ABSTRACT

This study was carried out to extract and quantify the lactic acid bacteriocins from ‘Ogiri’ a locally fermented food. A total of nine (10) samples of Ogiri were purchased from local vendors who hawk the product around the Umuahia market. The serial dilution technique was employed in the inoculation of the Ogiri samples of which each of the samples was diluted in the 10-fold serial dilution technique. 0.1ml of the respective dilution (10-6 and 10-7) were plated on the various agar plates and evenly spread over the entire plate using a flame sterilized glass rod. The inoculated plates were incubated at 35°C for 48hrs. Discrete colonies from the culture plates were picked with sterile wire loop and inoculated onto freshly prepared MRS agar plates. The pure isolates were identified following a four-step analysis the steps employed are cultural examination, microscopic examination, biochemical reaction and sugar utilization test. In the present investigation, the five LAB isolates obtained from various fermented food samples were identified after morphological, biochemical and sugar fermentation tests as: Bacillus subtilis, Bacillus lichenformis, L. plantarum, Enterobacter species and Streptococcus species. The lactic acid bacterial isolates (Bacillus subtilis, Bacillus lichenformis, L. plantarum, Enterobacter species and Streptococcus species) were tested for antibacterial activity against Escherichia coli and Staphylococcus aureus which served as test organisms The inhibitory effect demonstrated by the lactic acid bacterial isolates against these bacteria is an indication of possession of antibacterial activity. Various factors seemed to affect bacteriocin production as well as its activity. Maximum activity was noted at pH 2, and temperature 50°C. Bacteriocin production was strongly dependent on pH and temperature. The production of these bacteriocins was much higher at 50 ºC than at 40ºC, which suggested that the growth temperature also played an important role. Proximate Analysis of Ogiri Samples was carried out using the method described by Association of Official Analytical Chemist (AOAC). The result of the proximate analysis indicated that the dry matter content of the samples was high with a percentage value of (52.09%), followed by the moisture content  of (38.02%), fat content of (28.57%), protein content of (12.22%), carbohydrate content of (11.7%), fibre content of (9.39%) and Ash content of (7.41%). This study concluded that the bacteriocin antibiotic produced by lactic acid bacterial isolates demonstrated inhibitory effects against bacterial indicator organisms.

TABLE OF CONTENTS

Title          Page

Title Page   i

Declaration ii

Certification iii

Dedication iv

Acknowledgement v

Table of Contents vi

List of Tables ix

Abstract x

CHAPTER ONE

1.1 Introduction 1

1.4 Aims and Objectives 4

CHAPTER TWO

2.0 Literature Review 5

2.1 Lactic Acid Bacteria 6

2.2 Bacteriocins 7

2.2.1 Range Of Activity 8

2.3 Classification of Bacteriocins 9

2.3.1 Class I 9

2.3.2 Class II 10

2.3.3 Class III 11

2.3.4 Nisin 11

2.4 Bacteriocin-Like Substances 12

2.5 Isolation and Purification 13

2.6 Methods of Purification 14

2.6.1 Purification of Class I Bacteriocins 16

2.6.2 Purification of class II bacteriocins 17

2.6.3 Purification of class III bacteriocins 20

2.7 Bacterial Resistance To Bacteriocins 20

CHAPTER THREE

3.0 Materials and Methods 22

3.1 Sample Collection 22

3.1.1 Sterilization of Materials 22

3.1.2 Normal Saline Preparation 22

3.1.3 Media Preparation for Isolation of Lactic Acid Bacteria from the Ogi 

Samples 22 

3.2 Isolation of Lactic Acid Bacteria 23

3.2.1 Sub-culturing 23

3.3 Characterization and Identification of Lactic Acid Bacterial Isolates 23

3.3.1 Gram staining techniques 23

3.3.2 Motility test 24

3.3.3 Catalase test 24

3.3.4 Coagulase test 24

3.3.5 Methyl red test 25

3.3.6 Voges-proskaeur test 25

3.3.7 Indole test 26

3.3.8 Citrate test 26

3.3.9 Oxidase test 26

3.4 Extraction of Crude Bacteriocin 26

3.4.1 Determination of Bacteriocin Activity 27

3.4.2 Determination of the Effects of pH on the Activity of the Crude 

Bacteriocin Extract 27

3.4.3 Determination of the Effects of Storage Temperature on the Activity of the  Crude Bacteriocin

3.4.4 Indicator Bacterial 28

3.5 Quantification of the Bacteriocin Activity 28

3.5.1 Nisin Standardization Protocol 29

3.5 Proximate Analysis Of Ogiri Samples 29

3.5.1 Moisture Content Determination 29

3.5.2 Total Ash Determination 29

3.5.3 Crude Protein Determination 30

3.5.4 Fat Content Determination 31

3.5.5 Crude Fibre Determination 31

3.5.6 Carbohydrate Determination 32

CHAPTER FOUR

4.0 Results 33

4.1 The Total Viable Bacterial Count from Ogiri Samples 33

4.2 The Morphological Identification of Bacterial Isolates From Fermented Ogiri 33

4.3 The Biochemical Characterization of Lactic Acid Bacterial Isolates From

Fermented Ogiri 33

4.4 The Morphological Identification of Indicator Bacteria 33

4.5 Biochemical Characterization of the Indicator Bacteria 34

4.6 Antagonistic Activities of Bacteriocin Produced By Different Lactic Acid 

Bacterial Isolates 34

4.7 Quantification of the Bacteriocin Produced By Lactic Acid Bacteria Isolates 34

4.8 The Effect of pH On Antimicrobial Activity of Lactic Acetic Bacterial (Lab)

Bacteriocin 34

4.9 The Effect of Temperature on Antimicrobial Activity of Lactic Acetic Bacterial

(Lab) Bacteriocin 34

4.10 The Proximate Analysis of Fermenting Ogiri Samples 35

CHAPTER FIVE

5.0 Discussion and Conclusion 46

5.1 Discussion 46

5.2 Conclusion 47

References 48