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Investigation of Bacteria Associated with Australian Wine Grapes Using Cultural and Molecular Methods

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Investigation of Bacteria Associated with Australian Wine Grapes Using Cultural and Molecular Methods Investigation of bacteria associated with Australian wine grapes using cultural and molecular methods Sung Sook Bae A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy University of New South Wales Food Science and Technology School of Chemical Engineering and Industrial Chemistry Sydney, Australia 2005 i DECLARATION I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of materials which have been accepted for the award of any other degree or diploma at UNSW or any other education institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged. Sung Sook Bae ii ACKNOWLEDGEMENTS I owe a tremendous debt of gratitude to numerous individuals who have contributed to the completion of this work, and I wish to thank them for their contribution. Firstly and foremost, my sincere appreciation goes to my supervisor, Professor Graham Fleet. He has given me his time, expertise, constant guidance and inspiration throughout my study. I also would like to thank my co-supervisor, Dr. Gillian Heard for her moral support and words of encouragement. I am very grateful to the Australian Grape and Wine Research Development and Corporation (GWRDC) for providing funds for this research. Their support has truly been of great contribution to my education. The writing of a thesis can be a lonely and isolating experience; it is obviously not possible without personal support from numerous people. Thus, I would like to give my special thanks to Ai Lin, Peter, Hugh, Mi Hwa, Lidia, Maria and Pat for making nervous and sweating moments bright and cheerful with friendship and humour. Thanks are extended to the staff of Food Science and Technology who have provided assistance and have helped to make my association with the department a rewarding experience. I would like to express my love and gratitude to my parents, Yungsook, Sangsook, Joon Sung, Jung Mo and Mi Hyun who have full faith in my performance and for their unconditional support. Finally, I thank my beloved husband, Minseok for his unending support and love. iii TABLE OF CONTENTS DECLARATION ····························································································· i ACKNOWLEDGEMENTS ·············································································ii TABLE OF CONTENTS················································································ iii ABSTRACT ··································································································vii PUBLICATIONS AND PRESENTATIONS FROM THIS THESIS ················· ix CHAPTER 1 INTRODUCTION····································································1 CHAPTER 2 LITERATURE REVIEW 2.1 VITICULTURE ······················································································5 2.1.1 Grapes······························································································5 2.1.2 Grape cultivation········································································5 2.2 THE PROCESS OF WINE MAKING····················································10 2.2.1 Grapes·····························································································10 2.2.2 Crushing and pre-fermentation treatments······································10 2.2.3 Alcoholic fermentation·····································································12 2.2.4 Malolactic fermentation (MLF)·························································13 2.2.5 Post fermentation processes······················································ 14 2.3 ROLES OF MICROORGANISMS IN WINEMAKING···························15 2.3.1 Fungi ·····························································································15 2.3.2 Yeasts ·····························································································17 2.3.3 Bacteria ···························································································18 2.3.3.1 Lactic acid bacteria··························································18 2.3.3.2 Other bacteria····························································20 2.4 THE GRAPE SURFACE AS A MICROBIAL ECOSYSTEM ················20 2.4.1 The physical and chemical properties of grape surfaces·················22 2.4.2 Nutrients for bacterial growth on grape surfaces·····························24 2.4.3 Vineyard environmental factors·······················································27 2.4.4 Agrichemical influences···································································29 2.4.5 Sources of bacterial contamination of grapes ·································31 2.4.6 Bacterial properties ···································································33 2.5 BACTERIA ASSOCIATED WITH WINE GRAPES······························35 2.5.1 Lactic acid bacteria··········································································36 iv 2.5.1.1 Isolation from wines·························································36 2.5.1.2 Isolation from grapes and musts ·····································38 2.5.1.3 Isolation from winery environments·································40 2.5.2 Acetic acid bacteria ·········································································41 2.5.3 Other bacteria ··········································································43 2.6 METHODS FOR THE ANALYSIS OF BACTERIA ASSOCIATED WITH WINE GRAPES ··················································································44 2.6.1 Conventional microbiological analysis of wine grapes·····················44 2.6.2 Molecular strategies for monitoring bacterial communities··············46 2.6.3 Factors affecting the performance of molecular methods for analysis of bacteria ····································································48 2.6.3.1 DNA extraction································································48 2.6.3.2 PCR·················································································49 2.6.3.3 DGGE··············································································50 2.6.3.4 Interpretation of 16S rRNA sequence data ··················· 51 2.7 SUMMARY···························································································52 CHAPTER 3 BACTERIAL ECOLOGY OF WINE GRAPES DURING CULTIVATION AT SEVERAL VINEYARDS IN AUSTRALIA 3.1 INTRODUCTION··················································································53 3.2 MATERIALS AND METHODS·····························································55 3.2.1 Grape samples················································································55 3.2.2 Analysis of bacteria on grapes ························································60 3.2.3 Culture on agar media ·····································································60 3.2.4 Enrichment culture···········································································62 3.2.5 DNA extraction for bacterial identification and PCR-DGGE analysis ···························································································62 3.2.6 PCR amplification of DNA for sequence identification and PCR- DGGE analysis················································································63 3.2.7 Denaturing gradient gel electrophoresis (DGGE)····························65 3.2.8 Construction of DGGE validation marker·········································66 3.2.9 DNA sequencing·······································································67 3.2.10 Species identification········································································68 3.3 RESULTS·····························································································68 3.3.1 Climatological data for the vineyard regions in the years of 2001-2003 ·······················································································68 v 3.3.2 Population of bacteria on wine grapes during cultivation·················70 3.3.3 The diversity of bacterial species associated with wine grapes during cultivation·············································································74 3.3.3.1 Hunter Valley region, 2001-2002·····································74 3.3.3.2 Hunter Valley region, 2002-2003·····································77 3.3.3.3 Mudgee region, 2002-2003·············································87 3.3.3.4 Griffith region, 2002-2003················································92 3.3.4 Bacterial species associated with undamaged and damaged wine grapes·············································································94 3.3.5 Frequency of isolation of bacteria from wine grape samples···········99 3.4 DISCUSSION ······················································································101 3.4.1 Population and species diversity ····················································101 3.4.2 Analytical strategies for monitoring bacterial species associated with wine grapes··························································105
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