ANTIMICROBIAL ACTIVITY OF HONEY ON SOME PATHOGENIC FUNGAL ISOLATES

ABSTRACT

The antimicrobial activity of honey on some pathogenic fungal isolates was investigated. The aim of this study was to test the antimicrobial activity of two honey samples from Nigeria against some pathogenic fungal isolates. The two samples of honey were purchased from Nsukka, in Enugu State and National Root Crop Research Institute, Umudike and were tested against fungal pathogens which includes Aspergillus flavus, Aspergillus niger, Candida albicans and Fusarium spp. The antimicrobial effect of the honey samples were tested using the agar diffusion method. The Minimum Inhibitory Concentration (MIC) and the Minimum Fungicidal Concentration (MFC) of the samples were also determined. The results obtained showed different level of inhibition at various concentrations against the tested fungi isolates with the zones of inhibition increasing as the honeys’ concentration increases. Aspergillus niger showed the highest zone of inhibition among all the fungi isolates used in the study with a diameter zone of inhibition value at 22mm at 100% for honey sample from National Root Crop Research Institute, while the least zone of inhibition of 15mm was recorded against honey from Nsukka, Enugu state at 100% with Candida albicans. The Minimum Inhibitory Concentration result was 9mm, 11mm, 9mm and 8mm for Aspergillus flavus, Aspergillus niger Fusarium spp. and Candida albicans respectively, against honey from National Root Crop Research Institute, Umudike and 10mm, 9mm, 8mm and 7mm for Aspergillus flavus, Aspergillus niger, Fusarium spp. and Candia albicans respectively, for honey from Nsukka, Enugu State. The Minimum Fungicidal Concentration result was 40% - 80% against the fungal isolates for both samples of honey used in this study. The honey samples used in this study exhibited a broad spectrum of antifungal activities, which shows that honeys from Nigeria have an interesting level of antimicrobial effect and can be used as an effective agent for future development and advancement of antifungal drugs.

TABLE OF CONTENTS

Title page                                                                                                                                                          i

Certification                                                                                                                                                      ii

Dedication                                                                                                                                                         iii

Acknowledgements                                                                                                                                        iv

Table of contents                                                                                                                                            v

List of tables                                                                                                                                                     vi

Abstract                                                                                                                                                             vii

CHAPTER ONE

1.0                       Introduction                                                                                                                        1

1.1                       Aims of the study                                                                                                               3

1.2                       Objectives of the study                                                                                                     3

CHAPTER TWO

2.0                       Literature Review                                                                                                               4

2.1                       Physiochemical properties of honey                                                                             5

2.2                       Benefits of Honey                                                                                                               8

2.3                       Health Benefits of honey                                                                                                  8

2.3.1     Honey against infections of the skin                                                                                            9

2.3.2     Oral Health                                                                                                                                         9

2.3.3     Stomatitis and other oral lesions                                                                                                  10

2.3.4     Honey against eye diseases                                                                                                            10

2.3.5     Against Catheter Infection of CKD                                                                                                11

2.3.6     Honey in Gastroenterology                                                                                                            11

2.3.7     Ulcers and Gastritis                                                                                                                          12

2.3.9     Laxative effects and constipation                                                                                                 13

2.3.9     Against acute gastroenteritis in children                                                                                    14

2.3.10  Against alcohol abuse                                                                                                                      15

2.3.11  Treatment of Reflux Oesophagus and Heartburn                                                                     15

2.3.12  Hepatitis and liver health                                                                                                               15

2.3.13  Honey for good digestion                                                                                                               15

2.3.14  Honey Against Cough                                                                                                                      15

2.3.15  Honey and the Brain                                                                                                                        16

2.4                       Nutritional Benefits of Honey                                                                                         17

2.5                       Cosmetic Uses of Honey                                                                                                   18

2.6                       Home Remedies that uses Honey                                                                                  19

2.7                       Types of Honey according to Ayurveda                                                                        19

CHAPTER THREE

3.0                       MATERIALS AND METHOD                                                                                               22

3.1                       Sample Collection                                                                                                               22

3.2                       Test Microorganisms                                                                                                         22

3.3                       Sterilization of materials                                                                                                   22

3.4                       Media Preparation                                                                                                             22

3.5                       Preparation of the Honey samples                                                                                23

3.6                       Common Tests for Real Honey                                                                                        23

3.7                       Sub-culturing of the inoculums                                                                                       23

3.8                       Preparation of inoculum                                                                                                   24

3.9                      Determination of antifungal activity                                                                             24

3.10                    Determination of Minimum Inhibitory Concentration (MIC)                                           24

3.2.7     Determination of Minimum Fungicidal Concentration (MFC)                                 25

CHAPTER FOUR

4.0                       Results                                                                                                                                  26

CHAPTER FIVE

5.0                       Discussion, Conclusion and Recommendation                                                            36

5.1                       Discussion                                                                                                                            36

5.2                       Conclusion                                                                                                                            38

5.3                       Recommendation                                                                                                               38

References

LIST OF TABLES

Table 1:             Nutritional Value of Honey per 100g                                                                          18

Table 2:             The diameter (mm) zone of inhibition of the honey samples in against the tested fungal isolates.                            28

Table 3:             Inhibitory effect of undiluted (100%) honey on the fungal Isolates using gel diffusion method.                 29

Table 4:             Minimum Inhibitory Concentration (MIC) of the honey samples against the test organisms.                 30

Table 5:             Minimum Fungicidal Concentration (MFC) of the honey samples against the test organisms.   31

Table 6: Typical colony morphology of the test organisms on Sabouraud Dextrose Agar.              32

LIST OF FIGURES

Figure 1: Inhibitory Effect of undiluted honey                                                                      33

Figure 2: Minimum Inhibitory Concentration of the honey samples                              34

Figure 3: Minimum Fungicidal Concentration of the honey samples                             35

CHAPTER ONE

1.0       INTRODUCTION

Honey is an organic substance that is produced from many different floral sources. Pollen from flowers are collected by honeybees and taken back to their nest. When the nectar enters the stomach of bee, it immediately becomes honey. Enzymes from the head gland called diastase-invertase and enzyme from the thoracic gland (glucose oxidase), mixed with the nectar in order to enhance the flavour of honey (Stone et al., 1958). Honey has been used for its medicinal properties in many cultures, since in ancient times. Work carried out by Molan (Molan, 1999) reported that honey is becoming accepted as a reputable and effective therapeutic agent by practitioners of conventional medicine and by the general public. In today’s society, it is still being used and is slowly replacing some of the artificial products used to treat human. According to Bangroo and co- workers, (Bangroo et al., 2005) despite the recent advances in antimicrobial activities of artificial products, none has eliminated the problem of infection. In some countries, honey is used as an antiseptic therapeutic agent for treatment of burns, ulcers and other wounds.

Raw and unprocessed honey is a type valued for its nutrient and medicinal properties. Honey contains as much as eight substances that are important to good nutrient in human. It is a sweet viscous liquid that has high enzyme content. Honey also contains proteins, carbohydrates, hormones, organic acids and antimicrobial and antibiotic properties. The composition of honey varies according to the plant species on which the bee forages, but the main constituents are the same in all honeys (White et al., 1962). Honey consists mainly of carbohydrates of which glucose and fructose are most abundant. These two sugars strongly attract water and as such honey when applied to wounds, absorb water, drying it out so that the growth of bacteria and fungi are inhibited (these micro-organisms thrive in moist environment). Secondly raw honey contains enzyme called glucose oxidase that, when combined with water, produces hydrogen peroxide (Majno, 1975), a mild antiseptic. In addition, honey also contains antioxidants and flavonoids that may function as antibacterial agents. The mineral and vitamin content of honey is very low (about 0.02% of its weight) and given the low consumption of honey, it thus have no significant nutritional benefit to man. The pH of honey is low and ranges from 3.2 to 4.5 with the most predominant one being gluconic acid. These properties of honey are well established. Undiluted honey inhibits the growth of bacteria such as Staphylococcus aureus, certain gut pathogens and fungi such as Candida albicans (Obeseiki-Ebor et al., 1984). At a concentration of 30-50%, honey has been shown to be superior to certain conventional antibiotics in treating urinary tract infections (Molan, 1992). The exact mechanism of the anti-microbial effect of honey remains obscure. Low pH, osmotic disruption of pathogens and the presence of bactericidal substances, collectively called inhibine may all play a part.

Honey has been reported to be effective in the healing of infected postoperative wounds (Al-waili et al., 1999). It has also been reported to inhibit the growth of a lot of bacteria such as Bacillus cereus, Staphylococcus aureus, Salmonella dublin and Shigella dysenteriae (El-Sukhon et al., 2001). It has also been reported to inhibit the growth of anaerobic bacteriodes (Elbagoury et al., 1993).

The evidence for the existence of other antimicrobial factors is mainly that the peroxide-generating system does not account for all of the observed antibacterial activity, but there have also been some reports of isolation of antibacterial substances from honey that are not hydrogen peroxide. Furthermore, it has been found that heating honey, which inactivates the glucose oxidase, causes loss of activity against some species whilst it is retained against others (Efem et al., 1992). Although the stability of the enzyme varies in different honey, there have been reports of honey with stability well in excess of this variation, showing that there must be an additional antibacterial factor involved. The most direct evidence for the existence of non-peroxide antibacterial factors in honey is seen in the reports of activity persisting in honeys treated with catalase to remove the hydrogen peroxide activity.

 The purpose of this study was to investigate the anti-fungal action of honey against Candida albicans, Aspergillus niger, Aspergillus flavus and Fusarium spp. The experiment was taken up using pure natural honey rather than commercially processed and strained honey which loses up to half of its original vitamin content.

1.2       Aims and Objectives of the Study

The general aim of the research is to determine the antifungal abilities of the honey samples obtained from different locations in Nigeria

1.3       Objectives of the Study

·       To determine the antifungal activity of the honey samples against some pathogenic fungal isolates.

·       To determine the minimum inhibitory concentration.

·       To determine the minimum fungicidal concentration.

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