WINE MICROBIOLOGY. Practical Applications and Procedures. Kenneth C. Fugelsang Charles G. Edwards. Second edition

Transcription

1 WINE MICROBIOLOGY

2 WINE MICROBIOLOGY Practical Applications and Procedures Kenneth C. Fugelsang Charles G. Edwards Second edition

3 Kenneth C. Fugelsang Charles G. Edwards Department of Viticulture Department of Food Science and and Enology Human Nutrition California State University, Washington State University Fresno Pullman, WA Fresno, CA Library of Congress Control Number: ISBN-10: X e-isbn-10: ISBN-13: e-isbn-13: Printed on acid-free paper Springer Science+Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excepts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights springer.com

4 CONTENTS Preface to the Second Edition Acknowledgments Introduction xv xvii xix Section I: Grape and Wine Microorganisms Chapter 1: Yeasts INTRODUCTION REPRODUCTION Sexual Reproduction Asexual Reproduction TAXONOMY Candida Dekkera Hanseniaspora Issatchenkia Metschnikowia Pichia Saccharomyces Saccharomycodes 13 v

5 vi Contents Schizosaccharomyces Zygosaccharomyces NUTRITIONAL REQUIREMENTS Nitrogen Growth and Survival Factors METABOLISM Glucose Sulfur Odor/Flavor Compounds Glycosidases Mannoproteins 28 Chapter 2: Lactic Acid Bacteria INTRODUCTION TAXONOMY Lactobacillus Oenococcus Pediococcus NUTRITIONAL REQUIREMENTS METABOLISM Glucose Arginine Malate Mannitol and Erythritol Diacetyl and Other Odor/Flavor Compounds 41 Chapter 3: Acetic Acid Bacteria INTRODUCTION TAXONOMY NUTRITIONAL REQUIREMENTS METABOLISM Carbohydrates Ethanol 49 Chapter 4: Molds and Other Microorganisms INTRODUCTION ECOLOGY HABITATS TAXONOMY Aspergillus (Black Mold) Botrytis (Gray Mold) Penicillium (Blue-Green Mold) NUTRITIONAL REQUIREMENTS METABOLISM Glucose Mycotoxins Odor/Flavor Compounds 58

6 Contents vii 4.6 OTHER MICROORGANISMS Bacillus Clostridium Streptomyces 60 Section II: Vinifi cation and Winery Processing Chapter 5: Managing Microbial Growth INTRODUCTION PRESERVATIVES AND STERILANTS Sulfur Dioxide Dimethyl Dicarbonate Lysozyme Sorbic Acid Other Preservatives and Sterilants FILTRATION Perpendicular-Flow Filtration ( Nominal or Depth ) Perpendicular-Flow Filtration ( Absolute or Sterile ) Cross-Flow/Tangential Filtration 80 Chapter 6: Microbial Ecology During Vinification INTRODUCTION NON-SACCHAROMYCES AND SACCHAROMYCES YEASTS Grapes and Musts Alcoholic Fermentation Post-fermentation DEKKERA/BRETTANOMYCES Grapes and Musts Alcoholic and Post-fermentation LACTIC ACID BACTERIA Grapes and Musts Alcoholic and Malolactic Fermentations Post-fermentation ACETIC ACID BACTERIA Grapes and Musts Alcoholic and Post-fermentation MICROBIAL INTERACTIONS Saccharomyces and Oenococcus Saccharomyces and Lactobacillus Oenococcus, Pediococcus, and Lactobacillus Other Interactions 100 Chapter 7: Harvest and Pre-fermentation Processing INTRODUCTION HARVEST AND TRANSPORT 102

7 viii Contents 7.3 FRUIT QUALITY ASSESSMENT Soluble Solids ph and Titratable Acidity Microbial Spoilage MUST PROCESSING Enzymes Suspended Solids Pre-fermentation Maceration (Cold-Soak) Thermovinification Inert Gassing PROCESSING MICROBIALLY DETERIORATED FRUIT Enzymatic Browning Fermentation Difficulties Clarification Concerns Management Strategies JUICE STORAGE (MUTÉ) 113 Chapter 8: Fermentation and Post-fermentation Processing INTRODUCTION MUST SUPPLEMENTATION ALCOHOLIC FERMENTATION Historical Perspective of Starter Cultures Preparation of Starter Cultures Strain Selection Temperature Immobilized Yeast NATURAL FERMENTATIONS FERMENTATION PROBLEMS Sluggish/Stuck Fermentations Hydrogen Sulfide MALOLACTIC FERMENTATION Preparation of Starter Cultures Strain Selection Timing of Inoculation Use of Schizosaccharomyces POST-FERMENTATION PROCESSING Aging and Storage Adjustment of Volatile Acidity BOTTLING 137 Chapter 9: Winery Cleaning and Sanitizing INTRODUCTION SAFETY ISSUES WATER QUALITY PRELIMINARY CLEANING 141

8 Contents ix 9.5 DETERGENTS, CLEANERS, AND SURFACTANTS Alkali Acids Surfactants RINSES SANITIZERS Iodine Quaternary Ammonium Compounds Acidulated Sulfur Dioxide Peroxides Chlorine and Chlorinated Formulations Hot Water and Steam Ozone SANITATION MONITORING SCHEDULES AND DOCUMENTATION 151 Chapter 10: Quality Points Program INTRODUCTION DEVELOPING QP PROGRAMS PROCESSING AND FLOWCHART QUALITY FACTOR ANALYSIS Examples of Quality Factors Preventive Measures Critical Quality Points CRITICAL LIMITS MONITORING PROCEDURES CORRECTIVE ACTIONS RECORD-KEEPING AND DOCUMENTATION VERIFICATION 161 Chapter 11: Wine Spoilage INTRODUCTION SPOILAGE MICROORGANISMS Acetobacter Dekkera/Brettanomyces Film Yeasts Saccharomycodes Zygosaccharomyces WINE FAULTS Volatile Acidity Ethyl Carbamate Mousiness Post-MLF Bacterial Growth Geranium Odor/Tone Biogenic Amines Acrolein 176

9 x Contents Mannitol Ropiness Tartaric Acid Utilization 179 Section III: Laboratory Procedures and Protocols Chapter 12: Basic Microscopy INTRODUCTION MICROSCOPES Magnification Resolution Contrast Fluorescence Microscopy USING MICROSCOPES Components General Use Calibration PREPARING SMEARS From Liquid Media From Solid Media PREPARING WET MOUNTS PREPARING MOLD SLIDE CULTURES 192 Chapter 13: Media Preparation and Culture Techniques INTRODUCTION PHYSICAL/CHEMICAL REQUIREMENTS FOR CULTIVATION Carbon and Nitrogen Oxygen Hydrogen Ion Concentration (ph) Moisture and Water Activity Incubation Temperature and Conditions Selective Agents STERILIZATION OF LABORATORY MEDIA AND SUPPLIES Boiling Water Steam Sterilization Dry Heat Sterile Filtration Chemical Sterilization STORAGE OF PREPARED MEDIA MEDIA FOR YEASTS AND MOLDS Wort Medium Grape Juice Medium WL Medium Brettanomyces Medium A 207

10 Contents xi Brettanomyces Medium B Brettanomyces Medium C Brettanomyces Medium D Lysine Medium Zygosaccharomyces Medium MEDIA FOR LACTIC ACID BACTERIA Apple Juice Rogosa Medium Tomato Juice Glucose Fructose Malate Medium Heterofermentation-Arginine Broth MEDIA FOR ACETIC ACID BACTERIA Glucose Yeast Extract Carbonate Medium Mannitol Yeast Extract Peptone Medium Yeast Extract Peptone Ethanol Medium ASEPTIC TRANSFER TECHNIQUES Transfers from Solid to Solid Media Transfers from Solid to Liquid Media Transfers from Liquid to Solid Media ISOLATION OF MICROORGANISMS MAINTENANCE AND STORAGE OF CULTURES Preparation of Agar Slants and Stabs Glycerol Suspensions Freeze-drying (Yeasts) Freeze-drying (Lactic Acid Bacteria) 222 Chapter 14: Estimation of Population Density INTRODUCTION SAMPLING SAMPLE DILUTION DIRECT MICROSCOPIC COUNT Using a Microscope Counting Chamber Methylene Blue Ponceau-S Wolford s Stain DIRECT PLATING Pour Plates Spread Plates Membrane Filtration BIOLUMINESCENCE NEPHELOMETRY AND OPTICAL DENSITY 239 Chapter 15: Identification of Wine Microorganisms INTRODUCTION IDENTIFYING MICROORGANISMS 242

11 xii Contents 15.3 YEASTS AND MOLDS Assimilation of Carbon and Nitrogen Demonstration of Ascospores Demonstration of Mycelia/Pseudomycelia Fermentation of Carbohydrates BACTERIA Ammonia from Arginine Catalase Dextran from Sucrose Fermentation of Carbohydrates Gas from Glucose Gram Stain Ketogenesis Lactate from Glucose Malate Utilization (Monitoring MLF) Mannitol from Fructose Oxidation of Ethanol Oxidation of Lactate 271 Chapter 16: Other Technologies for Identification and Enumeration INTRODUCTION PHENOTYPICAL IDENTIFICATION Biolog System Fatty Acid Methyl Ester Analysis Protein Characterization Electrophoretic Characterization of Isozymes (Zymograms) Immunochemical Techniques Enzyme-Linked Immunosorbent Assay Immunochemical Fluorescence Microscopy Phylogenetic Analyses Polymerase Chain Reaction Gel Electrophoresis Quantitative Polymerase Chain Reaction Hybridization Probes Fluoresence In Situ Hybridization Ribonucleic Acid Analysis PROBE DETECTION SYSTEMS TaqMan Probes Molecular Beacons Scorpions Peptide Nucleic Acid Chemiluminescent In Situ Hybridization Probes 287

12 Contents xiii 16.6 OTHER MOLECULAR METHODS Restriction Fragment Length Polymorphisms Pulsed Field Gel Electrophoresis Additional Methods EXTRACHROMOSOMAL ELEMENTS (SATELLITES) 289 Chapter 17: Chemical and Physical Instabilities INTRODUCTION CRYSTALLINE INSTABILITIES Tartrates CRYSTAL-LIKE INSTABILITIES Cork Dust Diatomaceous Earth FIBROUS INSTABILITIES AMORPHOUS INSTABILITIES Protein Phenolics Glucans, Pectins, and Starch Metal Casse 300 Chapter 18: Laboratory Setup INTRODUCTION MICROSCOPE AND ph METER AUTOCLAVE CENTRIFUGE AND FILTERS INCUBATORS WATER BATHS GLASS AND PLASTICWARE MEDIA PHOTOMETERS LAMINAR FLOW HOODS MISCELLANEOUS 309 Chapter 19: Laboratory Safety INTRODUCTION INJURY AND ILLNESS PREVENTION PROGRAM Training Information Communication Eyewash Stations and Safety Showers Fire Alarms and Prevention First Aid Personal Protective Equipment EXAMPLES OF SAFETY ISSUES Biohazard and Chemical Waste Electricity 319

13 xiv Contents Heat and Steam Machinery Safeguards Storage Ultraviolet Light 320 Glossary 322 References 331 Index 381

14 PREFACE TO THE SECOND EDITION Organization of the large volume of material that needed to be included in Wine Microbiology was a difficult task as evidenced by the number of approaches attempted. The second edition is divided into three parts; Grape and Wine Microorganisms (Chapters 1 to 4), Vinification and Winery Processing (Chapters 5 to 11), and Laboratory Procedures and Protocols (Chapters 12 to 19). As subject areas frequently cross section or chapter boundaries, every effort was made to cross-reference related topics as a means to reduce difficulties in finding information. Section I, Grape and Wine Microorganisms, describes those microorganisms found in grape must, juice, and wines; namely yeasts, lactic and acetic acid bacteria, and molds. Here, taxonomy, metabolism, nutritional requirements, and potential impacts on wine quality are areas of focus. Section II, Vinification and Winery Processing, addresses on those microbiological issues of practical importance to the winemaker. Included here is a general discussion of microbial management followed by in-depth examination of microbial ecology. This section also describes general principles of sanitation (Chapter 9), implementation of a quality control program (Chapter 10), and specific wine spoilage issues (Chapter 11). xv

15 xvi Preface to the Second Edition Section III, Laboratory Procedures and Protocols, begins with an introduction to the use of the microscope (Chapter 12) and follows with methodologies used to enumerate and identify wine microorganisms (Chapters 13 through 16). Because organic and inorganic precipitates found in wine are often confused with microorganisms, methods of identification as well as photomicrographs of typical precipitates are included in Chapter 17. Chapters 18 and 19 provide insight into designing a wine microbiological laboratory and related safety issues. The section ends with a glossary of terms commonly used by microbiologists.

16 ACKNOWLEDGMENTS The authors would like to thank several individuals and organizations for their assistance in preparation of this book. First, we wish to extend thanks to California State University (Fresno, CA) and to Washington State University (Pullman, WA) for their support of this project. The authors also thank W.D. Edinger (Canandaigua Wine Company, Madera, CA), R. Morenzoni (Art of Winemaking, Modesto, CA), and B. Watson (Chemeketa Community College, Salem, OR) for technical review and S. Safren (Springer Science and Business Media) for her editorial assistance. Special thanks are extended to R.H. Dougherty and B.A. Rasco (Washington State University, Pullman, WA) for providing HACCP materials and technical advice as well as to Eiji Akaboshi, Peter Gray, Cheryl Mitchell, Henry Moore Jr., and Roy Thornton involved in obtaining and optimizing photomicrographs. Appreciation is given to the American Journal of Enology and Viticulture, American Public Health Association, Australian Journal of Grape and Wine Research, Blackwell Publishing, Elsevier Ltd., John Wiley & Sons Ltd., Journal of Bacteriology, Invitrogen Corporation, R. Pawsey, Springer Science and Business Media, WineBugs LLC, and Wines and Vines for granting copyright permissions to use figures and tables. xvii

17 xviii Acknowledgments The authors convey their gratitude to the wine industry, commercial suppliers, and university colleagues around the world. Without continual input, advice, and innovative ideas from many individuals, this project would not have been possible. Finally, special thanks are extended to undergraduate and graduate students, research associates, technicians, and other staff with whom the authors have worked. Through their efforts performing research and developing protocols, a much better understanding of wine microbiology has been gained by all.

18 INTRODUCTION The winemaking community worldwide continues to be a study of philosophical contrasts. On the one hand, there are those winemakers and wineries that emphasize the scientific segment of winemaking through adoption of new research findings and technologies. On the other hand, others prefer to embrace Old World traditions and thereby accentuate the artistic aspects associated with wine production. In writing this book, the objective was not to debate the relative merits and deficiencies of either philosophy but, rather, to create a reference that was useful to enologists as well as to researchers and students globally. Since publication of the first edition of Wine Microbiology in 1997, the volume of new information and concepts has dramatically increased. Perhaps one of the most intriguing developments in the past decade has been application of real-time molecular methods. Based on similarities at the gene level, these methods have evolved beyond esoteric laboratory exercises to the point where real-world problems can be solved through rapid identification of microorganisms. Another relatively new application has been the use of starter cultures of non-saccharomyces yeasts, which yield wines that differ not only in flavor and aroma profiles but also in structure. xix

19 xx Introduction Winemakers are also increasingly facing spoilage issues associated with Brettanomyces, Lactobacillus, Pediococcus, and Zygosaccharomyces, some of these being consequences of changes in viticultural practices (e.g., increased so-called hang-time). Even with the tremendous increase in available information, a comprehensive understanding regarding the role of individual microorganisms toward wine quality as well as the impact of complicated interactions between microorganisms and processing techniques is lacking. A good example would be Brettanomyces, probably the most enigmatic and controversial microorganism in the wine industry. Although initially thought of as a major threat, some winemakers are beginning to view Brettanomyces as a potential ally in the vintages of the new millennium. Hopefully, additional research and experience will provide winemakers with better microbiological control during vinification, which, in turn, will lead to a continued increase in wine quality. We sincerely hope that you find the second edition of Wine Microbiology informative and useful in your winery, laboratory, or classroom. If you have any feedback for the authors (potential errors, ideas for the third edition, and the like), please feel free to write or us. Cheers! Kenneth C. Fugelsang and Charles G. Edwards February 14, 2006