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ANTIBACTERIAL ACTIVITY OF LACTIC ACID BACTERIA AGAINST FOOD BORNE PATHOGENS

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Product Category: Projects

Product Code: 00007156

No of Pages: 110

No of Chapters: 1-5

File Format: Microsoft Word

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ABSTRACT

This research work revealed the antimicrobial compounds present in lactic acid bacteria isolated from some traditionally fermented foods, ogiugba and yogurt and their antimicrobial activity against some selected food pathogens. A total of fifteen food samples consisting 5 each of ogiugba 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 Lactobacillufermentum 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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