Handbook of Vanilla Science and Technology

Dr. Daphna Havkin-Frenkel, Department of Plant Biology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey, USA. Dr. Faith C. Belanger, Department of Plant Biology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey, USA.

• List of Contributors xixPreface xxiiiPart I Production of Vanilla – Agricultural Systems and Curing 11 Mexican Vanilla Production 3Juan Hernandez‐Hernández1.1 Introduction 31.1.1 The Mexican Vanilla Legend 41.2 Cultivation Methods 51.2.1 “Traditional”/Acahual 51.2.2 Intensive System (Monoculture) 61.2.3 Vanilla Cultivation in Existing Orange Groves 61.2.4 Shade Houses 71.3 Vanilla Propagation Techniques 81.3.1 Preparation and Disinfection of Cuttings 81.3.2 Establishing Cuttings – Timing 81.3.3 Establishing Cuttings – Planting 91.3.4 New Bud Formation and Root Growth 91.4 Irrigation 91.5 Nutrition 101.5.1 Mulch 101.5.2 Building Compost 101.6 Weed Control 111.7 Shade Management (Pruning of Support Trees) 111.8 Shoot Management – Looping 121.9 Shoot Management – Rooting 121.10 Main Vanilla Insect Pest 121.11 Main Vanilla Diseases 131.11.1 Anthracnose 141.11.2 Rust 141.11.3 Yellowing and Pre‐mature Fruit Drop 141.12 Flowering and Pollination 141.12.1 Percent of Flowering Plants 151.12.2 Natural Pollination 151.12.3 Hand Pollination 151.12.4 Quantity of Flowers to be Pollinated 171.12.5 Fruit Development 171.13 Harvesting 171.13.1 Harvesting Practices 181.13.2 Preventing Theft 181.14 Green Vanilla Commercialization 191.14.1 Prices 191.15 Curing 191.15.1 Yield Ratio of Green/Cured Vanilla 211.16 Grading 211.16.1 Packing 221.17 Buyers 231.18 Export Volume 231.19 Prices 231.20 Aromatic Profile 231.21 Summary 24References 242 Vanilla Diseases 27Juan Hernandez‐Hernández2.1 Introduction 272.2 Root and Stem Rot (Fusarium oxysporum f. Sp. Vanillae) 272.2.1 Description 272.2.2 Damage 282.2.3 Control 282.3 Black Rot (Phytophtora Sp.) 292.3.1 Description 292.3.2 Damage 292.3.3 Control 292.4 Anthracnose (Colletotrichum Sp.) 302.4.1 Description 302.4.2 Damage 312.4.3 Control 312.5 Rust (Uromyces Sp.) 312.5.1 Description 312.5.2 Damage2.5.3 Control 322.6 Rotting of Recently Planted Cuttings 322.6.1 Description 322.6.2 Damage 322.6.3 Control 332.7 Yellowing and Shedding of Young Fruits 332.7.1 Description 332.7.2 Damage 342.7.3 Control 342.8 Viral Diseases 352.8.1 Cymbidium Mosaic Virus (CYMV) 352.8.2 Vanilla Mosaic Virus (VMV) 352.8.3 Vanilla Necrosis Potyvirus (VNPV) 352.8.4 Odontoglossum Ringspot Virus (ORSV) 352.8.5 Prevention of Viral Diseases 362.9 Damage by Adverse Climatic Factors 362.9.1 Natural Pruning of the Apical Buds 362.9.1.1 Description 362.9.1.2 Damage 372.9.1.3 Control 372.10 Damage from Sunburn 372.10.1 Description 372.10.2 Damage 372.10.3 Control 382.11 Hurricanes 38References 393 Vanilla Production in Costa Rica 41Elida Varela Quirós3.1 Introduction 413.2 History of Vanilla Production in Costa Rica 423.2.1 The First Phase of Large‐scale Cultivation in Costa Rica 423.2.2 The Second Phase of Vanilla Cultivation in Costa Rica 423.2.3 The Third Phase 433.3 Vanilla Production – The Traditional System 453.4 Vanilla Production – The Intensive System 473.5 Propagation 483.6 Diseases and Pests 493.7 Vanilla Bean Processing 503.8 Conclusions 50References 514 Atypical Flowering of Vanilla planifolia in the Region of Junín, Peru 53Juan Hernández-Hernández4.1 Preparation of the “Mother” Plant (Cuttings) 544.2 Planting Method 544.2.1 Weed Control 554.2.2 Shoot Management – Looping 554.2.3 Shoot Management – Rooting 554.3 Nutrition 554.4 Irrigation 554.5 Pests, Disorders, and Diseases 574.5.1 Vanilla Pest 574.5.2 Diseases 574.5.3 Intense Solar Radiation 574.5.4 New Pest 574.5.5 New Disease 584.6 Flowering Period 594.6.1 Atypical Vanilla Bloom in Peru 594.7 Hand Pollination 604.8 Harvesting 614.9 Vanilla Curing 624.10 Final Comments 62References 635 Vanilla Production in the Context of Culture, Economics, and Ecology of Belize 65Nelle Gretzinger and Dawn Dean5.1 Introduction 655.1.1 Toledo Agriculture and Socio‐demographics Today 665.1.2 Maya Mountain Research Farm 665.1.3 Agro‐ecological Systems 675.1.4 Maya Mountain Research Farm Vanilla Cultivation and Introduction Project 685.1.5 The Belize Organic Vanilla Association 695.1.6 OVA Description and Goals 695.1.7 Innovative Vanilla Plantation Establishment Method Pioneered by OVA Members Nicasio and Ophelia Chee Sanchez 715.1.8 Wild/Relic Vanilla Stands in Toledo District 725.1.9 Possibility of Wild Superior or Useful Genotypes/Species 745.1.10 Dr Pesach Lubinsky’s Research in Belize and Regarding Vanilla tahitensis 745.1.11 Manche Chol 765.2 Discussion 78Acknowledgments 79References 826 Conservation and Sustainable Use of Vanilla Crop Wild Relatives in Colombia 85Nicola S. Flanagan, Paul Chavarriaga, and Ana Teresa Mosquera‐Espinosa6.1 Introduction 856.1.1 Low Genetic Diversity in the Vanilla Crop 856.1.2 The Importance of Crop Wild Relatives for Agriculture 856.2 Vanilla Crop Wild Relatives 866.2.1 Phylogenetic Diversity Within the Genus Vanilla 866.2.2 The Secondary Gene Pool for Vanilla 866.2.3 Vanilla Diversity in Colombia 876.3 Vanilla Species in the Wild 896.3.1 Vanilla Species are Rare in the Wild 896.3.2 Reproductive Biology of Vanilla Wild Species 916.3.2.1 Pollinators 916.3.2.2 Autogamy 916.3.3 Mycorrhizal Interactions 926.3.4 Further Interactions with the Microbiome 936.3.5 Bioclimatic and Biophysical Adaptations 946.4 Conservation of Vanilla Crop Wild Relatives 956.4.1 Threats to Conservation 956.4.2 Conservation In situ 966.4.3 Conservation Ex situ 966.4.4 Conservation Ex situ of the Vanilla Microbiome 986.4.5 Conservation of Circa situm and Sustainable Use 986.5 Biotechnological Approaches for Vanilla Genetic Resource Conservation and Utilization 1006.5.1 Characterization and Utilization of Genetic Diversity 1006.5.1.1 DNA Barcoding 1006.5.1.2 Genomic Characterization of Vanilla 1006.5.2 Application of Microorganisms in Vanilla Cultivation 1016.6 An Integrated Strategy for Conservation and Sustainable Use of Vanilla Crop Wild Relatives 1016.6.1 A Colombian National Strategy for Vanilla CWR 1016.6.2 International Strategy for Conservation of Vanilla CWR 102References 1027 The History of Vanilla in Puerto Rico: Diversity, Rise, Fall, and Future Prospects 111Paul Bayman7.1 Introduction 1117.2 Diversity of Wild Vanilla in Puerto Rico 1117.2.1 Species and Distributions 1117.2.2 Flowering, Pollination, and Fruit Set 1127.3 Rise and Fall: The History of Vanilla Cultivation in Puerto Rico 1127.4 Socioeconomic Factors Contributing to the Decline of Vanilla 1147.5 Diseases and Decline 1147.5.1 Fusarium Root and Stem Rot (RSR) 1157.5.1.1 The Pathogen 1157.5.1.2 Symptoms of RSR 1167.5.1.3 Other Fusarium Species 1167.5.2 Other Diseases and Pests 1167.5.3 Possible Solutions to RSR 1167.5.3.1 Biological Control 1167.5.3.2 Mycorrhiza 1177.5.3.3 Chemical Control 1177.5.3.4 Breeding 1177.5.3.5 Cultural Control 1177.6 Future Prospects 118Acknowledgments 118References 1188 Origins and Patterns of Vanilla Cultivation in Tropical America (1500–1900): No Support for an Independent Domestication of Vanilla in South America 121Pesach Lubinsky, Gustavo A. Romero‐González, Sylvia M. Heredia, and Stephanie Zabel8.1 Introduction 1218.1.1 I. Pre‐Cultivation, ca. 1500–1750s 1278.1.2 II. Papantla Monopoly, 1760s–1840s 1318.1.3 III. The Vanilla Revolution, 1850s–1900, “… and we’ve never looked back” 1358.2 The Vanilla Necklace 1368.3 Summary 138Acknowledgments 139References 1399 Vanilla Production in Australia 147Richard Exley9.1 Introduction 1479.2 History 1479.3 Species 1489.4 Climatic Regions of Australia Suitable for Vanilla 1489.5 Climatic Conditions in the Vanilla Growing Regions 1499.6 Soil and Nutrients 1509.7 Watering 1509.8 Fertilizing 1509.9 Propagation 1509.10 Support 1519.11 Light/Shade 1529.12 Spacing 1539.13 Training 1549.14 Flowering, Fruit Set, Growth, and Maturation 1549.14.1 Flowering 1549.14.2 Fruit Set (Pollination) 1549.14.3 Growth and Maturation 1559.15 Harvesting 1559.16 Curing 1559.16.1 Overview 155References 15610 Vanilla in Dutch Greenhouses: A Discovery – From Research to Production 157Filip van Noort10.1 Introduction 15710.1.1 Start of Research 15710.2 Review of Literature 15710.3 Flowering 15910.3.1 Greenhouse 16010.3.2 Sustainability 16010.4 Varieties 16110.5 Propagation 16110.5.1 Cultivation 16110.5.2 Growing Systems 16210.6 Feasibility and Conclusions 162References 16311 Establishing Vanilla Production and a Vanilla Breeding Program in the Southern United States 165Alan H. Chambers11.1 Introduction 16511.2 Southern Florida Climate 16511.2.1 Average Temperatures 16611.2.2 Average Rainfall 16611.2.3 Average Solar Radiation 16611.2.4 Major Weather Events 16811.3 Native and Naturalized Vanilla Species of South Florida 16911.3.1 V. dilloniana 16911.3.2 V. mexicana 16911.3.3 V. barbellata 16911.3.4 V. phaeantha 16911.3.5 V. planifolia 17111.4 Establishing Vanilla Production in Southern Florida 17311.4.1 Shade House Cultivation 17311.4.2 Tutor Tree Cultivation 17311.4.3 Substrate Considerations 17411.4.4 Local Economics and Niche Opportunities 17411.5 Vanilla Breeding 17511.5.1 Establishing a Vanilla Breeding Program in the United States 17511.5.2 Acquiring Diverse Vanilla Accessions 17611.5.3 Creating Diversity in Vanilla 17611.5.4 Identifying the Primary Gene Pool 17711.5.5 Target Traits 17711.5.6 A Case for a Publically Available Vanilla Genome 17811.6 Conclusions 178References 17812 In vitro Propagation of Vanilla 181Rebeca Alicia Menchaca García12.1 Methods 18212.1.1 In vitro Germination 18212.1.2 Tissue Culture 18212.2 Results and Discussion 18312.2.1 Germination 18312.2.2 Seed Maturity 18312.2.3 Time for Germination 18312.2.4 Scarification 18312.2.5 Tissue Culture 18312.2.6 Hybridization 18412.2.7 In vitro Germplasm Bank 18512.2.8 Repatriation and Recovery of Mexican Species 18512.2.9 Method of Ex vitro Adaptation 18612.2.10 Greenhouse Collection 18612.2.11 Social Linkage 18612.2.12 Human Resource Training and International Interaction 18712.3 Conclusions 187References 18813 Curing of Vanilla 191Chaim Frenkel, Arvind S. Ranadive, Javier Tochihuitl Vázquez, and Daphna Havkin‐Frenkel13.1 Introduction 19113.2 Botany of the Vanilla Pod 19213.2.1 Two Fruit Regions 19213.2.2 Fruit Components 19213.2.3 Fruit Anatomy 19313.2.4 Pollination Initiates Ovary and Fruit Development 19313.2.5 Mature Fruit 19413.3 On‐the‐vine Curing Process in a Vanilla Pod 19513.4 Off‐the‐vine Curing Process of Vanilla Beans 19613.4.1 Purpose of Curing 19813.4.2 Traditional Methods of Curing 19913.4.2.1 Killing 19913.4.2.2 Sweating 20013.4.2.3 Drying and Conditioning 20113.5 Activity of Hydrolytic Enzymes Occurring in a Curing Vanilla Pod 20213.5.1 Protease Activity 20213.5.2 Cell Wall Hydrolyzing Enzymes 20413.5.3 Glycosyl Hydrolases 20413.6 Activity of Oxidative Enzymes Occurring in a Curing Vanilla Pod 20913.7 Vanilla Products 21213.8 Summary and Conclusions 21213.9 Addendum: Commercial Curing Methods of Green Vanilla Bean 21313.9.1 Traditional Methods 21313.9.1.1 Mexican Curing Method 21313.9.1.2 The Bourbon Curing Method 21413.9.1.3 The Tahitian Curing Method 21413.9.1.4 Other Traditional Curing Methods 21413.9.1.5 Indonesian Curing of Vanilla Bean 21513.9.2 Refinement of Traditional Curing Methods 21513.9.3 Novel Curing Methods 215References 21614 Fair Trade – The Future of Vanilla? 223Richard J. Brownell Jr14.1 The Crisis 22314.2 The Farmer 22414.3 Fast Forward 22614.4 Fair Trade – Background 22614.4.1 Fair Trade Principles 22714.4.2 Vanilla and Fair Trade 22814.5 Commodity Cycles 22914.6 Issues 23014.6.1 The Price Differential 23014.6.2 Vanilla Quality 23114.6.3 Limited Availability 23114.6.4 Ensuring that Farmers are Paid the FT Price 23214.6.5 Consumer Acceptance 23214.7 Conclusions 23314.7.1 Update 2017 – Fair Trade Vanilla: Today 23314.7.2 Update 2017 – Fair Trade Vanilla: The Future 234Part II Authentication and Flavor Analysis 23715 Quality Control of Vanilla Beans and Extracts 239Arvind S. Ranadive15.1 Introduction 23915.2 Quality Control of Vanilla Beans 23915.2.1 Grading of Vanilla Beans 24015.2.1.1 Vanilla Grading in Mexico 24115.2.1.2 Vanilla Grading in Madagascar 24115.2.1.3 Vanilla Grading in Indonesia 24115.2.1.4 Vanilla Grading in Uganda 24115.2.1.5 Vanilla Grading in Tahiti 24215.2.2 Aroma of Vanilla Beans 24315.2.3 Moisture Content of Vanilla Beans 24615.2.4 Vanillin Content 24615.2.4.1 Vanilla Bean Extraction 24715.2.4.2 Vanillin Determination 24715.2.4.3 Vanillin Determination in Vanilla Extracts and Other Vanilla Products 24815.2.4.4 HPLC Method 24815.2.5 Microbial Contaminant Limits 24915.3 Quality Control of Commercial Vanilla Products 24915.3.1 Definition of Vanilla Products 24915.3.1.1 Vanilla Extracts 24915.3.1.2 Vanilla Flavoring 25015.3.1.3 Vanilla‐Vanillin Extract and Flavoring 25015.3.1.4 Concentrated Vanilla Extract and Flavoring 25015.3.1.5 Vanilla Oleoresin 25015.3.1.6 Vanilla Absolute 25015.3.1.7 Vanilla Powder And Vanilla‐Vanillin Powder 25115.3.1.8 Vanilla Tincture for Perfumery 25115.3.2 Vanilla Extract Quality Parameters 25115.3.2.1 Appearance: Color and Clarity 25115.3.2.2 Flavor 25115.3.2.3 Soluble Solids Content 25215.3.2.4 Vanillin Content 25215.3.2.5 Organic Acids – (Wichmann) Lead Number 25315.3.2.6 Resin Content 25315.3.2.7 Microbial Limits 25315.4 Determination of Authenticity of Vanilla Extracts 25415.4.1 Guidelines for Determination of Authenticity 25415.4.1.1 Evaluation of the Ratios Between Specific Components 25515.4.1.2 Isotope‐ratios Mass Spectrometry 25515.4.1.3 Site‐specific Quantitative Deuterium NmR 25515.4.2 Other Methods to Determine Authenticity 25615.4.2.1 Stable Isotope Ratio Analysis (SIRA) 25615.4.2.2 SNIF‐NMR Technique 25815.5 Summary 259Acknowledgment 259References 25916 Flavor, Quality, and Authentication 261Patrick G. Hoffman and Charles M. Zapf16.1 Introduction 26116.2 Vanilla Flavor Analyses 26216.3 Biochemistry and Genetic Research on Vanilla 26616.4 Vanilla Quality and Authentication Analyses 26716.4.1 Liquid Chromatographic Methods 26816.4.2 Isotopic Techniques 27216.4.3 Radiometric and Stable Isotope Ratio Analysis 27216.4.4 Nuclear Magnetic Resonance (NMR) 27416.4.5 Isotopic Techniques Summary 27416.4.6 Integrated and Miscellaneous Methodologies 27516.5 Conclusion 277References 27917 Volatile Compounds in Vanilla 285Stephen Toth, Keun Joong Lee, Daphna Havkin‐Frenkel, Faith C. Belanger, and Thomas G. Hartman17.1 Lexicon of Vanilla Aroma/Flavor Descriptors 285References 34518 A Comprehensive Study of Composition and Evaluation of Vanilla Extracts in US Retail Stores 349Daphna Havkin‐Frenkel, Faith C. Belanger, Debra Y.J. Booth, Kathryn E. Galasso, Francis P. Tangel, and Carlos Javier Hernández Gayosso18.1 History 34918.2 Uses of Vanilla in the Industry 34918.2.1 Household Products 35018.2.2 Dairy Products 35018.2.3 Ice Cream (Frozen Dairy Products) 35018.2.4 Yogurt 35018.2.5 Puddings 35118.2.6 Chocolate 35118.2.7 Confections 35118.2.8 Baked Goods 35118.2.9 Beverages 35118.2.10 Pet Products 35218.2.11 Pharmaceutical Products 35218.2.12 Oral Care 35218.2.13 Perfume 35218.2.14 Toys 35218.3 Major US Vanilla Companies 35318.4 Introduction to the Study 35318.5 Materials and Methods 35318.6 Results and Discussion 35418.6.1 Labeling of Retail Vanilla Extracts 35418.6.2 Flavor Components in the Retail Vanilla Extracts 35918.6.3 Total Phenol Content of the Retail Vanilla Extracts 36318.7 Conclusion and Recommendation 363References 36519 Vanilla in Perfumery and Beverage Flavors 367Felix Buccellato19.1 Earliest Recorded Use of Vanilla 367Reference 373Part III Biology of Vanilla 37520 Vanilla Phylogeny and Classification 377Kenneth M. Cameron20.1 Vanilloideae Among Orchids 38120.2 Diversity Within Vanilloideae 38120.2.1 Tribe Pogonieae 38220.2.2 Tribe Vanilleae 38320.3 Origins and Age of Vanilloideae 38420.4 Diversity Within Vanilla 38520.5 Systematic Conclusions and Implications 388References 38921 Molecular Analysis of a Vanilla Hybrid Cultivated in Costa Rica 391Faith C. Belanger and Daphna Havkin‐Frenkel21.1 Methods 39221.1.1 PCR Amplification, Cloning, and DNA Sequencing 39221.1.2 Phylogenetic Analysis 39321.1.3 Preparation of Vanilla Extracts 39321.2 Results and Discussion 393References 39922 Root Cause: Mycorrhizal Fungi of Vanilla and Prospects for Biological Control of Root Rots 403Paul Bayman, María del Carmen A. Gonzalez‐Chávez, Ana T. Mosquera‐Espinosa, and Andrea Porras‐Alfaro22.1 Introduction 40322.1.1 Orchids and Their Mycorrhiza 40322.1.2 The Fungi: Rhizoctonia and Related Taxa 40422.2 Phylogenetic Diversity of Mycorrhizal Fungi of Vanilla 40622.2.1 Methods 40622.2.2 Diversity of Mycorrhizal Fungi 40822.2.3 Fusarium 40922.2.4 Distribution of Mycorrhiza and Colonization of Roots 40922.2.5 Roots in Soil vs. Roots on Bark 41022.2.6 Differences in Mycorrhiza Among Agrosystems 41022.2.7 Limitations of Methods and Sources of Bias 41022.3 Mycorrhizal Fungi of Vanilla Stimulate Seed Germination and Seedling Growth 41122.3.1 Seedling Germination Experiments 41122.3.2 Seedling Growth and Survival Experiments 41122.4 Can Mycorrhizal Fungi Protect Vanilla Plants from Pathogens? 41422.4.1 Biocontrol of Plant Diseases Using Arbuscular Mycorrhizal Fungi 41422.4.2 Biocontrol of Plant Diseases Using Ceratobasidium 41522.4.3 Are Rhizoctonia Strains Used for Biocontrol also Potential Pathogens? 41622.4.4 Fusarium Species as Potential Biocontrol Agents to Protect Vanilla from Fusarium oxysporum Root Rots 41722.5 Conclusions 417References 41823 Enzymes Characterized From Vanilla 423Andrzej Podstolski23.1 L‐Phenylalanine Ammonia‐Lyse (Pal) and Cinnamate‐4‐Hydroxylase (C4h) 42323.2 Chain-shortening Enzymes 42423.3 4‐Coumaric Acid 3‐Hydroxylase (C3H) 42723.4 O‐Methyltransferase (OMT) 42823.5 Benzyl Alcohol Dehydrogenase (Bad) 42823.6 Glycosyltransferases (GTS) 42923.7 β‐Glycosyl Hydrolases and Curing 430References 43124 Vanillin Biosynthesis – Still not as Simple as it Seems? 435Richard A. Dixon24.1 Introduction 43524.2 Multiple Pathways to Vanillin Based on Biochemistry? 43824.3 Elucidation of Vanillin Biosynthesis via Molecular Biology? 440References 44225 Vanilla planifolia – The Source of the Unexpected Discovery of a New Lignin 447Fang Chen and Richard A. Dixon25.1 Introduction 44725.2 Identification of C‐lignin in V. planifolia 44925.3 Identification of Genes Potentially Involved in Lignin and Vanillin Biosynthesis 45125.4 C‐Lignin Biosynthesis in Other Plants 45225.5 Commercial Value of C‐Lignin as a Novel Natural Polymer 453References 454Part IV Biotechnological Production of Vanillin 45726 Biotechnology of Vanillin: Vanillin from Microbial Sources 459Ivica Labuda26.1 Introduction 45926.1.1 Why? 45926.1.2 How? 46026.2 Substrates 46026.2.1 Ferulic Acid (4‐Hydroxy 3‐Methoxy Cinnamic Acid) 46026.2.1.1 Non‐β‐oxidative Deacetylation (CoA‐dependent) 46226.2.1.2 β‐Oxidative Deacetylation (CoA‐Dependent) 46326.2.1.3 Non‐Oxidative Decarboxylation 46426.2.1.4 CoA‐Independent Deacetylation 46526.2.1.5 Side‐Chain Reductive Pathway 46626.2.2 Eugenol and Isoeugenol 46726.2.3 Lignin 46826.2.4 Sugars 46926.3 Microorganisms 47026.3.1 Bacteria 47026.3.1.1 Pseudomonas 47026.3.1.2 Streptomyces 47026.3.1.3 Bacillus 47126.3.1.4 Corynebacterium 47226.3.1.5 Escherichia coli 47226.3.1.6 Amycolatopsis sp. 47326.3.1.7 Lactic Acid Bacteria (LAB) 47326.3.1.8 Clostridium 47426.3.2 Fungi and Yeasts 47426.4 Processes 47726.4.1 Direct Bioconversion Process 47726.4.2 Bi‐Phasic Fermentation 48026.4.3 Mixed Culture Fermentation 48026.4.4 Continuous Fermentation with Immobilized Cells 48126.4.5 Enzymes 48126.4.6 Cofactors 48226.5 Downstream Processing and Recovery 48226.6 Conclusions 482References 483Index 489