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                                                                                                      28

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

LIST OF TABLES

CHAPTER ONE

1.1        INTRODUCTION

Lactic acid bacterial are important organisms recognized for their fermentative ability as well as their health and nutritional benefits (Adenike et al., 2007). They produce various compounds such as bacteriocin or bacteriocidal proteins during lactic acid fermentation (Moshood and Yusuf, 2013). Lactic acid bacteria are Gram positive bacteria, with low guanine and cytosine content, acid tolerant, non-sporulating, nonrespiring rod or cocci that are associated by common metabolic and physiological characteristics. Bacteriocins are produced by several Lactic acid bacteria strains and this is to the disadvantage of other spoilage and pathogenic microorganisms.

Bacteriocins (natural bio-preservatives) are proteinaceous toxins produced by antagonistic microorganisms to    inhibit or destroy undesired microorganisms in foods to enhance food safety and extend shelf life. Using bio preservatives in foods compared to chemical additives ensures natural, fresher and minimally processed foods. Fermented foods like buttermilk, curd, cheese, Koozh, which are lactic acid bacteria fermented products, have bacteriocins in themselves due to fermentation. Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). Bacteriocins are a heterogeneous group of anti-bacterial proteins that vary in spectrum of activity, mode of action, molecular weight, genetic origin and biochemical properties. Significantly however, the inhibitory activity of these substances is confined to Gram-positive bacteria and inhibition of Gram- negatives by these Bacteriocins has not been demonstrated, an observation which can be explained by a detailed analysis and comparison of the composition of Gram-positive and Gram-negative bacterial cell walls .In both types the cytoplasmic membrane which forms the border between the cytoplasm

and the external environment, is surrounded by a layer of peptidoglycan which is significantly thinner in Gram-negative bacteria than in Gram- positive bacteria. Gram-negative bacteria possess an additional layer, the so-called outer membrane which is composed of phospholipids, proteins and lipopolysaccharides (LPS), and this membrane is impermeable to most molecules. Nevertheless, the presence of Porins in this layer will allow the free diffusion of molecules with a molecular mass below 6ooDa. The smallest Bacteriocins produced by lactic acid bacteria are approximately 3kDa and are thus too large to reach their target, the cytoplasmic membrane. However, scientists have demonstrated that Salmonella species and other Gram-negative bacteria become sensitive to Nisin after exposure to treatments that change the permeability barrier properties of the outer membrane Lactic acid bacteria (LAB) are among the most important groups of microorganisms used in food fermentation where they play an essential role and a wide variety of strains are routinely employed as starter cultures in the manufacture of dairy, meat, vegetable and bakery products (Noopur et al., 2010; Hassanzadazar and Ehsani, 2013). One of the most important contributions of these microorganisms is the extended shelf life of the fermented products. Growth of spoilage and pathogenic bacteria in these foods is inhibited due to competition for nutrients and the presence of starter derived inhibitors such as lactic acid, hydrogen peroxide, diacetyl and bacteriocins (Noopur et al., 2010; Noordiana et al., 2013). Bacteriocins are extracellularly produced primary compounds of bacterial ribosomal synthesis which have a relatively narrow spectrum of bactericidal activity. They are active against other bacteria despite varying greatly in the chemical nature and mode of action. Bacteriocins have important advantage over the classical antibiotics in being easily degraded by the digestive enzymes without the risk of disruption of normal tract ecology. Bacteriocin producing LAB have the ‘generally recognized as safe’ (GRAS) status and have been shown to strengthen the barrier function of the gut microflora as well as promote the non-specific enhancement of the immune system of man and animals (Tome et al., 2008). Equally, research on the biochemical changes during the fermentation as well as the proximate composition and properties of the seeds have also received modest scientific attention (Aremu et al., 2007 and Odibo et al., 2008).

There is presently, paucity of scientific information on the ecological contribution of the LAB and bacteriocins for the safety and biopreservation of the food condiments. Bacteriocins are antimicrobial peptides or proteins produced by strains of diverse bacterial species. The antimicrobial activity of this group of natural substances against foodborne pathogens, as well as spoilage bacteria, has raised considerable interest for their application in food preservation (Noopur et al., 2010; Gong et al., 2010; Ana, 2012). In the past years, a lot of work has aimed to detect, purify and characterize bacteriocins, as well as their application in food preservation strategies. Application of bacteriocins may help reduce the use of chemical preservatives and/or the intensity of heat and other physical treatments, satisfying the demands of consumers for foods that are fresh tasting, ready to eat, and lightly preserved. In recent years, considerable effort has been made to develop food applications for many different bacteriocins using bacteriocinogenic strains (Ana, 2012; Adenike et al., 2007). Iman et al., (2014) focused in their study on the isolation and characterization of bacteriocin producing local lactic acid bacteria isolates, beside the activity of these strains against several spoilage and pathogenic bacteria, choosing the best isolate which has the best antibacterial activity.

As a result of the extent in which to which “Ogiri” is consumed, it is imperative to know how its preparation process is carried out, the step by step approach. “Ogiri” is prepared mainly locally, it becomes imperative to know the conditions, sanitary environment, whether it’s exposed to

contamination during the process of preparation and preservation. This study therefore examines the microbial quality of processed ‘Ogiri’. ‘’Ogiri‟ is the name used by igbos for the traditionally prepared fermented condiments based on vegetable proteins. It is obtained by fermenting melon seeds (Citrullus vulgaris), fluted pumpkin (Telferia occidentallis) and castor oil seeds (Ricimus communis) [4].These raw materials are used to create the different varieties of „ogiri‟ such „ogiri-egusi,‟ „ogiri-ugu‟, „ogiri-isi‟ and „ogiri-okpiye‟. Hence this work is aimed at evaluating the microbial quality in processed ‘’Ogiri’’.

1.2       Aims and Objectives

The aim of this study was to extract and quantify the lactic acid bacteriocins from ‘Ogiri’ a locally fermented food

The objectives are;

·       To isolate lactic acid bacteria from ‘Ogiri’ sample

·       To characterize the lactic acid bacteria involved in the fermentation process

·       Extraction of crude bacteriocin

·       Quantification of the bacteriocin activity

·       To determine the Proximate analysis of Ogiri samples

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