Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Antimicrobial effect of Manuka honey and Kanuka honey alone and in combination with the bioactives against the growth of Propionibacterium acnes ATCC 6919 A Thesis submitted in partial fulfilment of the requirements for the degree of Master of Food Technology Massey University Albany, New Zealand Qiong Wu June 2011 Abstract Background: Acne vulgaris is a chronic inflammatory disease of the pilosebaceous follicle. The Propionibacterium acnes (P. acnes) play a key role in inflammation and the formation of comedones. P. acnes has been reported to develop antibiotic resistance, which has generated some interest in developing natural antimicrobial compounds which forms the subject matter of this study. Recently, Manuka honey (leptospermum scoparium) has demonstrated strong antibacterial activities against a wide range of pathogens with distinct non-peroxide activity. Kanuka honey has also shown to be effective against many bacterial species. Many natural bioactives were reported to possess strong antibacterial activities, a few of which were studied against P. acnes. Therefore, the aims of this study were to investigate the antibacterial activity of Manuka honeys and Kanuka honey with and without catalase against the growth of P. acnes alone and to screen the antibacterial activities of five selected nature bioactives alone and in combination with honey against P. acnes in vitro. Methods: The growth of P. acnes was evaluated under aerobic and anaerobic conditions. P. acnes was cultivated in nutrient broth and fastidious anaerobic agar containing horse blood. Manuka honeys 20+, 15+ and 10+ UMF and Kanuka honeys were tested against the growth of P. acnes, ranging from 0.5 % to 12.5 % (w/v) with and without catalase under both aerobic and anaerobic conditions. The artificial honey was used as the control. Manuka tree essential oil (MTO), lavender essential oil (LO), green tea extract (GTE), olive leaves extract (OLE), propolis were screened using disc diffusion method, spectrophotometric assay, viable cell counts to determine the survival of P. acnes in the bioactives testing solutions. The combination creams of Manuka honey 10+ UMF (10 %, w/v) with bioactives were studied using viable cell count method to determine the viable cells of P. acnes. Results: P. acnes is capable of growing under both aerobic and anaerobic conditions. Manuka and Kanuka honeys exhibited antibacterial activity against the growth of P. acnes. Kanuka honey had similar antibacterial activity as Manuka honey 15+ UMF and Manuka honey 20+ UMF without catalase. MIC of Manuka honey 20+ UMF was 100 148.90 mg/mL; MIC of Manuka honey 15+ UMF was 125.81 mg/mL; MIC of 100 100 Manuka honey 10+ UMF was 144.43 mg/mL; MIC of Kanuka honey was 123.28 100 mg/mL. Manuka honeys possessed non-peroxide activity, but the antibacterial activity of Kanuka honey decreased significantly after the removal of hydrogen peroxide with MIC of 549.21 mg/mL. Artificial honey did not markedly inhibit the growth of P. 100 acnes. Among the five bioactives, only GTE and MTO had bactericidal ability. Honey creams with bioactives showed that cream containing 10 % honey and 1 % GTE caused about five log reductions in the bacterial cell numbers; in contrast, cream of honey (10 %) and MTO (0.125 %) resulted in about two log reductions. No bacterial cells (< 100 CFU/mL) were found in the creams containing honey (10 %), MTO (0.125 %) and GTE (1 %). Conclusion: Manuka honey exhibited antibacterial activity against the growth of the P. acnes. The antibacterial potency of the honey was significantly enhanced by the presence of bioactives in the emulsion cream. i A CKNOWLEDGEMENTS It has been a great experience for me to pursue my post-graduate study in Food Technology at Massy University. This thesis would not have been possible without the guidance and the help of several individuals who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study. I would like to thank Dr. Tony Mutukumira, my supervisor from Massey University, who has believed me and gave me unlimited support and guidance throughout the whole process of my study life. I would also like to thank, Dr Jonathan Stephens, Bilge Thompson, and Dr Ralf Schlothauer from Comvita New Zealand Ltd, who gave me this great opportunity to work on a commercial orientated project, unlimited supervision, and invaluable contribution. I also acknowledge Rachel Liu for her assistance in organising laboratory equipment and purchasing testing materials for the experiments. I also would like thank Chirag Ahuja, Comvita New Zealand Ltd, for helping me to prepare bioactives solutions. Finally, my special thanks go to Comvita New Zealand Ltd and Technology New Zealand for providing financial support. I am thankful to have Lei Jiao as my partner, who brings love, happiness and encouragement in my life and study, whose advice and support helped me accomplish everything. Most of all, I would like to thank my parents for their unconditional love. ii TABLE OF CONTENTS ABSTRACT ................................................................................................... i ACKNOWLEDGEMENTS ..............................................................................ii TABLE OF CONTENTS ............................................................................... iii LIST OF FIGURES .................................................................................... viii LIST OF TABLES ..................................................................................... xiii LIST OF APPENDICES .............................................................................. xiv ABBREVIATIONS ..................................................................................... xvi 1.0 INTRODUCTION ........................................................................................ 1 2.0 LITERATURE REVIEW ............................................................................... 7 2.1 Acne vulgaris ............................................................................................ 7 2.1.1 Grading of acne ........................................................................................ 8 2.1.2 Pathogenesis of acne vulgaris .................................................................. 9 2.2 Propionibacterium acnes (P. acnes) ...................................................... 12 2.2.1 Taxonomic classification ........................................................................ 12 2.2.2 Genomic structure .................................................................................. 12 2.2.3 Cellar and colonial morphology ............................................................. 13 2.2.4 Biofilm formation ................................................................................... 14 2.2.4 Biochemical characteristic of P. acnes .................................................. 15 2.2.5 Metabolism of P. acnes .......................................................................... 15 2.2.7 Incubation temperature of P. acnes ........................................................ 17 2.2.8 Media for cultivation the P. acnes ......................................................... 17 2.3 Treatment of acne ................................................................................... 17 2.4 Honey and healing .................................................................................. 21 2.4.1 Historical overview of the use of honey in medicine ............................. 21 iii 2.4.2 The characteristics of honey ................................................................... 25 2.4.3 Sensory and physical characteristics of honey ....................................... 26 2.4.4 Chemical characterises of honey ............................................................ 27 2.4.5 Antibacterial activity of honey ............................................................... 30 2.4.6 Manuka honey, a unique honey .............................................................. 32 2.4.7 Kanuka honeys ....................................................................................... 36 2.5 Bioactives and their antibacterial properties ........................................... 37 2.5.1 Manuka tree oil ....................................................................................... 37 2.5.2 Lavender oil ............................................................................................ 41 2.5.3 Green tea extract ..................................................................................... 47 2.5.4 Olive leaf extract and its antibacterial activity ....................................... 53 2.5.5 Propolis ................................................................................................... 57 2.6 Susceptibility methods for P. acnes ........................................................ 62 2.6.1 Various susceptibility test methods ........................................................ 62 2.6.2 Preparation of bioactives in susceptibility testing of P. acnes ............... 68 3.0 MATERIALS AND METHODS .................................................................... 70 3.1. The in vitro study of P. acnes ................................................................. 70 3.1.1 Bacterial strain and media ...................................................................... 70 3.1.2 Preparation of media............................................................................... 70 3.1.3 Preparation of the broth culture of P. acnes ........................................... 71 3.1.4 Analyzing the growth of P. acnes using microtiter plate reader ............ 72 3.2 The antibacterial activities of Manuka honeys and Kanuka honey ........ 73 3.2.1 Honey samples........................................................................................ 73 3.2.2 Spectrophotometric assay using microplate reader ................................ 74 3.3 Screening natural bioactives ................................................................... 76 3.3.1 Preparation of stock solutions of bioactives ........................................... 76 iv 3.3.2 Disc diffusion method ............................................................................ 78 3.3.3 Spectrophotometric assay ....................................................................... 82 3.4 Effects of bioactives and Manuka honey on killing rates of P. acnes .... 84 3.5 Evaluating the antibacterial activities of emulsion creams containing honey and bioactives .............................................................................. 85 3.5.1 Preparation of P. acnes inoculum .......................................................... 85 3.5.2 Preparation of cream containing honey and bioactives separately ........ 85 3.5.3 Preparation of cream samples containing honey and bioactives cooperatively .......................................................................................... 87 3.5.4 Evaluation of antibacterial ability of creams with honey and bioactives88 3.6 Statistical analysis .................................................................................. 88 4.0 RESULTS ................................................................................................. 89 4.1 The in vitro study of P. acnes ................................................................. 89 4.1.1 Characterisation of P. acnes when grown on the Fastidious Anaerobic agar plate ................................................................................................ 89 4.1.2 Growth of P. acnes in BHI broth ........................................................... 91 4.1.3 Gram stain of P.acnes ............................................................................ 92 4.1.4 Growth of P. acnes measured by microtiter plate reader ....................... 93 4.1.5 Correlation between the standard plate count and absorbance readings 95 4.2 The antibacterial activities of Manuka honey and Kanuka honey ......... 96 4.2.1 Growth kinetics of P. acnes in Manuka and Kanuka honeys under aerobic environment ............................................................................................ 96 4.2.2 Growth kinetics of P. acnes in the Manuka honey and Kanuka honey samples with catalase (non-peroxide) under aerobic environment ...... 101 4.2.3 Growth kinetics of P. acnes in Manuka and Kanuka honey samples .. 106 4.2.4 Growth (anaerobic) kinetics of P. acnes in the Manuka and Kanuka honey samples with catalase ........................................................................... 110 v 4.2.5 Growth of P. acnes in the artificial honey solution .............................. 115 4.2.6 Dose-response curves of P. acnes in Manuka and Kanuka honey samples .............................................................................................................. 118 4.2.7 Determination of the MICs of Manuka and Kanuka honey samples ... 122 4.3 Screening natural bioactives ................................................................. 125 4.3.1 Disc diffusion method .......................................................................... 125 4.3.2 Inhibition potential of bioactive against P. acnes using the spectrophotometer method ................................................................... 129 4.3.3 Antibacterial activity of five bioactives determined by the minimum bactericidal concentration ..................................................................... 139 4.3.4 Time-to-kill curves of GTE and MTO ................................................. 140 4.4 Evaluating the antibacterial activities of formulated emulsion creams containing honey and bioactives ........................................................... 143 4.4.1 Antibacterial activity of cream containing honey MTO, GTE separately .............................................................................................................. 143 4.4.2 Antibacterial activity of honey cream formula with and without bioactives .............................................................................................................. 146 4.4.3 Comparison of the antibacterial activity of honey formula with bioactives and commercial products ...................................................................... 148 5.0 DISCUSSION .......................................................................................... 149 5.1 The in vitro study of P. acnes ................................................................ 149 5.1.1 Colonial and bacterial cell morphology ............................................... 150 5.1.2 Biochemical characteristics of P. acnes grown under both aerobic and anaerobic environment ......................................................................... 151 5.2 The antibacterial activity of Manuka honey and Kanuka honey .......... 153 5.2.1 Spectrophotometric assay to determine the antibacterial activity of Manuka and Kanuka honeys ................................................................ 153 vi 5.2.2 Dose-response curves and MIC for determining the antibacterial activity of Manuka and Kanuka honeys ............................................................ 154 5.2.3 The kinetic bacterial growth of P. acnes in Manuka honey and Kanuka honey .................................................................................................... 155 5.2.4 The antibacterial activity of Manuka honey and Kanuka honey .......... 156 5.2.5 Non-peroxide activity of Manuka and Kanuka honeys ........................ 157 5.2.6 Effect of honey concentrations on the antibacterial activity of Manuka honey and Kanuka honey ..................................................................... 158 5.3 Screening natural bioactives for antibacterial activities ....................... 160 5.3.1 The antibacterial activity of manuka tree oil ........................................ 160 5.3.2 The antibacterial activity of lavender oil .............................................. 162 5.3.3 The antibacterial activity of green tea extract ...................................... 164 5.3.4 The antibacterial activity of olive leaf extract ...................................... 165 5.3.5 The antibacterial activity of Propolis ................................................... 166 5.4 Combined effects of honey and bioactives ........................................... 168 5.5 Evaluation of the antibacterial activity of formulated emulsion creams .... .............................................................................................................. 169 6.0 CONCLUSION ........................................................................................ 171 7.0 RECOMMENDATIONS ............................................................................ 174 8.0 REFERENCES ........................................................................................ 176 vii