ANTIMICROBIAL ACTIVITIES OF LACTIC ACID BACTERIA ISOLATED FROM LOCALLY FERMENTED FOODS

ABSTRACT

Fermented food are as those products that have been subjected to the effect of microbial enzymes, particularly amylases, proteases and lipases that causes biochemical transformation of polysaccharides, proteins and lipids to non‐toxic variety of desirable products with tastes, aromas and textures attractive to a consumer. Locally fermented foods are Ogi, Ogi, Ogiri and Ugba. The aim of this work was to investigate the antimicrobial activities of  lactic acid bacteria from locally fermented food. A total of 3 different fermented food which include ogiri, Ugba and ogi. Were purchased from the local market named Ndoro in Ikwuano L.G.A Abia State. The samples were collected using sterile bottle to the laboratory. One gram of the sample was mixed with 9ml of distilled water, 0.1ml was cultured on Mann Rogosa Sharpe (MRS) agar plates and incubated 370C for 24hours. After incubation the Average Total lactic acid bacterial (LAB) count from fermented food sample was observed The Ogi samples had total Lactic acid bacterial count range from 4.1 x 106 to 8.9 x 105, Ugba has colonies count range from 4.9 x 106 to 7.3 x 105, Ogiri has colonies count range from 4.8 x 106 to 8.8 x 106 respectively. Five lactic acid bacteria were identified as Streptococuss species, Lactococcus lactis, Lactobacillus brevis, Lactobacillus plantarum, and Lactobacillus lichenformis. The percentage occurrence of the isolates showed that Lactobacillus brevis and Lactobacillus Lichenformis (25.0%) has the highest percentage occurrence of lactic acid bacterial (LAB) isolates from traditional fermented food follow by streptococcus species, Lactobacillus plantarum and Lactobacillus lactis (16.7%).The five isolates were cultured in Mann Rogosa Sharpe (MRS) broth and incubated at 37oc, after incubation the cell free supernatant was obtained by centrifugation at 10,000 rpm for 15mins. The species of antimicrobial producing Lactic acid bacteria exhibited the highest inhibitiory activity against food borne pathogens for bacterial and fungal isolates. This study revealed the possibility of using bacteriocin as food biopreservative to control food spoilage and pathogenic organism.

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgements                                                                                                                iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          vii

Abstract                                                                                                                                  ix

CHAPTER ONE

CHAPTER TWO

2.0 Literature review                                                                                                  4

2.1 lactic acid bacteria                                                                                                            4

2.1.1   Taxonomy of lactic acid bacteria                                                    4

2.1.1.1 Streptococcus species                                                                                    5

2.1.1.2 Lactococcus species                                                                                       5

2.1.1.3 Enterococcus species                                                                                     7

2.1.1.4 Carnobacterium species                                                                                7

2.1.1.5 Tetragenococcus species                                                                               8

2.1.1.6 Vagococcus species                                                                                       8

2.1.1.7 Lactobacillus species                                                                                     9

2.2      Isolation and Identification of Lactic Acid Bacteria            12

2.3       Antimicrobial Agents                                                                                    12

2.4       Characteristics and Importance of LAB and Bacteriocin                              13

2.4.1.   Characteristics and Importance of LAB                                                         13

2.4.2.   Characteristics and Importance of Bacteriocin                                              15

2.5. Bacteriocin producing Lactic Acid Bacteria                                                       17

2.6. Fermented food                                                                                                    18

2.6.1 Traditionally Fermented Foods in Nigeria                                                        19

2.7       Overview of Pathogenic Bacteria                                                                  18

2.7.1    Listeria monocytogenes                                                                                  18

2.7.2    Escherichia coli O157:H7                                                                              19

2.7.3    Salmonella                                                                                                      19

CHAPTER THREE

3.0       Materials and Methods                                                                                   21

3.1       Materials                                                                                                         21

3.1.1    Sample collection                                                                                           21

3.1.2    Media preparation                                                                                          21                   

3.2     Microbiological serial dilution                                                                         21

3.3.      Isolation of Lactic Acid Bacteria (Lactobacillus spp.)                                   21

3.4       Collection and maintenance of pathogen cultures                                         22

3.5.      Identification of Lactic acid bacteria.                                                            22

3.6       Identification of Bacteria                                                                               22

3.6.1    Gram staining                                                                                                 22

3.6.2. Cultural Characteristics                                                                                                23

3.6.3. Morphological Characteristics                                                                                     23

3.6.3. Biochemical Characteristics                                                                                         23

3.6.3.1. Motility test                                                                                                               23

3.6.3.2. Catalase Test                                                                                                              23

3.6.3.3. Oxidase test                                                                                                               24

3.6.3.4. Indole Production                                                                                                      24

3.6.3.4. Methyl Red reaction                                                                                                  24

3.6.3.5. Voges Proskaur (VP) reaction                                                                                   25

3.6.3.6. Citrate utilization                                                                                                       25

3.6.3.7. Carbohydrate fermentation                                                                                       25

3.7 Antimicrobial activities of lactic acid bacteria                                                                26

CHAPTER FOUR

4.0       Results                                                                                                                        28

CHAPTER FIVE

5.0       Discussion, Conclusion and Recommendation                                                          33

5.1       Discussion                                                                                                                   33

5.2      Conclusion                                                                                                                   35

5.3       Recommendation                                                                                                        35

    References                                                                                                                          37-42

LIST OF TABLES

                  CHAPTER ONE

1.0     INTRODUCTION

  Lactic acid bacteria (LAB) are a group of Gram-positive bacteria that produce lactic acid as their main fermentation product (Mathur, 2005). Typical LAB members are Gram-positive, facultative anaerobic, catalase negative organisms with low G+C content. Most LAB have a long history of being consumed as part of traditional fermented foods and have been awarded the status of Generally Regarded As Safe (GRAS) by the Food and Drug Administration (FDA) (Ammor et al., 2007). The LAB group comprisnes the genera Lactobacillus, Streptococcus, Lactococus, Leuconostoc, Pediococcus, Aerococcus, Alloicoccus, Dolosigranulum, Enterococcus, Globicatella, Lactospaera, Oenococcus, Carnobacterium, Tetragenococcus, Vagoccus and Weissella (Ko and Ahn, 2000). Lactobacilli are naturally present or deliberately added as starter cultures in unpasteurized milk and dairy products such as cheeses, yogurts and fermented milks (Coeuret et al., 2004). LAB produces various compounds such as organic acids, diacetyl, hydrogen peroxide, and bacteriocin or bactericidal proteins during lactic fermentations (Hamilton-Miller, 2004). Bacteriocins are antimicrobial proteinaceous compounds that are inhibitory towards sensitive strains and are produced by both Gram-positive and Gram negative bacteria (Padmanabha et al.,2006). Bacteriocins are protein or peptides, which do not harm the producer strain but have lethal antibacterial activity against food spoilers and/or food borne pathogens (Rodriguez et al., 2009). Most of the bacteriocins from LAB have been isolated from species of the genus Lactobacillus (Klein et al., 1998). The bacteriocins from the lactic acid bacterial isolates generally recognized as safe (GRAS). Bacteriocins producedby these bacteria can inhibit pathogenic and spoilage microorganisms extending the shelf-life and enhancing the safety of food products (Grobben et al., 2008). Crude extract bacteriocin inhibited the E.coli and S. aureus (Abubakar and Arpah, 2015).Gram-negative bacteria produce a relatively narrow inhibiting bacteriocin, while Gram-positive bacteria produce relatively broad inhibiting bacteriocin (De Vuyst and Leroy, 2007). Many bacteriocins showed stable activity to the temperature of -20 to 100ºC, while it might be sensitive to pH. Bacteria may produce bacteriocin in synthetic media such as de Man Rogosa and Sharpe broth (MRS broth), Tryptone Glucose Extract Yeast (TGE), or other synthetic media. However, MRS broth is a common one (Todorov and Dicks, 2004). Bacteriocin is potentially used in the control of contaminant bacteria in the food industry, but the availability of bacteriocin is very limited and the price is very high, therefore to find LAB bacteriocin producers that can inhibit E.coli and S.aureus is still necessary.

Fermented foods are of great importance because they provide and preserve vast quantities of nutritious foods in a wide diversity of flavours, aromas and textures which enrich the human diet (Ijabadeniyi, 2007). Soomro (2012) reported that the number of traditional fermented foods in the world is estimated to be about 3,500.  These food products result from the activities of microorganisms which modify the flavour and texture and increase long term product stability in the process known as fermentation (Oyedeji, Ogunbanwo and Onilunde, 2013). During fermentation, the bacteria and fungi transform the raw material fermented food products. Fermentation has been reported to improve the digestibility of some grain legumes and enable their nutrient content such as protein and mineral (Adesokan et al., 2017).

Food spoilage is a metabolic process that causes foods to be undesirable or unacceptable for human consumption due to changes in sensory characteristics. Spoiled foods may be safe to eat, i.e. they may not cause illness because there are no pathogens or toxins present, but change in texture, smell, taste, or appearance cause them to be rejected. Some ecologists have suggested these noxious smells are produced by microbes to repulse large animals, thereby keeping the food resource for themselves (Sherratt et al., 2006). Antibiotics are at present restricted for use in foods and feeds, and bacteriocins are an interesting group of biomolecules with antimicrobial properties that may represent a good alternative in preventing food spoilage (Jack et al., 2005).

1.2       AIM AND OBJECTIVES OF THE STUDY

This study focuses on the antimicrobial activities of lactic acid bacteria isolated from locally fermented foods.

Specific Objectives include to;

1.     To isolate lactic acid bacteria from fermented food products.

2.     To screen and identify the lactic acid bacteria from fermented food products.

3.     To extract and evaluate the antimicrobial effect on pathogenic organisms.

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