Extraction And Quantification Of Lactic Acid Bacteria Bacteriocins From ‘Ogiri’; A Locally Fermented Food

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