ABSTRACT
This study aimed at isolating and identifying LAB from traditionally fermented foods in Nigeria (ogi, kunu zaki and akpu) and to evaluate their probiotic potentials invitro. Nine lactic acid bacteria were isolated from the selected sources. These isolates were then screened for their phenotypic and biochemical properties. The selected isolates were then identified molecularly using the 16SrRNA sequencing as Bacillus spp (3), Lactobacillus fermentum (3 strains) and Lactococcus lactis (2 strains) and their partial sequence were deposited in the Gene Bank. Among the LAB isolates that were identified, 3 isolates Lactobacillus fermentum CS19, Lactococcus lactis, and Lactobacillus fermentum exhibited invitro safety attributes and were able to survive inhibitory conditions like acid environment and 0.3%bile salt. Furthermore, they demonstrated bactericidal activity against some selected drug-sensitive and multi-drug resistant pathogens (Salmonella typi, Escherichia coli and Staphylococcus aureus) capable of causing infection in both gastro intestinal tract and skin. Lactobacillus fermentum CS19 demonstrated the highest antibacterial activity against the selected food pathogens with the highest activity being recorded Salmonella typhi (15.33±1.52mm) and Staphylococcus aureus (15.33±1.15mm). Among the selected isolates Lactobacillus fermentum CS19 demonstrated highest hydrophobicity and adhesion to mucin. However, this study suggests that the LAB isolates Lactobacillus fermentum CS19, Lactococcus lactis, and Lactobacillus fermentum are good probiotic strain and could be introduced in starter cultures in traditionally feremented foods.
TABLE OF CONTENTS
Page
Title
Page i
Declaration
ii
Dedication
iii
Certification
iv
Acknowledgments
v
Table
of Contents vi
List
of Tables ix
List
of Figures x
Abstract
xi
CHAPTER 1:
INTRODUCTION 1
1.1 Background of the Study 1
1.2 Statement of Problem 3
1.3 Justification of the Study
3
1.4 Aim
and Objectives of the Study 4
CHAPTER 2:
LITERATURE REVIEW 5
2.1 Lactic
Acid Bacteria 5
2.1.1 Classification
of lactic acid bacteria 6
2.1.2 Importance
of lactic acid bacteria 6
2.2 Probiotics 10
2.2.1 History
of probiotics 11
2.2.2 Essential
characteristics of probiotics 12
2.2.3 Mechanism of action 13
2.3 Criteria
for Choosing Probiotic Strains 16
2.4 Mucin
Biosynthesis 21
2.5 Interactions
of Lactic Acid Bacteria with Intestinal
Glycoconjugates 24
2.6 Mucin
Adhesion Factors and Adhesion Mechanisms in
Lactic
Acid Bacteria 26
2.7 Cell
Wall Anchored Proteins 28
2.8 Probiotics
and Gut Health 31
2.8.1 The gut microbiome 31
2.9 Probiotic
Attributes of Lactic Acid Bacteria 35
2.9.1
Acid tolerance 35
2.9.2 Bile
tolerance 36
2.9.3 Adherence property 38
2.9.4 Adhesion mechanisms of probiotics to
intestinal mucosa 40
2.9.5 Antagonism against pathogens 44
CHAPTER 3: MATERIALS AND METHODS 48
3.1 Materials 48
3.1.1 Sample collection 48
3.1.2
Isolation and screening media 49
3.1.3 Chemicals and reagents 49
3.1.4 Equipment resources 49
3.2 Methods 49
3.2.1 Isolation of LAB 50
3.2.2 Purification and storage of
cultures 50
3.2.3 Identification and
characterization of isolated LAB strains 53
3.2.4 Evaluation
of invitro probiotic characteristics of isolated
LAB strains 57
3.2.5
Auto-aggregation assay 58
3.2.7 Statistical analysis 60
CHAPTER 4: RESULTS
AND DISCUSSION 61
4.1 Results 61
4.1.1 Morphological
identification of isolates 61
4.1.2 Antibacterial activity of LAB isolates 63
4.1.3 Exopolysaccharide production of LAB isolates
65
4.1.4 Probiotic
characteristics of LAB isolates 67
4.1.5 Cell hydrophobicity of LAB isolates 69
4.1.6 Autoagreggation of isolates 71
4.1.7 Adherence properties of LAB isolates 73
4.1.8 Genotypic characterization results 75
4. 2 Discussion 78
CHPATER 5:
CONCLUSION AND RECOMMENDATIONS 81
5.1 Conclusion 81
5.2 Recommendations 81
References 83
Appendices 91
LIST OF TABLES
Page
2.1
The probiotic effect of LAB on the human host 9
2.2
Established health benefits with their mode of action 15
2.3
Desirable criteria for the selection of probiotics in
commercial
applications 18
2.4 Some receptors along with their
functions 39
4.1 Phenotypic Characterization of Isolates 62
4.2
Antimicrobial activity of LAB isolates 64
4.3 Exopolysaccharide production of LAB
isolates 66
4.4
Probiotic chracteristics of LAB isolates 68
4.5: Hydrophobicity of organisms 70
4.6 Autoaggregation of isolates 72
4.7 Adherence of LAB isolates to mucin 74
LIST OF FIGURES
Page
2.1 Guidelines
for the evaluation of probiotics for food use 20
2.3 Polymeric structure of mucin molecules 22
2.4 Cell
surface architechture of gram positive bacteria 27
2.5a Composition across the length of the
gastrointestinal tract 32
2.5b Longitudinal variations in microbial
compositions in the intestine 33
2.6 Schematic representation of
anti-adhesive properties of probiotics
and
prebiotics. 47
4.1 Phylogenetic tree showing the
evolutionary distance between
the
bacterial isolates 76
4.2 Agarose gel electrophoresis of the
16SrRNA gene
of some selected
bacterial isolates. 77
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
The ability of a
probiotic to adhere to the luminal epithelium and colonize the human
gastrointestinal system is considered a requirement for probiotic selection in
order to give health benefits and protection against gastrointestinal disorders
after ingestion (Gupta and Sharma, 2015). Adhesion is the effective attachment
of microorganisms to the intestinal epithelial cells to minimize or prevent
colonization of infections (Kesarcordi et
al., 2008). Probiotic strains must be able to travel through the upper
gastrointestinal tract (GIT) unharmed by colonizing and sticking to mucin or
epithelial cells originating from the gut and tolerating stomach acidity and
bile salt concentrations in order to generate therapeutic effects (Jamaly et al., 2011). Antibiotic capabilities
against enteropathogens through the synthesis of antimicrobial compounds, toxin
breakdown, nutritional competition, and immune system regulation are some of
the proposed mechanisms for ingested probiotic microorganisms to deliver potential
advantages to their host (Hirayama and Rafter, 2000; Rinkien et al., 2000).
However, among
other selection yardsticks for probiotic microorganisms, attachment
of lactic acid bacteria to epithelia of the intestine is regarded as crucial.
Beneficial microorganisms' ability to assemble and stick together to surfaces
such as epithelial chamber and mucosal layer aids in successful gut
colonization and the establishment of a barrier that prevents enteropathogenic
organisms from establishing an infection (Tareb et al., 2013).
According to Rozen
et al. (2004) carbohydrate
constituents on the mucosal layer are responsible for lactic acid bacteria
adhesion properties. Lactic acid bacteria create exopolysaccharides, which are
known to aid in adherence to surfaces. Exopolysaccharides are exocellular
polymers found on the surface of several lactic-producing strains and are
currently utilized as a preservative in the production of fermented dairy
products, source of natural thickener and stabilizing ingredients, and a number
of strains of Lactobacillus and Bfidobacteriun are responsible for
production of these polymers (Landerbjo et
al., 2002).
Microorganisms
have developed a wide spectrum of hydrophobic substances known as adhesins on
their cell surface, including proteins and less typically polysaccharide,
teichoic, and fatty acids, that enable them to adhere covalently to the surface
of gastrointestinal epithelial cells and mucosa (Gupta and Sharma, 2015). These
hydrophobic residues are the most important determinant in the adhesive
reaction between the organism and a suitable substrate, such as carbohydrate residues
on the epithelial surface, such as mannose, galactose, fructose, or
glycolipids, and the formation of biofilms by bacteria on both animate and
inanimate surfaces (Abdulla et al.,
2014). The hydrophobicity response is triggered by a complicated interaction
between negatively charged, positively charged, hydrophobic, and hydrophilic
components of bacterial surfaces.
As a result, the
purpose of this study is to see if autoaggregation ability, cell surface
adhesion, hydrophobicity, and exopolysaccharide productivity tests can be used
to screen lactic acid bacteria isolated from fermented traditional foods for
adhesive properties suitable for commercial and industrial applications.
1.2 STATEMENT OF PROBLEM
Several food-borne
diseases are caused by the growth of pathogenic microorganisms in the food.
There is an elevated proclivity to infections of the gastrointestinal tract among
children and adult due to the infancy of the gut
defense obstacle, between the internal and external environments and the
constant challenge from potentially pathogennic factors in the external
environment.
More specifically,
the mucus coating epithelia cells is one of the first surfaces encountered by
infected germs in the human gut, and it's thought to be a key site for
bacterial adherence and colonization. Mucus is constantly degraded, yet new
mucin glycoproteins are constantly produced. Bacteria that can attach to mucus
but not reach epithelial cells may thus be removed from the mucosal surface by
destroyed mucin and washed away with luminal contents. This could help to
explain why most probiotic bacteria have a transitory colonization pattern.
Again, in the
context of the food industry, there is a need for the choosing of beneficial
microorganisms that can be applied as probiotics and starter cultures in
various fermentation processes, as well as essential criteria that form the
foundation for the formulation of nutraceutical products that provide human
health benefits.
1.3 JUSTIFICATION OF THE STUDY
Lactic acid
bacteria (LAB) can be established in a variety of areas, including human and
animal mucosal surfaces, in addition fermented foods. Many types have been
employed in food bioprocessing and are also known as probiotic organisms, which
offer a variety of health benefits. However, further information is needed on
the ability of isolated probiotic strains to overcome the stomach environment,
the existence of bile salts, and to resist the flux of gastrointestinal content
by "adhesion".
Attachment to host
tissue is required for various gastrointestinal passages, however a competitive
exclusion paradigm based on binding site competition has evolved. As a result,
determining the adhesion potentials of probitic Lactic acid bacteria isolates
is critical in the development of functional foods and nutraceuticals based on the use of probiotics to improve the well-being of
the gut. Therefore, the focus of this study will be on how probiotic lactic
acid bacteria strains attach to gastric mucus and epithelial cell lines, in
addition to how well they colonize the human gut.
1.4 AIM
AND OBJECTIVES OF THE STUDY
The
purpose of this research is to assess the probiotic properties of prospective
Lactic Acid Bacteria (LAB) strains from traditional fermented foods.
Specific objectives are;
1. Isolation,
screening and characterization of LAB isolates from traditionally fermented
foods.
2. Screening
of LAB isolates for probiotic characteristics.
3. Examination
of surface adhesion properties of isolated LAB.
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