

CFU/mL harian) selama 28 hari. Populasi LAB yang tinggi telah dijumpai dalam

sampel tahi dan usus. Kajian ini telah menunjukkan bahawa strain Lb. plantarum

dapat hidup dan mengkolonisasikan saluran usus. Ketinggian vilus dan kedalaman

krypt telah meningkat dengan lebih nyatanya (P < 0.05) daripada kumpulan kontrol.

Tambahan pula, Lb. plantarum RI11 dan RG11 telah mengurangkan paras kolesterol

serum tikus sebanyak 24.4 % (P < 0.05) dan 19.3 % (P < 0.01) masing-masing.

Dalam kajian haiwan seterusnya, tikus telah diberi enam bakteriosin (800 AU/mL

harian) yang dihasilkan oleh Lb. plantarum selama 28 hari. Bakteriosin telah

mengurangkan populasi Enterobacteriaceae dalam usus sementara mengekalkan

populasi LAB dalam usus. Secara umunya, semua bakteriosin telah meningkatkan

kedalaman krypt usus kecil dan sekum dengan nyatanya (P < 0.05). Sementara,

bakteriosin RS5, RI11 dan RG11 telah mengurangkan paras kolesterol serum tikus

dengan nyatanya sebanyak 21.9 % (P < 0.05), 15.6 % (P < 0.05) dan 15.2 % (P <

0.01) masing-masing. Kajian terkini ini telah menunjukkan bahawa strain-strain Lb.

plantarum dan bakteriosin yang dihasilkan telah mempamerkan kesan probiotik

dalam kajian in vitro dan in vivo. Walaubagaimanapun, kesan probiotik in adalah

spesifik atau terhad kepada strain-strain Lb. plantarum dan bakteriosin yang dikaji.

viii

ACKNOWLEDGEMENTS

I would like to express my most sincere gratitude and appreciation to my supervisor,

Assoc. Prof. Dr. Foo Hooi Ling for her invaluable guidance, patience and constant

encouragement throughout my study. I am also immensely grateful for the advice

and support given by the members of my supervisory committee, Assoc. Prof. Dr.

Loh Teck Chwen, Prof. Dr. Raha Abdul Rahim, Prof. Dr. Mohd. Hair Bejo and Prof.

Dr. Gulam Rusul Rahmat Ali.

I owe my deepest gratitude to Assoc. Prof. Dr. Abdul Rahman Omar, who have

provided me the facility for carried out cell tissue culture work and I am also

indebted to Mr. Kufli C. N., Mr. Saparin Demin, Mr. Zakaria Md. Shah, Mr.

Saipuzaman Ali, Mr. Rosli Aslim and Mrs. Aluyah Marzuki for their excellent

technical assistance.

I am heartily thankful to friends and staff in UPM who have supported me in various

aspects during the completion of this study. Last but not least, this thesis would not

have been possible without the love and supports from Boon Chin and my family.

Thanks God for everything!

ix

I certify that a Thesis Examination Committee has met on 24th

February, 2010 to

conduct the final examination of Lim Yin Sze on her thesis entitled “Probiotic

characteristics of bacteriocinogenic Lactobacillus plantarum strains isolated

from Malaysian foods” in accordance with the 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 degree of

Doctor of Philosophy.

Members of the Thesis Examination Committee were as follows:

Raja Noor Zaliha Raja Abd. Rahman, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Norhani Abdullah, PhD

Professor



(Internal Examiner)

Shuhaimi Mustafa, PhD

Associate Professor



(Internal Examiner)

Rickey Yoshio Yada, PhD

Professor

Department of Food Science

University of Guelph

Canada

(External Examiner)

______________________________

BUJANG BIN KIM HUAT, PhD

Professor and Deputy Dean

School of Graduate Studies


Date:

x

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

accepted as fulfilment of the requirement of the requirement for the degree of Doctor

of Philosophy. The members of the Supervisory Committee were as follows:

Foo Hooi Ling, PhD

Associate Professor

Faculty of Biotechnology and Molecular Sciences


(Chairman)

Raha Abdul Rahim, PhD

Professor

Faculty of Biotechnology and Molecular Sciences


(Member)

Mohd. Hair Bejo, PhD

Professor

Faculty of Veterinary Medicine


(Member)

Gulam Rusul Rahmat Ali, PhD

Professor

School of Industrial Technology

Universiti Sains Malaysia

(Member)

________________________________

HASANAH MOHD. GHAZALI, PhD

Professor and Dean

School of Graduate Studies


Date: 13th

May 2010

xi

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.

______________

LIM YIN SZE

Date:

xii

TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENTS viii

APPROVAL ix

DECLARATION xi

LIST OF TABLES xvi

LIST OF FIGURES xviii

LIST OF APPENDICES xx

LIST OF ABBREVIATIONS xxii

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 4

2.1 Probiotic 4

2.1.1 Definition of probiotic 4

2.1.2 Selection criteria of probiotic 5

2.1.3 Microorganisms used as probiotic 10

2.2 Lactobacillus plantarum as probiotic 13

2.2.1 Systematic and physiology of Lb. plantarum 14

2.2.2 Antimicrobial potential of Lb. plantarum 15

2.3 Health benefits of probiotic 19

2.3.1 Modulation of the intestinal microflora 19

2.3.2 Prevention of gastrointestinal diseases 22

2.3.3 Cholesterol lowering effect 27

2.3.4 Modulation of the immune system 28

2.3.5 Anticarcinogenic effect 29

2.4 Industrial perspective of probiotic 31

2.4.1 Probiotic in foods 33

2.4.2 Probiotic in pharmaceutical products 34

2.4.3 Probiotic in animal feeds 37

2.5 Safety considerations and future development of probiotic 38

2.5.1 Quality assurance of probiotic 40

2.5.2 Regulatory issues of probiotic 41

2.5.3 Future development of probiotic 41

3 ANTIMICROBIAL POTENTIAL OF LACTOBACILLUS

PLANTARUM STRAINS AND THEIR BACTERIOCINS

43

3.1 Introduction 43

3.2 Methodology 44

3.2.1 Preparation of bacterial cultures 44

3.2.2 Competitive exclusion study of Lb. plantarum strains and

pathogens

45

3.2.3 Antimicrobial activity of bacteriocins against pathogens 47

xiii

3.2.4 Statistical analysis 48

3.3 Results 49

3.3.1 Growth of Lb. plantarum strains and pathogens in mono-

culture assay

49

3.3.2 Competitive exclusion study of Lb. plantarum strains and

pathogens

50

3.3.3 Antimicrobial activity of bacteriocins against various

bacteria

67

3.4 Discussion 71

3.4.1 Competitive exclusion ability of Lb. plantarum strains

against pathogens

71

3.4.2 Antimicrobial ability of bacteriocins against various

bacteria

73

3.5 Conclusion 76

4 IN VITRO PROBIOTIC CHARACTERISATION OF

LACTOBACILLUS PLANTARUM STRAINS

77

4.1 Introduction 77

4.2 Methodology 78

4.2.1 Preparation of washed bacterial cell suspension 78

4.2.2 In vitro upper gastrointestinal transit tolerance assessment 78

4.2.3 In vitro bile tolerance assessments 79

4.2.4 In vitro cholesterol assimilation assessment 82

4.2.5 In vitro intestinal epithelial adhesion assessment 83


4.3 Results 87

4.3.1 Effect of in vitro upper gastrointestinal transit on viability

of Lb. plantarum strains

87

4.3.2 In vitro bile salt deconjugation ability of Lb. plantarum

strains

94

4.3.3 In vitro cholesterol assimilation ability of Lb. plantarum

strains

100

4.3.4 In vitro intestinal epithelial adhesion ability of Lb.

plantarum strains

101

4.4 Discussion 110



111

4.4.2 Effect of bile on viability of Lb. plantarum strains 114

4.4.3 In vitro cholesterol assimilation ability of Lb. plantarum

strains

117


plantarum strains

118

4.5 Conclusion 122

xiv

5 IN VIVO STUDY OF RATS FED WITH LACTOBACILLUS

PLANTARUM STRAINS

123

5.1 Introduction 123

5.2 Methodology 124

5.2.1 Animal 124

5.2.2 Bacteria preparation 124

5.2.3 Experimental design 124

5.2.4 Bacteriological analysis of faecal samples 125

5.2.5 Bacteriological analysis of intestinal samples 126

5.2.6 Histological analysis of intestinal samples 126

5.2.7 Determination of concentration of short chain fatty acids in

faecal samples

127

5.2.8 Determination of serum cholesterol level 128


5.3 Results 130

5.3.1 Growth performance of rats 130

5.3.2 Microbial population in faecal and intestinal samples 133

5.3.3 Intestinal villous height and crypt depth 141

5.3.4 Concentration of short chain fatty acids in faecal samples 144

5.3.5 Serum cholesterol level of rats 144

5.4 Discussion 147


5.4.2 Faecal and intestinal microflora 148




5.4.6 Summary of the probiotic effects of Lb. plantarum strains 158

5.5 Conclusion 159

6 IN VIVO STUDY OF RATS FED WITH BACTERIOCINS 160

6.1 Introduction 160

6.2 Methodology 161

6.2.1 Animal 161

6.2.2 Preparation of bacteriocin 161

6.2.3 Experimental design 162

6.2.4 Bacteriological analysis of faecal samples 163

6.2.5 Bacteriological analysis of intestinal samples 163

6.2.6 Histological analysis of intestinal samples 163

6.2.7 Determination of concentration of short chain fatty acids in

faecal samples

164

6.2.8 Determination of serum cholesterol level 164


6.3 Results 165


6.3.2 Microbial population in faecal and intestinal samples 165




xv

6.4 Discussion 182


6.4.2 Faecal and intestinal microflora 184




6.4.6 Summary of the probiotic effects of bacteriocins 190

6.5 Conclusion 191

7 GENERAL DISCUSSION AND SUMMARY 192

7.1 Antimicrobial potential of Lb. plantarum strains and their

bacteriocins

192

7.2 In vitro probiotic characterisation of Lb. plantarum strains 196



196

7.2.2 In vitro bile tolerance, deconjugation and cholesterol

assimilation ability of Lb. plantarum strains

198


plantarum strains

200

7.3 In vivo study of rats fed with Lb. plantarum strains 202

7.4 In vivo study of rats fed with bacteriocins 205

7.5 Recommendation for future research 208

REFERENCES 210

APPENDICES 237

BIODATA OF STUDENT 260

xvi

LIST OF TABLES

Table Page

2.1 Key and desirable criteria for the selection of probiotics in

commercial application

8

2.2 Microbes used as probiotics 12

2.3 Commercial strains that are currently sold as probiotic 32

2.4 Probiotics and so-called ‘probiotics’ that patients are using in

Canada

36

2.5 Organisations that involved in establishing standards for probiotic

bacteria in commercial products

39

3.1 Generation time of Lb. plantarum and pathogen strains grown as

monoculture in nutrient broth

49

3.2 Antimicrobial activity of bacteriocins against Gram-positive and –

negative bacteria

70

4.1 Qualitative measurement of bile salt deconjugation by Lb.

plantarum strains

96

4.2 Quantitative measurement of bile salt deconjugation by Lb.

plantarum strains

99

5.1 Effect of direct fed of Lb. plantarum on body weight of rats 131

5.2 Effect of direct fed of Lb. plantarum on total feed intake, total live

weight gained and feed conversion ratio in rats

132

5.3 Effect of direct fed of Lb. plantarum on the population of lactic acid

bacteria in faecal samples

134

5.4 Effect of direct fed of Lb. plantarum on the population of

Enterobacteriaceae bacteria in faecal samples

135


bacteria in different intestinal regions after week-4 trial

137

5.6 Effect of direct fed of Lb. plantarum on the population of

Enterobacteriaceae bacteria in different intestinal regions after

week-4 trial

139


bacteria and Enterobacteriaceae bacteria in intestinal digesta after

week-4 trial

140

xvii

5.8 Effect of direct fed of Lb. plantarum on villous height of different

small intestinal regions after week-4 trial

142

5.9 Effect of direct fed of Lb. plantarum on crypt depth of different

small and large intestinal regions after week-4 trial

143

5.10 Effect of direct fed of Lb. plantarum on concentration of short

chain fatty acids in Week-4 faecal samples

145

5.11 Effect of direct fed of Lb. plantarum on serum cholesterol level of

rats after feeding trial

146

6.1 Effect of direct fed of bacteriocins on body weight of rats 166

6.2 Effect of direct fed of bacteriocins on total feed intake, total live

weight gained and feed conversion ratio in rats

167

6.3 Effect of direct fed of bacteriocins on the population of lactic acid

bacteria in faecal samples

168

6.4 Effect of direct fed of bacteriocins on the population of

Enterobacteriaceae bacteria in faecal samples

169


bacteria in different intestinal regions after week-4 trial

173

6.6 Effect of direct fed of bacteriocins on the population of

Enterobacteriaceae bacteria in different intestinal regions after

week-4 trial

174


bacteria and Enterobacteriaceae bacteria in intestinal digesta after

week-4 trial

175

6.8 Effect of direct fed of bacteriocins on villous height of different

small intestinal regions after week-4 trial

177

6.9 Effect of direct fed of bacteriocins on crypt depth of different small

and large intestinal regions after week-4 trial

178

6.10 Effect of direct fed of bacteriocins on concentration of short chain

fatty acids in week-4 faecal samples

180

6.11 Effect of direct fed of bacteriocins on serum cholesterol level of rats

after feeding trial

181

xviii

LIST OF FIGURES

Figure Page

3.1 Competitive exclusion assay of B. cereus B43 and Lb. plantarum

strains: (a) UL4, (b) TL1, (c) RS5

52

3.2 Competitive exclusion assay of B. cereus B43 and Lb. plantarum

strains: (a) RI11, (b) RG11 and (c) RG14

53

3.3 Competitive exclusion assay of L. monocytogenes L55 and Lb.

plantarum strains: (a) UL4, (b) TL1, (c) RS5

56


plantarum strains: (a) RI11, (b) RG11 and (c) RG14

57

3.5 Competitive exclusion assay of S. typhimurium S1000 and Lb.


59



60

3.7 Competitive exclusion assay of Staph. aureus S942 and Lb.


62



63

3.9 Competitive exclusion assay of E. coli E30 and Lb. plantarum


65



66

3.11 Formation of inhibition zone by bacteriocins against various Gram-

positive and -negative bacteria

69

4.1 In vitro simulated gastric transit of Lb. plantarum strains: (a) UL4,

(b) TL1, (c) RS5, at pH 2, 3 and 4

88

4.2 In vitro simulated gastric transit of Lb. plantarum strains: (a) RI11,

(b) RG11, (c) RG14, at pH 2, 3 and 4

89

4.3 In vitro simulated small intestinal transit of Lb. plantarum strains:

(a) UL4, (b) TL1, (c) RS5, at pH 8, with and without presence of

bile salt

92

xix

4.4 In vitro simulated small intestinal transit of Lb. plantarum strains

(a) RI11, (b) RG11, (c) RG14, at pH 8, with and without presence

of bile salt

93

4.5 Tolerance of Lb. plantarum strains: (a) UL4, (b) TL1, (c) RS5, (d)

RI11, (e) RG11, (f) RG14, in the presence of bile salt at different

concentrations

95

4.6 Qualitative assay of bile salt deconjugation by Lb. plantarum UL4 97

4.7 Cholesterol assimilation ability of Lb. plantarum strains 100

4.8 Cell surface hydrophobicity of Lb. plantarum strains 101

4.9 Adhesion percentage of (i) Lb. plantarum strains or (ii) L.

monocytogenes L55 to Caco-2 cell line by using three adhesion

assays

103

4.10 Adhesion percentage of (i) Lb. plantarum strains or (ii) Staph.

aureus S942 to Caco-2 cell line by using three adhesion assays

106

4.11 Adhesion percentage of (i) Lb. plantarum strains or (ii) E. coli E30

to Caco-2 cell line by using three adhesion assays

107

4.12 Adhesion percentage of (i) Lb. plantarum strains or (ii) S.

typhimurium S1000 to Caco-2 cell line by using three adhesion

assays

108

4.13 Adhesion percentage of (i) Lb. plantarum strains or (ii) B. cereus

B43 to Caco-2 cell line by using three adhesion assays

109

xx

LIST OF APPENDICES

Appendix Page

3.1 Competitive exclusion assay of B. cereus B43 and Lb.


237

3.2 Competitive exclusion assay of B. cereus B43 and Lb.

plantarum strains: (a) RI11, (b) RG11, (c) RG14

238



239



240



241



242



243



244


strains: (a) UL4, (b) TL1, (c) RS5

245


strains: (a) RI11, (b) RG11, (c) RG14

246

4.1 Cholic acid standard curve 247

4.2 Cholesterol standard curve 247

4.3 In vitro simulated gastric transit of Lb. plantarum strains at pH 2 248



4.6 In vitro simulated small intestinal transit of Lb. plantarum

strains at pH 8, without addition of bile salt

249

4.7 In vitro simulated small intestinal transit of Lb. plantarum

strains at pH 8, with addition of bile salt

249

5.1 Hematoxylin and eosin staining 250

xxi

5.2 Chromatogram of short chain fatty acids standard 251

5.3 Cholesterol standard curve 251

5.4 Effect of direct fed of Lb. plantarum strains on concentration of

acetic acid in faecal samples

252


propionic acid in faecal samples

252


iso-butyric acid in faecal samples

253


butyric acid in faecal samples

253


iso-valeric acid in faecal samples

254


n-Valeric acid in faecal samples

254


n-Caproic acid in faecal samples

255


heptanoic acid in faecal samples

255

6.1 Effect of direct fed of bacteriocins on concentration of acetic

acid in faecal samples

256

6.2 Effect of direct fed of bacteriocins on concentration of propionic


256

6.3 Effect of direct fed of bacteriocins on concentration of iso-

butyric acid in faecal samples

257

6.4 Effect of direct fed of bacteriocins on concentration of butyric


257

6.5 Effect of direct fed of bacteriocins on concentration of iso-

valeric acid in faecal samples

258

6.6 Effect of direct fed of bacteriocins on concentration of n-Valeric


258

6.7 Effect of direct fed of bacteriocins on concentration of n-

Caproic acid in faecal samples

259

6.8 Effect of direct fed of bacteriocins on concentration of heptanoic


259

xxii

xxii

LIST OF ABBREVIATION

AU Arbitrary unit

CFS Cell free supernatant

cm Centimetre

CFU Colony forming unit

CE Competitive exclusion

Da Dalton

d Day

oC Degree Celsius

ETB Enterobacteriaceae bacteria

EMB Eosin methylene blue medium

g G – force

GIT Gastrointestinal tract

GT Generation time

h Hour

HCl Hydrochloric acid

kg Kilogram

K2HPO4 di-Potassium hydrogen phosphate buffer

LAB Lactic acid bacteria

L Litre

LB Lubria medium

MRS De Man, Rogosa and Sharpe medium

mM Millimolar

mg Milligram

mL Millilitre

xxiii

mm Millimetre

min Minute

mol Mole

µg Microgram

µL Microlitre

NaCl Sodium chloride

NaOH Sodium hydroxide

nm Nanometre

OD Optical density

PBS Potassium buffered saline

% Percent

s Second

sp. Species

subsp. Subspecies

SCFA Short chain fatty acid

TS Tryptic soy medium

v/v Volume/volume

w/v Weight/volume

Documents

UNIVERSITI PUTRA MALAYSIA PROBIOTIC …psasir.upm.edu.my/id/eprint/9834/1/FBSB_2010_1_A.pdfinhibited the Pediococcus acidilactici 4-46, Enterococcus faecalis N-103, Enterococcus faecium