ABSTRACT
This research work revealed the antimicrobial compounds present in lactic acid bacteria isolated from some traditionally fermented foods, ogi, ugba and yogurt and their antimicrobial activity against some selected food pathogens. A total of fifteen food samples consisting 5 each of ogi, ugba and yogurt were screened for lactic acid bacteria. Nine organisms were isolated from the food samples and characterized both phenotypically and genotypically using PCR amplification and 16SrRNA DNA sequencing which revealed that 2 of the isolates were Lactococcus spp, 5 Lactobacillus spp, 1 Bacillus spp and 1 Streptococcus spp. The genotypic characterization revealed that the lactic acid bacteria isolates Lactococcus lactissubsp lactis 0711XYBLS, Lactobacillus fermentum CS19, Lactococcus lactis and Lactobacillus fermentum were closely related at 99% evolutionary distance. Lactococcus lactis subsp lactis 0711XYBLS and Lactobacillus fermentum CS19 were utilized for the sole purpose of this research to determine their antimicrobial potentials against selected food pathogens. The lactic acid bacteria isolates produced antimicrobial metabolites diacetyl, hydrogen peroxide and lactic acid at varying degrees. The selected latic acid bacteria isolates were tested against food pathogens and their clear zones of inhibitions were noted. Lactococcus lactis subsp lactis 0711XYBLS isolated from ogi showed the highest Gram positive and Gram negative food pathogens ranging from 18.67 ± 1.15 to 12.67 ± 0.57 and 16.33 ± 0.56 to 8.00 ± 6.08 respectively where as Lactobacillus fermentum CS19 showed Antimicrobial activity for Gram positive food pathogens at 16.00 ± 1.00 to 8.67 ± 1.15 and 15.33 ± 1.15 to 8.67 ± 0.57 in Gram negative food pathogens. A great statistical significant difference was recorded between the pathogens at a significant level of p≤0.05. However, both organisms portrayed a great deal of antimicrobial activity against the selected food pathogens and can serve as novel antimicrobial agents. These potentials can be harnessed in food industries on a large scale as biopresevatives instead of the use of chemical preservation which may pose detrimental health risk on its consumers.
TABLE OF CONTENTS
Page
Title
Page i
Declaration ii
Dedication iii
Certification iv
Acknowledgements 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 Problem
Statement 3
1.3 Justification
of the Study 4
1.4 Aim
and Objectives of the Study 4
CHAPTER 2: REVIEW OF LITERATURE 6
2.1 Lactic
Acid Bacteria 6
2.2 Applications
of Lactic Acid Bacteria 8
2.3 Anti-Metabolites
from Lactic Acid Bacteria 15
2.3.1 Low molecular mass antimicrobial substances 17
2.3.2 High molecular mass antimicrobial substances 19
2.4 Bacteriocin-Producing
Lactic Acid Bacteria in Food Industry 23
2.4.1 Application of nisin in food industry 24
2.5 Empirical
Reviews of Antimicrobial Properties of Lactic
Acid Bacteria 25
CHAPTER 3: MATERIALS AND METHODS 31
3.1 Materials
31
3.1.1 Test
organisms 31
3.1.2 Collection
of fermented food samples 31
3.1.3 Sample and media preparation 31
3.2 Methods 31
3.2.1 Isolation of lactic acid bacteria from fermented food samples 31
3.2.2 Morphological and microscopic characterization of the bacteria 32
3.2.3 Gram staining 32
3.2.4 Biochemical characterization of the bacteria 32
3.2.5 Screening of lactic acid bacteria 34
3.2.6 Genotypic characterization of isolates 34
3.2.7 Determination of lactic acid produced by lactic acid
bacteria isolates 36
3.2.8 Determination of hydrogen peroxide production by lactic acid
bacteria isolates 37
3.2.9 Determination of diacetyl produced by the lactic acid
bacteria isolates 37
3.2.10 Evaluating antimicrobial
properties of different bacterial strains
using
the agar well diffusion test 38
3.2.11 Statistical
analysis 38
CHAPTER 4: RESULTS AND DISCUSSION 39
4.1 Results 39
4.1.1 Morphological, microscopic and biochemical identification
of isolates 39
4.1.2 Genotypic characterization of isolates 41
4.1.3: Screening for metabolites
produced by lactic acid bacteria 43
4.1.4 Antimicrobial activity of lactic acid bacteria isolates
against selected food pathogens 45
4.2 Discussion
47
CHAPTER 5: CONCLUSION AND
RECOMMENDATIONS 50
5.1 Conclusion 50
5.2 Recommendations
50
References 51
Appendices 61
LIST OF TABLES
Page
4.1 Phenotypic
characterization of isolates 40
4.2 Metabolites production by isolates 44
4.3 Antibacterial
activity of lactic acid bacteria isolates against selected
food borne pathogens 46
LIST OF FIGURES
Page
2.1 Anti-metabolites from lactic acid bacteria 16
4.1
Phylogenetic tree showing the
evolutionary distance between the
bacterial isolates 14
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF
THE STUDY
Food contamination, food poisoning
and food borne- illnesses is a major public health concern in developing
countries, hence the awareness about food safety and hygiene has increased over
the years. Every year 40,000 people die due to food borne diseases (WHO, 2015)
although the number of people that suffered from food borne diseases are higher
in the under developed and developing countries, the number is nonetheless
significantly large in the western countries, such as the USA (Rane,
2011). The common symptoms of these diseases include
vomiting, bloating, stomach ache, flatulence, and excessive fluid discharge
through feces. These diseases are due to wide range of pathogens which include
bacteria, virus, protozoa, and parasites. The popular ones are Salmonella spp, Vibrio cholera, Campylobacter,
Helicobacter pylori, Esherichia coli, Gardia lambia and many more (Newell et al., 2010).
The pathogens come in contact with
food during preparation and preserving time. Improper food preparation includes
cooking with contaminated water, improperly washed vegetables, fruits and raw
meat and fish with blood especially. Some of the pathogens die while cooking,
whereas some stay alive and cause disease in the enteric and other parts of the
body. Moreover, cooking for a short time also allows pathogens to grow and
strive in the food products. Also food in restaurants and particularly food
from road side are the most causes of food borne diseases (Rane,
2011).
In recent years, people have become
more aware of the functions “probiotic organisms”, particularly lactic acid
bacteria, play in fermented foods and the bioactive chemicals they generate. In
contrast, lactic acid bacteria have several antibacterial properties in
fermented foods. This is mainly due to the production of bacteriocins, organic
acids, ethanol, Hydrogen peroxide, diacetyl and reuterin (Cintas et al., 2001). The most common
anti-microbial agent produced by lactic acid bacteria is bacteriocin (Deegana et al., 2006). Lactic acid bacteria play
an important role in food industry by increasing nutritional values of food and
food safety (Adeyemo and Onilude, 2013). The antimicrobials produced by lactic acid
bacteria have been used widely as bio-preservatives and shelf life extender and
has found application in many industries and various commercial purposes.
During the recent years health-conscious consumers are looking for natural
foods without chemical preservatives that will fit in their healthy lifestyle.
Bio-preservation refers to extended shelf life and enhanced safety of foods
using microorganisms or their metabolites (Ross et al., 2002). In fermentations when lactic acid bacteria are
present, it not only promotes the sensory characteristics of the finished
product, but also the safety of the microorganisms (Leroy et al., 2004). Lactic acid bacteria generate lactic- and
acetic-acids, propionic, sorbic, benzoic-acids, hydrogen peroxide, diacetyl,
ethanol, phenolic- and proteinaceous-compounds as well as antibacterial
substances like bacteriocins.
However, some LAB strains can
produce antimicrobial substances other than bacteriocins. Increased interest in
developing natural antimicrobial agents that may help the safety of food items
has risen in recent years due of their unique bacteriocin-like inhibitory
substances (BLIS) capabilities. These antimicrobial compounds from lactic acid
bacteria lack amino acid sequence characterization while possessing necessary
bacteriocin capabilities. There has been much research on the bacteriocins produced
by lactic acid bacteria, and are generally regarded as safe (GRAS) since they
provide a useful new method of preventing pathogens in food. Perhaps preventing
microbial contamination by natural and microbiotic agents might prevent food
waste, which could in turn lead to reduced numbers of food poisoning cases
(Galvez et al., 2008). Lactic acid
bacteria have antagonistic effects on food borne pathogenic and spoilage
microorganisms example inhibition of Bacillus
subtilis which contaminates bread and causes spoilage, survival of Escherichia coli 0157:H7 in dairy
products which is a potential health hazards because of the link with dairy
cattle and raw milk (Saad et al.,
2001).
Lactic acid bacteria constitute a
group of genus that has the following common features: cocci, rods and a basic
composition of DNA below 50 mol% G+ C. Most of them are Gram-positive, mesophilic,
and can grow at temperatures between 5°C and 45°C, provided that they have
access to oxygen. In addition, they are unable to oxidize or break down
nitrates and do not produce indole or hydrogen sulfide. The group is made up of
several different types of bacteria, such as Lactobacillus, Lactococcus, Leuconostoc, Carnobacterium, Streptococcus,
Enterococcus, Aerococcus, Bifidobacterium, and Pediococcus (Doyle et al.,
2006). It is recognized that lactic acid bacteria serve several roles, such as
antitumor activity, lowering cholesterol, reducing lactose intolerance,
activation of the immune system, and keeping infections out (Reid, 2006).
Based on these functions, different
kinds of Lactic acid bacteria have been developed as probiotics, and the market
volume of probiotics has rapidly increased. In recent years Lactic acid
bacteria have been playing important role in the food and feed fermentation and
preservation either as the natural microflora or as starter culture added under
controlled conditions. This is due to the fact that they have been recognized
as GRAS (Generally Recognized as Safe) microorganisms (Reid, 2006).
1.2 PROBLEM
STATEMENT
Infections
caused by microbes that contaminate the food supply are a frequent reminder of
the complex food web that links us with animal, plant and microbial populations
around the world. While all these are at risk, the consequences are most servers in the vulnerable populations of the
very young, elderly and those with comprised immune serotypes typhi are sustained
in human reservoirs and contaminate the food supply via the excrete of infected
humans. Some pathogens persist in the environment, or in multiple host, and can
contaminate the food we eat via pathways that reflect the variety of ecosystem
that make up our food supply microbial contaminations of food can be due to raw
materials which are naturally contaminated by microorganisms originating from
air, water, soil, animal/human carrier or linked to food processing and work
environment. Deficient handling and manufacturing processes or practices can
contribute to an increase in the presence of microbial indicators such as lack
of hygiene and sanitation. Improper food production procedures have induced
microbial proliferation, biofilm formation and cross-contamination with
consequences on both microbiological safety and quality of food. These
phenomena have resulted in reduction of shelf-life, food spoilage and spread of
food borne pathogen the most common pathogen causing food borne disease in
humans belonging to genera Salmonella,
Campylobacter, Vibro, Clostridium, besides some specific species such as Listeria monocytogenes, Bacillus cereus,
Yersina enterocolitica, and Escherichia
coli. However, there is a striking need to device means of identifying
these pathogens in food and also strategizing possible means of eliminating
them in food for the safety of both humans and animals in general.
1.3 JUSTIFICATION
OF THE STUDY
The food
borne out breaks that have occurred in the last decades highlighted the importance
of development and implementation of preventive measures aimed at reducing
biological hazards. The identification of new food safety problems can be
accelerated by important improvement in surveillance and response. These new
surveillance tools captures information about infections in humans and animals
and contamination of food, providing important information that is integrated
across sectors. Lactic acid bacteria found in food may serves as bacterocin
potentially promoting host specific health due to their probiotic nature.
Lactic acid bacteria predominate the micro flora of fermented food which
produces an array of antimicrobial substances such as organic acid, diacetyl
acetone, hydrogen peroxide, reuterin and bacterocin. The antimicrobial produced
makes them suitable for food preservation. The study is however carried out to
ascertain the full benefit of Lactic acid bacteria found in food and the best
way of utilizing them in promoting human health.
1.4 AIM AND
OBJECTIVES OF THE STUDY
The aim of
the study is to evaluate the antibacterial activity of LAB against food borne pathogens.
Specific objectives of the study include:
1. Isolation and identification of LAB
based on physiological, biochemical and 16SrRNA sequence analysis.
2. Determination of the antimicrobial
activity against some selected food borne pathogens (Shigella spp, Staphylococcus aureus, Eschericha coli, Bacillus cereus,
Pseudomonas aeruginosa, Salmonella typhi.).
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