EVALUATION OF ANTIMICROBIAL ACTIVITIES OF CRUDE BROTHS OF LACTIC ACID BACTERIA ISOLATED FROM FERMENTED FOODS.

ABSTRACT

Five samples each of Ogi, Ogiri, Ugba and Fermented Yoghurt were collected from different sites at Ahiaeke in Abia State, from which forty-eight organisms were isolated using spread plate method and belong to four genera and six species: Lactobacillus plantarum, Lactobacillus salivarius, Lactococcus lactis, Leuconostoc mesentroides, Streptococcus thermophilus and Lactobacillus sakei. Agar well diffusion method was used to test the isolated lactic acid bacteria on Escherichia coli, Staphylococcus aureus and Salmonella typhi. The lactic acid bacterial were found to exhibit antibacterial activity against the pathogenic food organisms. The inhibition diameter obtained ranged between 6mm to 28mm. the highest inhibition was obtained from Lactobacillus sakei against Escherichia coli (28mm) and the smallest inhibition was obtained with Streptococcus thermophilus against Salmonella typhi (6mm), and no inhibition obtained with Streptococcus thermophilus against Staphylococcus aureus. The antimicrobial activity is attributed to bacteriocin, hydrogen peroxide, hypothiocyanate, lactic acid and fatty acid. Lactic acid bacterial have potential application for assuring and improving food quality and safety.

TABLE OF CONTENTS

Title page                                                                                                                                i

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgement                                                                                                                  iv

Table of contents                                                                                                                    v

Lists of table’s                                                                                                                        viii

Abstract                                                                                                                                  ix

CHAPTER ONE

1.1  Introduction                                                                                                                1

1.2 Aim and Objectives                                                                                                                     2                                         

CHAPTER TWO

2.1  Literature review                                                                                                        3

2.2 Classification of lactic acid bacterial                                                                                4

2.3 Sources of lactic acid bacterial                                                                                                  5

2.3.1 Conditions for the growth of lactic acid bacterial                                                                     6 

2.4 Mode of action of lactic acid bacterial                                                                                     8

2.5 Uses of lactic acid bacterial                                                                                                       9

2.6 Lactic acid bacterial and food fermentation                                                                     10

2.6.1 Dairy products                                                                                                                          11

2.6.2 Vegetables                                                                                                                                 12

2.7 Lactic acid bacterial as spoilage organisms                                                                            13

2.7.1 Wine spoilage                                                                                                                           14                                              

2.7.2 Beer spoilage                                                                                                                             14

2.7.3 Milk spoilage                                                                                                                 15

2.8 lactic acid bacterial as probiotic                                                                                               15

2.8.1 Anticancer effects                                                                                                                     16

2.8.2 Principal factors contributing to antimicrobial effect of lactic acid bacterial                      17

2.9 Bacteriocins                                                                                                                      17

2.9.1 Classification of bacteriocins                                                                                                  17

2.9.2 Synthesis of bacteriocins                                                                                               20

2.10 Test Organisms                                                                                                                   21

2.10.1 Escherichia coli                                                                                                                21

2.10.2 Staphylococcus aureus                                                                                                     22

2.10.3 Salmonella typhi                                                                                                             22

CHAPTER THREE

3.0  Materials and methods                                                                                                24

3.1 Sample collection                                                                                                             24

3.2Pathogenic organisms and Re-idetification                                                                      24

3.2.1 Methyl Red Test                                                                                                            24

3.2.2 Indoles Test                                                                                                                   24

3.3 Media preparation                                                                                                             25

3.3.1 De Man Rogosa Sharpe (MRS) Broth                                                                           25

3.3.2 Mueller- Hinton Agar                                                                                                    25

3.3.3 Nutrient Agar                                                                                                                 25

3.3.4 Salmonella shigella Agar (SSA)                                                                                     25

3.4 Sample analysis                                                                                                                 26

3.5 Isolated of lactic acid bacterial                                                                                         26

3.6 Identification and characterization of isolates                                                                  26

3.6.1 Macroscopy                                                                                                                   26

3.6.2 Microscopy                                                                                                                    27

3.6.2.1 Gram stain                                                                                                                  27

3.6.2.2 Motility Test                                                                                                               27

3.6.3 Biochemical Test                                                                                                           27

3.6.3.1 Catalase                                                                                                                       27

3.6.3.2 Oxidase                                                                                                                       28

3.6.3.3 Coagulase                                                                                                                   28

3.6.3.4 Carbohydrate fermentation                                                                                         28

3.7 Preparation of MRS Lactic acid Bacteria Broth                                                              28

3.7.1 Preparation of Antimicrobial sensitivity disc with lactic acid bacteria Isolate            29

3.7.2 Preparation of control disc using Amoxylcillin                                                            29

3.8 Selection of bacteriocin producing lactic acid bacterial                                                   29

3.9 Screening for Antimicrobial Activity                                                                               30

CHAPTER FOUR

1.0  Results                                                                                                                              31

CHAPTER FIVE

5.1 Discussion                                                                                                                          36

5.2 Conclusion                                                                                                                               38

5.3 Recommendation                                                                                                              38

Reference

LISTS OF TABLES

Table 1:         Identification and characterization of isolated lactic acid bacterial                  32

Table 2:         Sources and distribution of isolates                                                                  33          

Table 3:         Re-identification of pathogenic Test organisms                                              34

Table 4:         Inhibition of pathogenic organisms by lactic acid bacterial                            35

CHAPTER ONE

1.1       INTRODUCTION

Technologies on processing and preservation of food products, which help in maintaining its nutritional values besides ensuring safety issues are the area of current food research (Kabuki et al., 2007). Many chemicals are being used for inactivation of food borne pathogens so as to preserve food products for long duration (Baliet et al., 2011). Lactic acid bacteria produce various compounds such as organic acids, diacetyl, hydrogen peroxide and bacteriocin or bactericidal proteins during lactic fermentations (Oyetayo et al., 2003). The antimicrobial effect of LAB has been appreciated by man from more than 10,000 years and has enabled him to extend the shelf life of many foods through fermentation processes (Sarika et al., 2010). Control of both pathogenic and spoilage microbe in a variety of foods is important to guarantee food quality and safety.

Lactic acid bacteria exert strong antagonistic activity against many microorganisms including food spoilage organisms and pathogens. This technique is used as an alternative to chemical additives for increasing self-life storage and enhancing safety of food by using natural micro flora and their antimicrobial products (Stiles, 1996). Lactic acid bacteria are believed to be safe because, they have been long established as the normal flora in fermented food; thus, they have great potential for use in bio preservation. The preserving effects of LAB are due to the production of antimicrobial agents such as organic acids, hydrogen peroxide and bacteriocin or related substances (Cocolin et al., 2007). Bacteriocins are proteinaceous compounds that mainly inhibit closely related species (Klaenhammer, 1993). Although some bacteriocins have been shown to possess the ability to inhibit the actions of unrelated genera such as Clostridia, Listeria, Enteropathogenicbacteria and Gram-negative bacteria. Most bacteriocins are heat stable and sensitive to certain proteolytic enzymes. The bacteriocins produced inhibited food spoilage and pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Bacillus cereus, Bacillus subtilis, Listeria monocytogens and Clostridium perfringens which are recalcitrant to traditional food preservation method (Bizani et al., 2002). Several bacteriocins from Gram-positive bacteria are very effective, have broad inhibitory spectra and may be used as antimicrobial agents for various practical applications (Torodov et al., 2007). These bacteriocins are harmless against its own producers because the secretory bacterial strains have genetically pre-determined genes of immunity (Abada, 2008). The use of lactic acid bacterial bacteriocins in the food industry can help to reduce the addition of chemical preservatives as well as the intensity of heat treatments, resulting in foods which are more naturally preserved and richer in organoleptic and nutritional properties.

1.2           Aim and Objectives

This project work aimed as to isolate Lactic acid bacteria that produce antimicrobial substances belonging to bacteriocin type able to inhibit the bacteria which causes food poisoning and spoilage.

i.               Isolation and identification of crude Lactic acid bacteria isolated from fermented foods

ii.              Determination of the antimicrobial activity of crude Lactic acid bacteria.

iii.            Evaluation of antibiotic susceptibility of Lactic acid bacteria.

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