INVITRO INHIBITORY EFFECT OF BACTERIOCIN FROM LACTIC ACID BACTERIA FROM UGBA (PENTACLETHRA MACROPHYLA BENTH) AGAINST CARROT (DAUCUS CARROTA) SPOILAGE BACTERIA

ABSTRACT

This work studied the in vitro inhibitory effect of bacteriocin from lactic acid bacteria (LAB) from Ugba against spoilage bacteria of carrot (Daucus carrota). Seven bacteria species were isolated from randomly selected spoilt carrot tubers from five (5) locations. The isolates and their respective occurrences Staphylococcus aureus (100%), Pseudomonas spp (75%), Proteus spp (40%), Serratia spp (80%), Bacillus spp (80%), Corynebacterium spp (15.3) and Escherichia coli (40%). Two lactic acid bacteria, Lactobacillus fermenti and Lactobacillus plantarum were isolated from Ugba and their crude bacteriocin extracts were used for tests. Result of pathogenecity test of the spoilt carrot bacteria isolates, showed only three isolates, Corynebacterium, Pseudomonas and Serratia species were confirmed to be pathogens of fresh healthy carrot tubers. The inhibitory effects of the crude bacteriocin extracts of the lactic acid bacteria isolates showed inhibitions of varied sizes against the pathogens in vitro. L. casei crude extracts generated inhibition zones of diameters in the range of 11.67mm (Corynebacterium) to 13.67mm (Serratia) while L. fermenti cause inhibitions of 10.67mm (Pseudomonas) to 12.67mm (Serratia). The inhibitory of L. plantarium crude bacteriocin extracts was in the range of 10.67mm (Corynebacterium) to 13.67mm (Serratia). The inhibitory activities of the lactic acid bacteria extracts were all lower than that of a standard antibiotic (control) which ranged between 18.67mm (Serratia) 22.0mm (Pseudomonas). The potentials of the crude bacteriocin extracts from lactic acid bacteria controlling carrot spoilage bacteria was observed in spite of the relatively low level of inhibitions.

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgement                                                                                                                  v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          vii

Abstract                                                                                                                                  ix

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aim and Objectives                                                                                                    3

CHAPTER TWO

2.0       Literature Review                                                                                                       5

2.1       Importance and Description of the African Oil Bean Tree                                        5

2.2       Preparation of Ugba                                                                                                    5

2.3       Lactic Acid Bacteria                                                                                                   6

2.3.1    Taxonomical Classification of Lactic Acid Bacteria                                                 7

2.3.2    Occurrence of Lactic Acid Bacteria in Nature                                                           7

2.4       Bacteriocins                                                                                                                8

2.4.1    Antimicrobial Property by Bacteriocin Production                                                    9

2.4.2    Range of Activity                                                                                                       10

2.5       Classification of Bacteriocins                                                                                    11

2.5.1    Class I                                                                                                                         11

2.5.2    Class II                                                                                                                        12

2.5.3    Class III                                                                                                                      13

2.5.4    Nisin                                                                                                                           13

2.6       Bacteriocin-Like Substances                                                                                      14

2.7       Isolation and Purification                                                                                           15

2.8       Methods of Purification                                                                                              16

2.8.1    Purification of Class I Bacteriocins                                                                           18

2.8.2    Purification of class II bacteriocins                                                                            19

2.8.3    Purification of class III bacteriocins                                                                          22

2.9       Bacterial Resistance to Bacteriocins                                                                          22

2.10     Bacterial Pathogens Associated with Fruit Contamination                                       22

2.10.1  Campylobacter                                                                                                           20

2.10.2  Pathogenic Escherichia coli                                                                                       23

2.10.3  Salmonella spp                                                                                                            23

2.10.4  Shigella spp                                                                                                                24

2.10.5  Staphylococcus                                                                                                           25

2.10.6  Vibrio                                                                                                                          25

2.10.7  Listeria monocytogenes                                                                                              26

2.10.8  Sporeformers                                                                                                              26

2.11     Sources of Contamination                                                                                          27

2.11.1  Production Environment                                                                                            27

2.11.2  Postharvest Handling                                                                                                  28

2.11.3  Human Hygiene                                                                                                          29

2.12     Pathogen Prevention                                                                                                   30

2.12.1  Processing Strategies                                                                                                  30

2.12.2  Handling Parameters                                                                                                  32

2.12.3  Alternative Technologies                                                                                           33

CHAPTER THREE

3.0       Materials and Methods                                                                                               35

3.1       Source of Materials                                                                                                    35

3.2       Sample Preparation                                                                                                    35

3.3       Isolation of Bacteria                                                                                                   35

3.4       Characterization of Isolates                                                                                        36

3.4.1    Characterization of Bacteria Isolates                                                                          36

3.4.2    Microscopic Features                                                                                                 36

3.5       Identification of Isolates                                                                                             36

3.5.1    Gram Staining                                                                                                            37

3.5.2    Biochemical Reaction Tests                                                                                       37

3.5.2.1 Sugar Utilization Tests                                                                                               38

3.5.2.2 Catalase Test                                                                                                               38

3.5.2.3 Indole Test                                                                                                                  38

3.5.2.4 Citrate Utilization Test                                                                                               38

3.5.2.5 Hydrogen Sulphide (H2S) Production Test                                                               39

3.5.2.6 Voges-Proskauer Test                                                                                                 39

3.5.2.7 Urease Test                                                                                                                 39

3.5.2.8 Methyl Red Test                                                                                                         39

3.5.2.9 Oxidase Test                                                                                                               40

CHAPTER FOUR

4.0       Results                                                                                                                        41

CHAPTER FIVE

5.0       Discussion, Conclusion and Recommendations                                                        47

5.1       Discussion                                                                                                                   47

5.2       Conclusion                                                                                                                  49

5.3       Recommendations                                                                                                      50

            References

LIST OF TABLES

LIST OF FIGURES

CHAPTER ONE

1.0       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 (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 (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 (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 (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.

1.1       AIM AND OBJECTIVES

To determine the inhibitory effect of bacteriocins from Lactic acid bacteria from Pentaclethra macrophyla (UGBA) against carriot (Darius carota) spoilage bacteria, while the specific objectives are;

·       To isolate Lactic acid bacteria from fermented Ugba

·       To isolate some pathogenic bacteria species from spoilt carrots

·       To extract bacteriocins from Lactic acid bacteria isolates.

·       To determine the antibacterial activity of the bacteriocins against the isolated carrot (Darius carota) spoilage bacteria.

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