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University of Ghana College of Basic and Applied UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES DEPARTMENT OF NUTRITION AND FOOD SCIENCE CHEMICAL COMPOSITION AND ANTIMICROBIAL EFFICACY OF ESSENTIAL OIL FROM XYLOPIA AETHIOPICA FRUIT PODS BY DZESI KWAME TORKU (10551784) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL FOOD SCIENCE DEGREE JULY 2017 DECLARATION I declare that this thesis was carried out by me under supervision in the Department of Nutrition and Food Science, University of Ghana, Legon. I take full responsibility for whatever has been reported here. Related work by others, which served as a source of information has been duly acknowledged by reference to the authors. -------------------------------------------- ------------------------------ TORKU DZESI KWAME DATE (STUDENT) ---------------------------------------------- ----------------------------- DR ANGELA PARRY-HANSON KUNADU DATE (PRINCIPAL SUPERVISOR) -------------------------------------------- ------------------------------ DR MAAME YAAKWAAH BLAY ADJEI DATE (CO - SUPERVISOR) -------------------------------------------- ------------------------------ PROF. FIRIBU KWESI SAALIA DATE (CO - SUPERVISOR) i DEDICATION This research work is dedicated to God Almighty, my dad Mr Benjamin Sefakor Cudjo Torku (of blessed memory), all my siblings, my mother Comfort Yevuyibor Stella Afua (Mrs. Torku), my bosom friend Mr Alexander Kwame Gavu and all who supported me financially during my study. ii ACKNOWLEDGEMENT I am grateful to God Almighty for his favour granted me throughout my research work. I earnestly appreciate my supervisors, Dr Angela Parry-Hanson Kunadu, Dr Maame Yaakwaah Blay Adjei and Prof. Firibu Kwesi Saalia for their intellectual support and motivation without which I could not have completed my research work. I am happy to have benefited from the Essential Oils Project at the Department of Nutrition and Food Science, University of Ghana, a project funded by the Office of Research, Innovation and Development, University of Ghana. I also thank Mr Samuel Quarcoo, CEO of Quin Organics (Cape Coast) for accepting partnership to assist in the extraction of essential oils used in the project. I appreciate the support of CSIR – Food Research Institute for donating bacterial strains for the research work. To the staff of Nutrition and Food Science Department, Sensory Evaluation Laboratory (UG- NFS), Microbiology Research Laboratory (UG-NFS) and Bacteriology Unit of Noguchi Memorial Institute, I say, I am grateful for your support. iii ABSTRACT Antimicrobial properties of essential oils (EOs) from plants have historically been explored and traditionally used as antiseptics and medicines. Recent studies on EOs of selected edible plants have shown great potential for inhibition of spoilage and pathogenic microorganisms that occur in foods. Food applications of essential oils have however been limited due to solubility issues, unaffordable extraction technologies, low yield and strong flavours etc. Indigenous spices are commonly used in traditional foods to add flavour. Limited studies have shown that spices such as Xylopia aethiopica may have antimicrobial properties. The objective of this study was the evaluate the chemical composition of Xylopia aethiopica fruit pods and assess its antimicrobial efficacy against prevalent foodborne pathogens in laboratory media and in food systems. The study assessed the chemical composition of the oil extracts using Gas Chromatograph-Mass Spectrometry (GC-MS) and determined the in vitro antimicrobial potential of the essential oil at 10%, 25%, 40%, 55%, 70% and 85% concentrations on Escherichia coli, Salmonella enterica serovar Typhimurium and Staphylococcus aureus using the modified Kirby-Bauer disk diffusion method. Also, overall sensory differences between spiked (2 µl/ml essential oil) and unspiked samples of bissap extract and ice kenkey were assessed using the triangle test. The study further assessed the antimicrobial effects of essential oils in ice kenkey and bissap extract through a microbiological challenge test, using a cocktail of organisms comprised of E. coli, S. Typhimurium and S. aureus. The study revealed that X. aethiopica essential oil was composed of 105 different chemical compounds out of which 104 were identified. EO constituents worked synergistically to inhibit microorganisms, producing a clear zone referred to as the zone of inhibition. The highest EO concentrations to S. Typhimurium (17.00±1.00 mm), S. aureus (13.33+0.58 mm) and E. coli (11.67 ± 0.58 mm) were 70%, 85% and 85% EO respectively. The positive control, Gentamycin (200 µg) was inhibitory to all organisms, E. coli 20.33 ± 0.58 mm, S. Typhimurium 22.33 ± 1.15 mm and S. iv aureus 23.67 ± 0.58 mm in ascending order, with zones of inhibition higher than those reported for the various EO concentrations. Essential oils of X. aethiopica in vitro could not inhibit any of the organisms at the least concentration (10%) tested on microorganisms. There was a significant sensory difference between EO spiked and unspiked samples of bissap extract and ice kenkey (p<0.05). Over 192hr of challenge studies, EO could not significantly influence pH in bissap and ice kenkey samples (p>0.05.) S. aureus and E. coli counts in ice kenkey spiked with EO under ambient conditions reduced for 323 (0hr) to 1 (192hr) and 41 (0hr) to 0 (192hr). In bissap samples kept under ambient and refrigerated conditions, growths of S. aureus, E. coli and S. Typhimurium were generally not recorded between the 0 hr to 192 hr challenge test. The antimicrobial efficacy of EO was generally higher in bissap extract than ice kenkey. v TABLE OF CONTENTS DECLARATION ........................................................................................................................ i DEDICATION ........................................................................................................................... ii ACKNOWLEDGEMENT ....................................................................................................... iii ABSTRACT .............................................................................................................................. iv TABLE OF CONTENTS .......................................................................................................... vi LIST OF TABLES .................................................................................................................... xi LIST OF FIGURES ................................................................................................................. xii LIST OF PLATES ................................................................................................................... xii CHAPTER ONE ........................................................................................................................ 1 1.0 INTRODUCTION ............................................................................................................ 1 1.1 Background of the study .................................................................................................. 1 1.2 Problem statement and Justification ................................................................................. 3 1.3 General objectives ............................................................................................................ 5 1.4 Specific Objectives ........................................................................................................... 5 CHAPTER TWO ....................................................................................................................... 7 2.0 LITERATURE REVIEW ................................................................................................. 7 2.1 History of spices ............................................................................................................... 7 2.2 Xylopia aethiopica (Negro pepper, Ethiopian pepper) ..................................................... 7 2.2.1 Scientific classification ................................................................................................. 9 2.2.2 Pre-and Post-harvest treatments and Usage of X. aethiopica...................................... 10 2.3 The need for novel and natural sources of antimicrobials ............................................. 10 2.4 Essential oils (Eos), their nature and applications.......................................................... 12 2.4.1 Variations in essential oils........................................................................................... 13 vi 2.4.2 Adulteration of essential oil ........................................................................................ 14 2.5 Extraction of essential oils – conventional and modern methods and technologies ...... 15 2.5.1 Solvent extraction methods ..................................................................................... 16 2.5.1.1 Extraction using organic solvents ..................................................................... 16 2.5.2 Other conventional methods of solvent extraction .................................................. 18 2.5.2.1 Steam distillation .............................................................................................. 18 2.5.2.2 Supercritical carbon dioxide ............................................................................. 19 2.5.2.3 Cold expression or pressing .............................................................................
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