TABLE OF CONTENT
Title Page
Certification
Dedication
Acknowledgement
Abstract
Table of Content
List of Abbreviations
List of Tables
List of Figures
List of Pictures
List of Plates
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1.1. Introduction
1.2. Statement of Problem
1.3. Aim of the Study
1.4. Objectives of the Study
1.5. Literature Review
1.5.1. Ancient use of Honey as a Medicine
1.5.2. Honeys as Modern Medicine
1.5.3. Some Physical and Chemical Properties of Honey
1.5.4. Factors contributing to Antimicrobial Properties of Honey
1.5.4.1. Osmotic Effect
1.5.4.1. Acidity
1.5.4.3. Hydrogen Peroxide Production
1.5.4.4. Non-Peroxide Components
1.5.4.5. Antioxidant Activity
1.5.5. Therapeutic Properties of Honey
1.5.5.1. Antimicrobial Activity
1.5.5.2. Anti-Inflammatory Activity
1.5.5.3. Anti-Oxidant Activity
1.5.5.4. Boosting of Immune System
1.5.6. Clinical Conditions for Treatment with Honey
1.5.6.1. As Remedy for Diarrhoea
1.5.6.2. As Medicine for Gastric Ulcers
1.5.6.3. As Medicine for Canine Recurrent Dermatitis
1.5.6.4. As Immune Inducer
1.5.6.5. As Anti-diabetic Agent
1.5.6.6. Antimutagenic and Antitumor Activity
1.5.6.7. As Treatment for Arthritis
1.5.6.8. As Skin Disinfectant
1.5.6.9. The Action of Honey in Wound Healing
1.5.7. Factors that affect Antimicrobial Activity of Honey
CHAPTER TWO: MATERIALS AND METHODS
2.0.Collection of Honey Samples
2.1.Extraction Procedure
2.2.Physico-Chemical Analyses
2.2.1. Determination of pH
2.2.2. Moisture Content
2.2.3. Determination of Electrical Conductivity and Total Dissolved Solids
2.2.4. Determination of Colour Characteristics
2.2.5. Determination of Colour Intensity (Abs450)
2.2.6. Determination of the Total Sugar Contents
2.2.7. Reducing and Non-Reducing Sugar Contents
2.2.8. Determination of Hydroxymethyl Furfural (HMF)
2.2.9. Determination of Free, Total, and Lactone Acidities
2.2.10. Determination of Protein Contents
2.2.11. Determination of the Total Phenolic Contents
2.2.12. Determination of the Total Flavonoid Content
2.2.13. Determination of Proline Content
2.2.14. Determination of Ascorbic Acid Content
2.3. Antimicrobial Activity Tests
2.3.1. Preparation of Honey and Solutions
2.3.2Collection of Test Microorganisms
2.3.3 Preparation of Standard Inocula
2.3.4 Antimicrobial Susceptibility Testing of Honey
2.3.5Determination of Minimum Inhibitory Concentration (MIC)
2.3.6Determination of Minimum Biocidal Concentration
2.3.7Determination of Non-Peroxide Antimicrobial Activities of Honeys
2.4. Statistical Analyses
CHAPTER THREE: RESULTS
3.1. Results of the Physicochemical Analyses
3.1.1. pH of the Honey Samples
3.1.2. Moisture Content
3.1.3. Electrical Conductivity
3.1.4. Total Dissolved Solids (TDS)
3.1.5. Colour Characteristics and Colour Intensity (ABS450)
3.1.6. Total Sugars, Reducing Sugars and Sucrose Contents
3.1.7. Determination of HMF Concentrations
3.1.8. Total, Free and Lactone Acidities of the different Honey Samples
3.1.9. Protein Contents of the Honey Samples
3.1.19. Total Polyphenol Content
3.1.11. Flavonoid Content
3.1.12. Proline Content
3.1.13. Ascorbic Acid Content
3.1.14. Correlation among some Physicochemical Parameters
3.2. Antimicrobial Activity screeningof the Honey Varieties
3.2.1. Antimicrobial Activity of Apis mellifera Honey Samples
3.2.2. Antimicrobial Activity of Hypotrigona sp. Honey Samples
3.2.3. Antimicrobial Activity of Melipona sp. Honey Samples
3.3. Mean Antibacterial Activities of the Different Honey Varieties
3.4. Minimum Inhibitory Concentration of investigated Honey Samples
3.4.1. MIC of Apis mellifera Honey Samples
3.4.2. MIC of Hypotrigona sp. Honey Samples
3.4.3. MIC of Melipona sp. Honey Samples
3.4.4. MIC of non-peroxidase Activity of the Honey Varieties
3.5. Mean MIC of Apis mellifera, Hypotrigona sp. and Melipona sp. Honeys
3.6. Minimum Biocidal Concentration of investigated Honey Varieties
3.6.1. MBC of Apis mellifera Honey Samples
3.6.2. MBC of Hypotrigona sp. Honey Samples
3.6.3. MBC of Melipona sp. Honey Samples
3.6.4. MBC of non-peroxidase Activities of the Honey Varieties
3.7. Mean MBC of Apis mellifera, Hypotrigona sp. and Melipona sp. Honeys
3.8. Mean of non-peroxidase MIC and MBC
3.9. MICs and MBCs of control drugs
CHAPTER FOUR: DISCUSSION
4.1. Discussion
4.1.1. Physicochemical Properties of the Honeys
4.1.2. Antibacterial Activities of the HoneyVarieties
4.2. Conclusion
4.3. Recommendations
REFERENCES
APPENDICES
ABSTRACT
Honey has been used traditionally for ages to treat infectious diseases. Antimicrobial activity of honey is complex due to the involvement of multiple bioactive compounds. The physico-chemical and antimicrobial properties of honey varieties from Apis mellifera and stingless bees,Hypotrigona sp. (Okotobo - Igbo) and Melipona sp. (Ifufu - Igbo) were studied using International Honey Commission protocols and microbiological methods (agar-well diffusion and broth microdilution) respectively. A total of nine honey samples (3 from each) were used. The physico-chemical analyses of the honey varieties showed that the honeys had mean pH range of 3.73±0.08 - 4.24±0.20. Honey samples from Hypotrigona sp. had the highest mean moisture (17.50 ± 0.80 %), total dissolved solids (370.01 ± 22.51 ppm), hydromethylfurfural (16.58 ± 0.37 mg/kg), total acidity (35.57 ± 0.42me q/kg), protein content (16.58 ± 0.37 g/kg)and phenol content (527.41 ± 3.60 mg/kg). Melipona sp. honey had the highest average flavonoids (86.39 ± 4.69 mg/kg), total sugar (80.71 ± 1.37 % (g/100g) and reducing sugar (75.64 ± 1.99 % (g/100g) contents. There were no statistically significant differences between the mean pH, electrical conductivity and protein contents of A. mellifera and Melipona sp. honeys (p< 0.05). Several strong correlations were observed among some of the physicochemical properties of these honey varieties. In the initial antimicrobial activity testing, Hypotrigona sp. honey samples had statistically the highest mean inhibition zones diameter (mm) against MDR Staphylococcus aureus (7.14 ± 4.11), Klebsiella pneumonia (7.92 ± 3.96),Pseudomonas aeruginosa ATCC 25783 (9.77 ±4.58), MDR S. enterica (6.96 ± 4.03), and Aspergillus niger (10.12 ± 5.51).The minimum inhibitory concentrations (MICs) of the honey varieties from A. mellifera, Hypotrigona sp. and Melipona sp. ranged from 6.3 – 25.0%, 3.1 – 12.5% and 6.3 – 25.0% (v/v) respectively. There were no statistically significant differences between the mean MICs of A. mellifera, Hypotrigona sp. and Melipona sp.honeys against P. aeruginosa ATCC 25783 (7.64 ± 2.76, 7.28 ± 4.14 and 8.33 ± 3.31 % v/v respectivel y).Hypotrigona sp. honey had the least mean MICs (4.15 ± 1.58 – 11.11 ± 2.76 % v/v) against most of the test organisms.The minimum biocidal concentration (MBC) of the honey varieties fromA. mellifera, Hypotrigona sp. and Melipona sp. against the test organismsvaried from 6.3
– 50%, 3.1 – 25% and 12 – 50% (v/v) respectively. T here were no statistically significant differences between the mean MBCs of the honey varieties against
Klebsiella pneumonia(p = 0.669),P. aeruginosa ATCC 25783 (p = 0.977), A. niger(p
= 0.688) and C. albicans (p = 0.168).The honey varieties had exceptional levels of hydrogen peroxide-dependent activity, and non-peroxide activity against the test organisms. This research has also shown that the honey varieties varied significantly in their physicochemical and antimicrobial properties. ‘Okotobo’ and ‘ifufu’ honeys that are both not consumed as widely as regular bee honeyhave shownto contain bioactive compounds and have antimicrobial properties similar to those of regular bee honey.
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1.1. INTRODUCTION
Traditional medicine has been used to treat infections since the origin of
mankind and honey is one of the oldest medicines considered as a remedy for microbial infections (Cooper et al., 2009). It was not until late 19th century that researchers discovered that honey has natural antimicrobial qualities (Zumla and Lulat, 1989). Resistance to antibiotics continues to rise and few new therapies are on the horizon, there is further increased interest in the antimicrobial potency of honey (Fahim et al., 2014). Previous studies showed that honey hadremarkable antimicrobial activity against fungi, bacteria,viruses and protozoa(Molan, 1992; Sherlock et al., 2010; Mohapatra et al., 2011; Fahim et al., 2014).
Honey is a natural sweet mixture produced by honey insects from the nectar of flowers or from living parts of plants. The insect transform the nectar into honey by combining this mixture with substances of their own. The mixture is then regurgitated, dehydrated and stored in the waxy honeycomb inside the hive to ripen and mature for further use (Iurlina and Fritz, 2005). Honey is composed mainly of carbohydrates, smaller amount of water and a great number of minor components. Sugars are the main constituents of honey, constituting of about 95%. Honey characterization is based on the determination of its chemical, physical or biological properties (Gomes et al., 2010).
Even though honey is produced worldwide, its composition and antimicrobial activity can be variable, and are dependent primarily on their botanical origin, geographical and entomological source (Maryann, 2000). Other certain external factors, such as harvesting season, environmental factors, processing and storage condition, also play important roles (Gheldof and Engeseth, 2002). Entomologically, the honey variety produced by honey bees (the genus Apis) is one most commonly referred to, as it is the type of honey collected by most beekeepers and consumed by most people in Nigeria. Honeys produced by other insects (stingless insects) have different properties (Sherlock et al., 2010).
Antimicrobial activity of honey is highly complex due to the involvement of multiple compounds and also due to large variations in the concentrations of these compounds among honeys. It depends on osmotic effect (sugar concentration), hydrogen peroxide, and low pH, as well as more recently identified compounds, methyl glyoxal and antimicrobial peptide, bee defensin-1 (Fahim et al., 2014).
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