IN VITRO ANTIMICROBIAL ACTIVITIES OF HIBISCUS SABDARIFFA AND MORINGA OLEIFERA ON TOMATO SPOILAGE ORGANISMS.

ABSTRACT

The invitro antimicrobial activities of Moringa oleifera and Hibiscus sabdariffa on tomato spoilage organisms was carried out. Spoiled tomato samples were purchased from four different markets in Umuahia Abia State and calyces of Hibiscus sabdariffa was purchased from the market, and also leaves of Moringa oleifera were gotten fresh from Moringa tree. The tomato samples were examined for the organisms which causes spoilage of tomato and the mean bacterial and fungal counts were gotten. The bacterial isolates were Escherichia coli and Bacillus sp. While the fungal isolate was Rhizopus sp. The colonial count of E. coli isolated from this study was in the range of 5.0 x 105 cfu/g to 1.5 x 106cfu/g while that of Bacillus ranged from 1.2 x03 to 1 5.0 x 104cfu/g. The mean fungal count was in the range of 4.7 x 105 to 2.8 x105. Moringa oliefera and Hibiscus sabdaffla leaf extract were shown to have some degree of antimicrobial properties on the tomato isolates by means of producing a zone of clearance around the growth of the organisms on nutrient plates. The aqueous extract of H. sabdariffa showed maximum activity against Bacillus species with a diameter zone of clearing of 20mm. The ethanol extract of both plants also recorded remarkable activity with zones of inhibition ranging from 6mm to 10mm for the Moringa extracts. The aqeous extract of Hibiscus sabdariffa had the lowest active concentration (MIC) against Rhizopus, E. coli, and Bacillus at 125mg/ml, 62.5mg/ml and 62.5mg/ml respectively. Plants have an almost limitless ability to synthesize aromatic substances. In many cases, these substances serve as plant defence mechanisms against predation by microorganisms.

TABLE OF CONTENTS

Title page                                                                                                                                i

Certification                                                                                                                            ii

Dedication                                                                                                                              iii

Acknowledgement                                                                                                                  iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          ix

Abstract                                                                                                                                  x

CHAPTER ONE

INTRODUCTION

1.0 Introduction                                                                                                                      1

1.1 Spoilage microorganisms and their adverse effects on fruits and vegetables                   2

1.2 Adverse effects                                                                                                                 3

1.3 Aim and objectives                                                                                                           4

CHAPTER TWO

LITERATURE REVIEW

2.0 Tomatoes                                                                                                                                                  5

2.1 Importance and uses of tomatoes                                                                                     5

2.2 Microorganisms associated with tomato spoilage and their effects                               6

2.3 Hibiscus sabdariffa (botanical and morphological description)                                      7

2.4 Phytochemistry of Hibiscus sabdarriffa                                                                           8

2.5 Biological and pharmacological activities of H. Sabdariffa                                            9

2.5.1 Antimicrobial activity of H. Sabdariffa                                                                        9

2.5.2 Antioxidant activity of H. Sabdariffa                                                                            10

2.5.3 Cancer-preventive activity of H. Sabdariffa                                                                 10

2.5.4 Uses of H. Sabdariffa                                                                                                    10

2.6 Moringa  oleifera (botanical and morphological description)                                         11

2.7 Phytochemistry of M. oleifera                                                                                          12

2.8 Biological and pharmacological activities of M.  oleifera                                               13

 2.8.1 Antimicrobial Effects of Moringa  oleifera                                                                 13

2.8.2 Antioxidant properties of Moringa  oleifera                                                                 13

2.8.3 Anti-cancer properties of M. oleifera                                                                            14

2.8.4 Uses of Moringa  oleifera                                                                                             14

CHAPTER THREE

Materials and Methods                                                                                                           16

3.0 Sample collection                                                                                                             16

3.1 Preparation of extracts                                                                                                      16

3.2 Sterilization of materials                                                                                                  16

3.3 Preparation of media                                                                                                        17

3.4 Isolation and Enumeration of Microorganisms                                                                17

3.4.1 Isolation of bacteria                                                                                                       17

3.4.2 Isolation of fungi                                                                                                           18

3.5 Purification (subculture) of bacterial isolates                                                                  18

3.6 Purification of fungal isolates                                                                                          18

3.7 Characterization and identification of isolates                                                                 19

3.7.1 Gram staining                                                                                                                19

3.7.2 Motility test                                                                                                                   20

3.8 Biochemical tests                                                                                                                                                              21

3.8.1 Catalase test                                                                                                                                                                   21

3.8.2 Coagulase test                                                                                                                                                                21

3.8.3 Oxidase test                                                                                                                                                                   21

3.8.4 Urease test                                                                                                                                                                     21

3.8.5 Indole test                                                                                                                                                                      21

3.8.6 Methyl red- voges proskauer test                                                                                                                                  21

3.8.7 Citrate utilizatrion test                                                                                                                                                   22

3.8.8 Sugar fermentation tests                                                                                                                                                22

3.9 Determination of minimum inhibitory concentration                                                      22

3.9.1 Determination of bactericidal concentration (mbc)                                                      23

3.9.2 Antimicrobial susceptibility testing (invitro)                                                                23

CHAPTER FOUR

Results                                                                                                                                    24

CHAPTER FIVE

Discussion, Conclusion and Recommendation                                                                      35

5.0 Discussion                                                                                                             35

5.1 Conclusion                                                                                                                        37

5.2 Recommendation                                                                                                             37

References                                                                                                                              38

LIST OF TABLES

CHAPTER ONE

INTRODUCTION

1.0 INTRODUCTION

Anti- microbial agents are substances that kill microorganisms or inhibit their growth. They are widely employed to cure bacterial and fungal diseases. Anti-microbial agents that reversibly inhibit growth of bacteria are called bacteriostatic where as those with irreversible lethal action on bacteria are known as bactericidal (Rajesh and Rathan, 2008).

Ideally, anti-microbial agents disrupt microbial processes or structures that differ from those of the host. They may damage pathogens by hampering cell wall synthesis, inhibiting microbial protein and nucleic acid synthesis, disrupting microbial membrane structure and function or blocking metabolic pathways through inhibition of key enzymes (Willey et al., 2008).

Before an anti-microbial agent is accepted for use in human it must demonstrate most, if not all, of the following properties:

1.     Selective toxicity (it should act  on bacteria without damaging the host tissue)

2.     It should be bactericidal rather than bacteriostatic.

3.     It should be effective against a broad range of bacteria.

4.     It should remain active in plasma body fluids etc.

5.     It should be stable and preferably water soluble.

6.     Desired level should be reached rapidly and maintained for adequate period of time and should not give rise to resistance in microorganisms.

7.     It should have a long shelf life and should not be expensive (Rajesh and Rathan, 2008).

Spoilage refers to any change in the condition of food in which the food becomes less palatable or even toxic, these changes may be accompanied by alterations in taste, smell, appearance or texture (Berdeque et al, 2005).

Food spoilage refers to various changes in which the food becomes less palatable or even toxic to consumers. These changes may be accompanied by alterations in taste, smell, appearance or texture. Numerous microbial defects of agricultural crops are characterized by the types of micro-organisms responsible for their deterioration (Akinmusire, 2011).

1.1 SPOILAGE MICROORGANISMS AND THEIR ADVERSE EFFECTS ON FRUITS AND VEGETABLES

Spoilage microorganisms can be introduced to the crop on the seed itself, during crop growth in the field, during harvesting and post-harvest handling or during storage and distribution (Barth et al., 2009).

Those types of soil-borne spoilage microbes that occur on produce are the same spoilage organisms that are present on harvesting equipment, on handling equipment, packaging house, and storage facility and on food contact surfaces throughout the distribution chain (Barth et al., 2009).

The main sources of microorganisms in vegetables are soil, water, air and other environmental sources and can include some plant pathogens. Fresh vegetables are fairly rich in carbohydrate (5% or more), low in proteins (about 1-2%); except for tomatoes have high _PH. The presence of air, high humidity and higher temperature during storage increases the chances of spoilage. The common spoilage defects are caused by molds belonging to genera Pennicillium, Phytophora, Alternaria, Aspergillus. Among the bacteria genera species from Pseudomonas, Erwinia, Bacillus and Clostridium are important. Microbial vegetables spoilage is generally described by the common term rot, along with changes in appearance, such as black rot, gray rot, pink rot, soft rot, stem-end rot (Hozbor et al., 2006).

A fruit is the edible part of a mature ovary of a flowering plant. When matured they are either fleshy or dry. Fleshy fruits are classified into berry (e.g tomato), drupes (e.g almond) and pomes (e.g apple). Dry fruits are classified in dehiscent and indehiscent fruits (Jay 2000).

Fruits and vegetable are vital source of nutrients to human beings. They give the body the necessary vitamins, fats, minerals and oil in the right proportions for human growth and development. They however, have serious challenges to their existence which includes: changes in climatic condition, pests, and microbial attack. Over the years, there has been an increase in the need to identify and isolate the microorganisms associated with the spoilage as a way of finding a means of controlling it (Akinyele and Akinkunmi, 2012).

Spoilage microorganisms can be introduced to the crop on the seed itself, during crop growth in the field, during harvesting and post-harvest handling or during storage and distribution (Barth et al, 2009).

Fruits are affected by a wide array of microorganisms causing its decay. Susceptibility of fruits and vegetables is largely due to differential chemical composition such as _PH and moisture contents and are associated with greater predisposition to microbial spoilage.

1.2 ADVERSE EFFECTS

Bacterial spoilage first causes softening of tissues as pectins and its degradation. Starches and sugars are metabolised next and unpleasant odours and flavour develop along with lactic acid and ethanol. Molds belonging to several genera including Rhizopus and Botrytis causes vegetable rot.

In fact, healthy fruits have many microbes on their surfaces but can usually inhibit their growth until after harvest. Ripening weakens cell walls and decreases the amounts of antifungal chemicals in fruits, and physical damage during harvesting causes breaks in outer protective layers of fruits that spoilage organisms can exploit. Molds are tolerant of acidic conditions and low water activity are involved in spoilage of fruits. Penicillium and Rhizopus are frequently isolated from spoiled fruits (Calvo et al., 2007).

1.3 AIM

To investigate the antimicrobial effect of extracts of Moringa oleifera and Hibiscus sabdariffa on Organisms that cause tomato spoilage.

1.4 OBJECTIVES

1.     To isolate and identify micro-organisms associated with spoilage of tomatoes.

2.     To investigate the effects of Hibiscus saabdariffa and Moringa oleifera extracts on tomatoes spoilage organisms.

3.     To determine the minimum inhibitory concentrations and minimum bactericidal concentration of extracts of Moringa oleifera and Hibiscus saabdariffa.

4.     To measure the zone of inhibition of extracts against tomatoes spoilage organisms.

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