EVALUATION OF THE ANTIMICROBIAL POTENTIALS OF LACTIC ACID BACTERIA METABOLITES AGAINST TOMATO PASTE SPOILAGE MICROORGANISMS

 ABSTRACT

Tomato is one of the most popular consumed vegetable crops worldwide mainly as fresh or processed products. The antimicrobial potentials of lactic acid bacteria metabolites against tomato paste spoilage microorganisms were evaluated.  Cow milk and Tomato samples were purchased. The tomato samples were processed into tomato paste. Standard microbiological methods were used in isolation, microbial count and identification of organisms. Cow milk samples were used for the isolation of lactic acid bacteria. Antimicrobial effects of the isolated lactic acid bacteria metabolites were carried out on the spoilage isolates from tomato paste samples using the agar well diffusion method, while shelf-life study was done on the tomato paste samples using the lactic acid bacteria metabolites as biopreservative agents for a period of 14 days. The results showed that total viable count ranged from 2.81 x 107Cfu/ml - 1.3 x 106Cfu/ml, the coliform count from 6.5 x 104Cfu/ml – 1.1 x 104Cfu/ml, while the fungal count was from 3.0 x 104Cfu/ml – 1.5 x 104Cfu/ml. The bacterial isolates from the tomato samples were Bacillus sp and Pseudomonas sp, while the fungal isolates were Rhizopus sp, Saccharomyces sp, Fusarium sp, Apergillus sp and Mucor sp. Lactic acid bacteria isolates from the cow milk samples were Lactobacillus fermentum M6, Lactobacillus fermentum LF-12 and Lactobacillus fermentum M8.The strains of Lactobacillus fermentum were able to grow at different temperature, pH and NaCl concentration and also produced antimicrobial compounds with Lactobacillus fermentum LF-12 recording the highest lactic acid (0.98 g/l), diacetyl (1.77 g/l) and hydrogen perioxide (1.60 g/l). The metabolites from strains of Lactobacillus fermentum were all potent against the spoilage microorganisms with zones of inhibition ranging from 2 mm to 12 mm. There were significant differences (p<0.005) in the activities of the metabolites of the strains of Lactobacillus fermentum. The result of the shelf-life study revealed a great reduction in bacterial load and growth over a period of 14 days of storage. This shows that strains of Lactobacillus fermentum can be used as bioperservative agents in preserving tomato paste as an alternative to chemical preservatives.

TABLE OF CONTENTS

Title Page                                                                                                                    i

Declaration                                                                                                                  ii

Certification                                                                                                                iii

Dedication                                                                                                                  iv

Acknowledgements                                                                                                    v

Table of Contents                                                                                                       vi

List of Tables                                                                                                              ix

List of Figures                                                                                                             x

Abstract                                                                                                                      xi

CHAPTER 1: INTRODUCTION                                                                          1

1.1       Statement of Problem/Justification                                                                 2

1.2       Aim                                                                                                                 2

 1.3      General Objective                                                                                           2

1.4       Specific Objectives                                                                                         2

CHAPTER 2: LITERATURE REVIEW                                                              4

2.1       Spoilage Microorganisms of Tomatoes                                                           5

2.2       Lactic Acid Bacteria                                                                                       9

2.2.1    Taxonomy of lactic acid bacteria (LAB)                                                        10

2.2.2    Role of lactic acid bacteria in fermented food                                               11

2.2.3    Potential probiotic benefits                                                                             16

2.2.4    The mechanism of action of lactic acid bacteria                                             16

2.2.5    Selection criteria of lactic acid bacteria                                                          17

2.2.6    Antimicrobial activity of lactic acid bacteria species                                      18

2.2.7    Antimicrobial compounds produced by lactic acid bacteria                           19

2.2.7.1 Diacetyl                                                                                                           20

2.2.7.2 Hydrogen peroxide                                                                                         21

2.2.7.3 Reuterin                                                                                                          22

2.2.7.4 Bacteriocin                                                                                                      22

CHAPTER 3: MATERIALS AND METHODS                                                   25

3.1        Sample Collection                                                                                           25

3.2        Preparation of Media and Diluents                                                                 25

3.3        Preparation of Tomato Paste Samples and Isolation of Spoilage

      Organisms                                                                                                       25

3.4        Determination of Microbial Population                                                          26

3.5        Characterization and Identification of Microbial Isolates                              26

3.5.1    Gram staining test                                                                                           26

3.5.2    Spore staining                                                                                                 26

3.5.3    Motility test                                                                                                    27

3.5.4    Biochemical characterization of bacteria isolation                                          27

3.6        Identification of Fungal Isolates                                                                    27

 3.7      Isolation and Characterization of Lactic Acid Bacteria (LAB) from

            Cow Milk Samples                                                                                          28

3.7.1        Cultural isolation and characterization of lactic acid bacteria                        28

3.7.2        Molecular identification of the lactic and bacteria                                         29

3.7.3        Determination of the effect of NaCl concentration on the growth of

lactic acid bacteria isolates                                                                              29

3.7.4        Determination of the effect of temperature on the growth of lactic acid

bacterial isolates                                                                                              29

3.7.5        Molecular identification of the lactic acid bacteria                                        29

3.8          Test Microorganisms Isolates from Tomato Samples                         31

3.9          Preparation of Cell-Free Filtrate of Lactic Acid Bacteria                              31

3.10      Determination of Lactic Acid Bacteria Metabolites                                       32

3.11      Antimicrobial Activity of Lactic Acid Bacteria Metabolites Against

    Spoilage Organisms                                                                                        33

3.12     Shelf Life/Biopreservative Study of Tomato Paste                                        34

3.13     Statistical Analysis                                                                                          34

CHAPTER 4: RESULT AND DISCUSSION                                                       35

4.1        Result                                                                                                              35

4.2        Discussion                                                                                                       51

CHAPTER 5: CONCLUSION AND RECOMMENDATION                           55

5.1       Conclusion                                                                                                      55

5.2       Recommendation                                                                                            55

References

Appendices

LIST OF TABLES              

4.1       The cultural and biochemical characteristics of the bacterial             

             isolates from the tomato samples                                                                   36

4.2       The total microbial loads of isolates from the tomato samples                                   in colony-forming units per milliliter (cfu/ml)                                          38

4.3       The colonial and microscopic morphology of the fungal                                            isolates from the tomato samples                                                                       40

4.4       The cultural and biochemical characteristics of lactic acid                             42

                bacteria isolates from cow milk samples

4.5       Antimicrobial metabolites produced by strains of lactobacillus fermentum  46

4.6       The antimicrobial sensitivity pattern of the metabolites of lactic acid

            bacteria isolated from cow milk against some selected tomato spoilage                   organisms (mm)                                                                                    48

4.7       The shelf life study of the tomato paste samples inoculated with                  50        the metabolites of lactic acid bacteria on the bacterial population                                              

LIST OF FIGURES

2.1       Tomato plants and parts                                                                                  4

2.2       Differentiation of species according to the recent taxanomy                         10

4.1       The result of molecular analysis of the lactic acid bacteria                             44

 from the diary product (cow milk) sample

CHAPTER 1

INTRODUCTION

Tomato (Solanum lycopersicum) is one of the most popular and extensively consumed vegetable crops worldwide. The health benefits of tomatoes are attributed to the abundant antioxidant components present in tomatoes (Willcox et al., 2003), such as lycopene and provitamin A, ascorbic acid, vitamin E and other flavonoids. Tomatoes are consumed mainly as fresh or processed products. Tomatoes are processed for wide ranges of products such as canned and sun-dried tomatoes, ketchups, pastes, purees, salads, sauces, soups, and juice products, supplying significant sources of vitamins and minerals to the consumers (Tsen et al., 2008).

 Lycopene (a carotenoid), an essential component of tomato with potent antioxidant activity, has been reported and accumulating evidence has shown an inverse correlation between the consumption of tomato products rich in lycopene and the risk of several types of cancer and cardiovascular disease (Talvas et al., 2010). Studies have shown that lycopene level in plasma increased only after the consumption of red tomato paste and purified lycopene (Talvas et al., 2010). It has also been documented that the absorption of lycopene is greater from the processed tomatoes than from fresh tomatoes, since processing breaks down the tomato cell matrix and makes lycopene more available.  Fermentation used with a purpose of the extension of shelf life and enhanced safety of foods by the use of natural or controlled microbiota and/or antimicrobial compounds is an approach to the solution of food preservation that has gained increased attention in recent years. Many lactic acid bacteria (LAB) strains are able to produce protein compounds, such as antimicrobial peptides (bacteriocins), and organic acids (lactic and acetic), hydrogen peroxide with efficient antimicrobial effect (Cleveland et al., 2001; DeVuyst and Leroy, 2007).

1.1       STATEMENT OF PROBLEM/JUSTIFICATION

Tomato is one of the highly nutritious food ingredients used in the preparation of food all over the world (Ogunniyi and Oladejo, 2011; Ogunbanwo et al., 2014).  Its  utilization  as  an  ingredient  in  vegetable  salads, other  dishes  and  its  processing  into  different  products  like puree,  ketchups  and  juice  is  well  documented (Ogunbanwo et al., 2014).  Nutritionally, it contains a  large  amount  of  water,  niacin,  calcium  and vitamins  especially  A,  C, and  E  which  are  important  in  the metabolic  activities  of  man  and  protects  the  body  against diseases (Bernard et al., 1999).

The high  water  content  in  tomatoes  makes  it very  susceptible  to  spoilage  bacteria  and  fungi  during storage,  harvesting  and  transportation  (Spadaro  and Gullino,  2004). Because of this use of lactic acid bacteria could help to increase the shelf life as well as reduce the pathogens of the fermented tomato paste during processing and storage.

1.2       AIM

This work is designed to evaluate the antimicrobial potentials of lactic acid bacteria metabolites against tomato paste spoilage microorganisms.

 1.3      GENERAL OBJECTIVE

To evaluate the antimicrobial potentials of metabolites of lactic acid bacteria isolated from diary product (cow milk) on tomato paste.

1.4     SPECIFIC OBJECTIVES

1.      To isolate and identify the Lactic acid bacteria isolates from the dairy product (cow milk) using phenotypic and molecular characteristics

2.      To isolate and characterize spoilage microorganisms of tomato paste samples.

3.      To assay the antimicrobial activities of metabolites of the lactic acid bacteria isolated from cow milk on the spoilage organisms of tomato paste.

4.      To check the bacterial load of tomato paste samples during the period of shelf-life study.

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