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
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Shuhaimi Mustafa, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(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
Universiti Putra Malaysia
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
Universiti Putra Malaysia
(Chairman)
Raha Abdul Rahim, PhD
Professor
Faculty of Biotechnology and Molecular Sciences
Universiti Putra Malaysia
(Member)
Mohd. Hair Bejo, PhD
Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(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
Universiti Putra Malaysia
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
4.2.6 Statistical analysis 86
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
4.4.1 Effect of in vitro upper gastrointestinal transit on viability
of Lb. plantarum strains
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
4.4.4 In vitro intestinal epithelial adhesion ability of Lb.
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
5.2.9 Statistical analysis 129
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
5.4.1 Growth performance of rats 147
5.4.2 Faecal and intestinal microflora 148
5.4.3 Intestinal villous height and crypt depth 153
5.4.4 Concentration of short chain fatty acids in faecal samples 155
5.4.5 Serum cholesterol level of rats 157
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
6.2.9 Statistical analysis 164
6.3 Results 165
6.3.1 Growth performance of rats 165
6.3.2 Microbial population in faecal and intestinal samples 165
6.3.3 Intestinal villous height and crypt depth 176
6.3.4 Concentration of short chain fatty acids in faecal samples 179
6.3.5 Serum cholesterol level of rats 179
xv
6.4 Discussion 182
6.4.1 Growth performance of rats 183
6.4.2 Faecal and intestinal microflora 184
6.4.3 Intestinal villous height and crypt depth 188
6.4.4 Concentration of short chain fatty acids in faecal samples 188
6.4.5 Serum cholesterol level of rats 189
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
7.2.1 Effect of in vitro upper gastrointestinal transit on viability
of Lb. plantarum strains
196
7.2.2 In vitro bile tolerance, deconjugation and cholesterol
assimilation ability of Lb. plantarum strains
198
7.2.3 In vitro intestinal epithelial adhesion ability of Lb.
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
5.5 Effect of direct fed of Lb. plantarum on the population of lactic acid
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
5.7 Effect of direct fed of Lb. plantarum on the population of lactic acid
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
6.5 Effect of direct fed of bacteriocins on the population of lactic acid
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
6.7 Effect of direct fed of bacteriocins on the population of lactic acid
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
3.4 Competitive exclusion assay of L. monocytogenes L55 and Lb.
plantarum strains: (a) RI11, (b) RG11 and (c) RG14
57
3.5 Competitive exclusion assay of S. typhimurium S1000 and Lb.
plantarum strains: (a) UL4, (b) TL1, (c) RS5
59
3.6 Competitive exclusion assay of S. typhimurium S1000 and Lb.
plantarum strains: (a) RI11, (b) RG11 and (c) RG14
60
3.7 Competitive exclusion assay of Staph. aureus S942 and Lb.
plantarum strains: (a) UL4, (b) TL1, (c) RS5
62
3.8 Competitive exclusion assay of Staph. aureus S942 and Lb.
plantarum strains: (a) RI11, (b) RG11 and (c) RG14
63
3.9 Competitive exclusion assay of E. coli E30 and Lb. plantarum
strains: (a) RI11, (b) RG11 and (c) RG14
65
3.10 Competitive exclusion assay of E. coli E30 and Lb. plantarum
strains: (a) RI11, (b) RG11 and (c) RG14
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.
plantarum strains: (a) UL4, (b) TL1, (c) RS5
237
3.2 Competitive exclusion assay of B. cereus B43 and Lb.
plantarum strains: (a) RI11, (b) RG11, (c) RG14
238
3.3 Competitive exclusion assay of L. monocytogenes L55 and Lb.
plantarum strains: (a) UL4, (b) TL1, (c) RS5
239
3.4 Competitive exclusion assay of L. monocytogenes L55 and Lb.
plantarum strains: (a) RI11, (b) RG11, (c) RG14
240
3.5 Competitive exclusion assay of S. typhimurium S1000 and Lb.
plantarum strains: (a) UL4, (b) TL1, (c) RS5
241
3.6 Competitive exclusion assay of S. typhimurium S1000 and Lb.
plantarum strains: (a) RI11, (b) RG11, (c) RG14
242
3.7 Competitive exclusion assay of Staph. aureus S942 and Lb.
plantarum strains: (a) UL4, (b) TL1, (c) RS5
243
3.8 Competitive exclusion assay of Staph. aureus S942 and Lb.
plantarum strains: (a) RI11, (b) RG11, (c) RG14
244
3.9 Competitive exclusion assay of E. coli E30 and Lb. plantarum
strains: (a) UL4, (b) TL1, (c) RS5
245
3.10 Competitive exclusion assay of E. coli E30 and Lb. plantarum
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
4.4 In vitro simulated gastric transit of Lb. plantarum strains at pH 3 248
4.5 In vitro simulated gastric transit of Lb. plantarum strains at pH 4 248
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
5.5 Effect of direct fed of Lb. plantarum strains on concentration of
propionic acid in faecal samples
252
5.6 Effect of direct fed of Lb. plantarum strains on concentration of
iso-butyric acid in faecal samples
253
5.7 Effect of direct fed of Lb. plantarum strains on concentration of
butyric acid in faecal samples
253
5.8 Effect of direct fed of Lb. plantarum strains on concentration of
iso-valeric acid in faecal samples
254
5.9 Effect of direct fed of Lb. plantarum strains on concentration of
n-Valeric acid in faecal samples
254
5.10 Effect of direct fed of Lb. plantarum strains on concentration of
n-Caproic acid in faecal samples
255
5.11 Effect of direct fed of Lb. plantarum strains on concentration of
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
acid in faecal samples
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
acid in faecal samples
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
acid in faecal samples
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
acid in faecal samples
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