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(Gryllus Bimaculatus and Scapsipedus Icipe) Used As a Source of Protein for Food

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(Gryllus Bimaculatus and Scapsipedus Icipe) Used As a Source of Protein for Food ANALYSIS OF MICROBIAL LOAD AND DIVERSITY IN CRICKETS (GRYLLUS BIMACULATUS AND SCAPSIPEDUS ICIPE) USED AS A SOURCE OF PROTEIN FOR FOOD TITLE GATHERU JEDIDA WANJIRU (B.Ed. Sci) I56/27235/2014 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE (MICROBIOLOGY) IN THE SCHOOL OF PURE AND APPLIED SCIENCES OF KENYATTA UNIVERSITY JULY 2019 ii DECLARATION This thesis is my original work and has not been presented for award of a degree in any other university or for any other award. Signature………………………… Date ………………… Gatheru Jedida Wanjiru REG. NO. I56/27235/2014 Department of Microbiology APPROVAL BY THE SUPERVISORS We confirm that the work recorded in this thesis was carried out by the candidate under our supervision. Dr. James Nonoh Signature…………… Date……………… Microbiology Department, Kenyatta University, Kenya Dr. Julius Maina Signature…………… Date……………… Food Science Department, Jomo Kenyatta University, Kenya Dr. Fathiya Khamis Signature…………… Date……………… Scientist, Plant Health Division, International Centre for Insect Physiology and Ecology iii DEDICATION This work is dedicated to the Almighty God for giving me life. To my family especially my husband Nicholas Kinyua, my sons Quincy Murimi, Adrian Gatheru and Edison Nyagah for the love, patience and understanding they showed me during the entire study without which this work could not have seen the light of the day. iv ACKNOWLEDGEMENTS I greatly appreciate and acknowledge Kenyatta University (KU) for the opportunity to be a student and the International Centre of Insect Physiology and Ecology (ICIPE) for the financial and technical support accorded to me during my study. My sincere gratitude goes to all individuals who in one way or another contributed to the successful completion of this work. I am especially deeply indebted to Dr(s) Subbi, Komi, Samira and Ekesi for the opportunity, unlimited resources and ambient workspace without which this work would not have been possible. My supervisors Dr. James Nonoh, Dr. Julius Maina and Dr. Fathiya Khamis for their expert and splendid selfless academic guidance, wise counsel, patience, unwavering concern, persistent encouragement, immense support and inspiration throughout the research and thesis development. Dr. Tanga for assisting in analysis of the work and Mr. Levi Ombura, for his tireless technical advice and invaluable support he accorded me during this study, I also wish to acknowledge all the scientists and technologists at the International Centre of Insect Physiology and Ecology (ICIPE) who supported me each in their own way to complete this work. Above all, I thank the almighty God for giving me life and blessing this work from the beginning to the end. v TABLE OF CONTENTS TITLE .................................................................................................................................. i DECLARATION ................................................................................................................ ii DEDICATION ................................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................... iv TABLE OF CONTENTS .................................................................................................... v LIST OF FIGURES ............................................................................................................ x LIST OF PLATES ............................................................................................................. xi LIST OF APPENDICES ................................................................................................... xii ABBREVIATIONS AND ACRONYMS ........................................................................ xiii ABSTRACT ..................................................................................................................... xiv CHAPTER ONE: INTRODUCTION ................................................................................. 1 1.1 Background ................................................................................................................ 1 1.2 Statement of the problem ........................................................................................... 5 1.3 Justification ................................................................................................................ 5 1.4 Hypotheses ................................................................................................................. 6 1.5 Objectives .................................................................................................................. 7 1.5.1 General objective ................................................................................................ 7 1.5.2 Specific objectives .............................................................................................. 7 1.6 Significance of the study ............................................................................................ 7 CHAPTER TWO: LITERATURE REVIEW ..................................................................... 8 2.1 Depending on edible insects to improve livelihoods ................................................. 8 2.2 Nutritional value of edible insects ............................................................................. 9 2.3 Insect farming for food ............................................................................................ 11 2.4 Major groups of edible insects ................................................................................. 12 vi 2.5 Insects consumption patterns ................................................................................... 13 2.6 Microbes associated with edible insects .................................................................. 14 2.6.1 Bacterial hazards ............................................................................................... 14 2.6.2 Fungal hazards .................................................................................................. 17 2.7 Identification of microbial contaminants of insects ................................................. 18 2.7.1 Fungal identification ............................................................................................. 19 2.7.2 Bacterial identification .......................................................................................... 19 2.8 Accepted microbial levels in raw and processed meat ............................................ 20 2.9 Processing procedures .............................................................................................. 20 2.9.1 Freezing ............................................................................................................ 20 2.9.2 Heating.............................................................................................................. 21 2.9.3 Drying ............................................................................................................... 21 2.10 Storage of edible insects ........................................................................................ 21 CHAPTER THREE: MATERIALS AND METHODS ................................................... 23 3.1 Study sites ................................................................................................................ 23 3.2 Traps ........................................................................................................................ 24 3.3 Sample rearing ......................................................................................................... 24 3.4 Sample preparation .................................................................................................. 26 3.5 Isolation of bacteria and fungi ................................................................................. 27 3.6 Determination of microbial load of crickets ............................................................ 27 3.7 Morphological characterization ............................................................................... 28 3.8 Harvesting of cells for molecular characterization .................................................. 28 3.9 Molecular characterization ....................................................................................... 29 3.9.1 Bacterial DNA extraction ................................................................................. 29 3.9.2 Fungal DNA extraction .................................................................................... 29 vii 3.9.3 Polymerase Chain Reaction (PCR) ................................................................... 29 3.9.4 Gel electrophoresis ........................................................................................... 30 3.9.5 Purification of PCR products ............................................................................ 30 3.9.6 Sequencing........................................................................................................ 31 3.10 Data Analysis ......................................................................................................... 31 CHAPTER FOUR: RESULTS ......................................................................................... 33 4.1 Morphological characteristics of fungal Isolates ..................................................... 33 4.2 Morphological characteristics of bacterial isolates
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