AN ASSESSMENT ON ANTIBACTERIAL ACTIVITTY OF EXOPOLYSACCHARIDES PRODUCING LACTIC ACID BACTERIA FROM NUNU

ABSTRACT

Several strains of lactic acid bacteria secrete extracellular polysaccharides in favourable environment such as milk. Exopolysaccharides (ESP) is used to describe extracellular polysaccharides either attached as capsule with bacterial cell wall or liberated into the medium as ropy polysaccharides. LAB exopolysaccharides is economically important because it impacts functional effects on food and confer beneficial health effects on the consumers such as antioxidant, cholesterol lowering effects and immune modulation. Lactic acid bacteria have been used for thousands of years in dairy products and food industries, they play crucial role in food fermentation processes. A wide variety of strains are routinely used as starter cultures to manufacture dairy products. These bacteria produce Lactic acids, hydrogen peroxides and several enzymes during fermentation. Growth of spoilage and pathogenic bacteria in the fermented food are inhibited due to the production of antimicrobial substances by LAB as their competition for nutrients. Test confirmed all the isolates to be gram positive, catalase negative, non-motile and non-spore forming rods and cocci in shape. Carbohydrates fermentation test confirmed Lactobacillus acidophilus, Lactococcus lactis, Lactobacillus fermentum, Leuconostoc messentoriodes as the probable organisms. These LAB all produced exopolysaccharides with mucoid and ropy colonies. These exopolysaccharides produced were used for the antimicrobial activity against staphylococcus aureus and Escherichia coli. The results showed zones of inhibition on EXO A, EXO L, EXO M and EXO F for Staphylococcus aureus as 18mm, 28mm, 25mm, 20mm and for   E. coli 20mm, 25mm, 30mm, and 22mm respectively which implied the ESP inhibited the growth of the test organisms.  

TABLE OF CONTENTS

Title page                                                                                                                                i

Certification                                                                                                                            ii

Dedication                                                                                                                               iii

Acknowledgement                                                                                                                  iv

Table of content                                                                                                                      v

List of table                                                                                                                             vii

Abstract                                                                                                                                   viii

CHAPTER ONE:      INTRODUCTION

1.2       Lactic Acid Bacteria (Lab)                                                                                         1

1.3       Aim and Objectives of Study                                                                                     2

CHAPTER TWO:    LITERATURE REVIEW

2.1       Fermented Foods                                                                                                        3

2.2       Purpose and Benefits of Food Fermentation                                                                                                                                      3

2.3       Lactic Acid Bacteria (Lab)                                                                                         4

2.4       What Is Lactic Acid Bacteria?                                                                                    4

2.5       History of Lab                                                                                                             5

2.6       Exopolysccharides                                                                                                      6

2.7       Exopolysaccharide Producing Lab                                                                             7

2.8       Types of Exopolysaccharide                                                                                      7

2.9       Exopolysaccharide Production, Isolation And                                                          

Characterization                                                                                                         8

CHAPTER THREE:     MATERIALS AND METHODS

3.1       Source of Material                                                                                                      9

3.2       Sample Preparation                                                                                                    9

3.3       Media Preparation                                                                                                      9

3.4       Preparation of MRS Broth                                                                                          10

3.5       Isolation of LAB                                                                                                         10

3.6       Colony Count of Isolates                                                                                            11

3.7       Identification of Isolates                                                                                             11

3.8       Determination of Colony Features                                                                             12

3.9       Microscopy                                                                                                                 12

3.10     Gram Staining                                                                                                            12

3.11.1  Catalase Test                                                                                                              13

3.11.2 Motility Test                                                                                                               13

3.11.3  Coagulase Test                                                                                                           13

3.12     Growth at Different Temperature                                                                              14

3.13     Growth at Different _PH                                                                                              14

3.13.1 Growth at _PH 3                                                                                                            14

3.13.2 Growth at PH 9                                                                                                            14

13.14   Salt Tolerance Test                                                                                                     15

3.15     Sugar Fermentation Tests                                                                                           15

3.16     Test Organisms                                                                                                           15

3.17     Anti-Bacterial Activity of EPS Producing Lab                                                          15

3.18     Screening for ESP Production                                                                                    16

3.19     Extraction of Exopolysaccharides                                                                              16

CHAPTER FOUR:      RESULTS                                                                                          17

CHAPTER FIVE:     DISCUSSION, CONCLUSION AND RECOMMENDATION       

5.0       Discussion                                                                                                                   22

5.1       Conclusion                                                                                                                  23

5.2       Recommendation                                                                                                       23

REFERENCES                                                                                                                       24

LIST OF TABLES

TABLE                           TITLE                         PAGE

Table 4.1:        Morphological, Physiological and Biochemical Characteristics of Isolated Genera of LAB                                                18

Table 4.2:        Carbohydrates Fermentation of Isolated Genera of LAB                                    19

Table 4.3         Shows the Lactic Acid Bacteria Produced                                                     20

Table 4.4:        Showing the Antibacterial Activity of Exopolysaccharides 

Producing LAB on Some Selected Pathogens                                               21

CHAPTER ONE

INTRODUCTION

Nunu is a spontaneously fermented milk (yoghurt-like) product in Nigeria and other west Africa including Ghana and Burkinafaso. Unlike other African fermented milk products where milk of goats, sheep, and carmels is used. Nunu is solely prepared from cow milk. The traditional processing of Nunu involves collecting fresh cow milk into containers and then allowing it to ferment for a day or two days at ambient temperature. Nunu is yoghurt -like in taste (a sharp acid taste) and it can be taken alone or with Fura. (Owusu-Kwarteng et al., 2012 ). Like many other spontaneously fermented foods in Africa, the production of Nunu is largely home based and the fermentation is spontaneous.

1.2 LACTIC ACID BACTERIA (LAB)

  Lactic acid bacteria (LAB)are Gram positive, fastidious, acids tolerant, generally nonsporullating, catalase negative, devoid of cytochrome, and non-respiring rods or cocci that are associated by their common metabolic and physiological characteristics that produce lactic acid as a major sole product of fermentative metabolism ( Holzapfel et al., 2001). Lactic acid bacteria have been used for the fermentation of food and feed products since ancient days and today their major applications are still in the food and feed industry as starter cultures  (Boomee et al., 2003).

Lactic acid bacteria produce variety of antimicrobial compounds such as ethanol, formic acid, acetone, hydrogen peroxide, diacetyl and bacteriocins which confer preservatives ability on them as a natural competitive means to overcome other micro-organisms sharing the same niche (Oliveira et al., 2008). Recently, there has been a great demand for lactic acid as it can be used as a monomer for the production of the biodegradable polymer polylactic acid (PLA), which is an alternative to synthetic polymers derived from petroleum resources (Datta et al., 1995).

Lactic acid bacteria are food grade organisms, possessing the generally recognised as safe (GRAS) status, and can secrete exopolysaccharide (EPS). LAB ESP has economically important functional effects to the consumer (Welman and Maddox,  2003).

LAB have arisen a great deal of attention as a novel approach to control pathogens in foodstuffs. Bacteriocins are antimicrobial proteinaceous compounds that are produced by both Gram positive and Gram negative bacteria. The antimicrobial effect of LAB has been appreciated by man for more than 10,000 years and has enabled him to extend the shelf life of many foods through fermentation processes. Reports from literatures have shown and indicated important health benefits of ESP to include antioxidant, cholesterol lowering effects and immune modulation

1.3       AIM AND OBJECTIVES OF STUDY

The aim of this project work was to study the antibacterial activity of exopolysaccharide producing lactic acid bacteria from nunu against some pathogenic bacteria.

Objectives of the study  

·       To isolate lactic acid bacteria from Nunu.

·       To characterize and identify the LAB isolates from nunu.

·       To extract and to screen the EPS from the LAB isolates.

·       To determine the activity of the EPS against selected bacteria species including E. coli and Staphylococcus aureus.

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