Beer AnalysisEdited byH. E Linskens and J. F. Jackson

Contributors

S. Aastrup M. J. Allison M. Bohl Pedersen B. E. ChristensenJ. A. Delcour S. Donhauser H. Eriksen C. GjermansenR. vanHeeswijck B. Jende-Strid L. P. Jepsen K. G. JorgensenA. K. Keiding G. C. J. Muts T. Peppard U. RasmussenM.I.G. Roncero P. Sigsgaard J. Strating L. C.Verhagen

With 97 Figures

Springer-VerlagBerlin Heidelberg New YorkLondon Paris Tokyo

Contents

Raw Material

Barley and Malt AnalysisG. C.J. MUTS

1 General Introduction 32 Barley Analysis 4

2.1 Visual Inspection 42.2 Mechanical Analysis 42.3 Germination Tests 42.4 Chemical Analysis 62.5 Varietal Identification 92.6 Micro-Malting 10

3 Malt Analysis 103.1 Wort Production and Analysis 103.2 Modification Analysis 123.3 Chemical Analysis 143.4 Enzyme Analysis 14

4 Further Reading 17References 17

Reversed Phase HPLC as an Aid for the Identification of Barley CultivarsM. J. ALLISON (With 5 Figures)

1 Introduction 221.1 Reversed-Phase High Performance Liquid Chromatography . . . 23

1.1.1 The RP-HPLC System 241.1.2 Sample Preparation 241.1.3 Hordeins 251.1.4 Albumins and Globulins 251.1.5 Glutelins 25

1.2 Hordein Identification 261.3 Repeatability of Hordein Elution 27

1.3.1 RP-HPLC Separations of Hordeins 291.3.2 RP-HPLC Separation of Albumins and Globulins 311.3.3 RP-HPLC Separation of Glutelins 32

References 33

XII Contents

The Application of Immunofluorescence for DetectingSpecific Proteins in Barley SeedsU. RASMUSSEN (With 7 Figures)

1 Introduction 352 Immunofluorescence : 36

2.1 Fluorescent Dyes 362.2 Staining Methods. . 37

2.2.1 The Direct Method with Fluorochrome-LabelledPrimary Antibodies 38

2.2.2 The Indirect Method with Fluorochrome-LabelledSecondary Antibodies 38

2.2.3 Use of Biotin-Labelled Primary Antibodies 382.2.4 Use of Biotin-Labelled Secondary Antibodies 39

3 Materials, Protocols and Comments 403.1 Production and Purification of Antibodies 403.2 Conjugation of Antibodies with Fluorochromes 41

3.2.1 Protocol for FITC Conjugation 413.2.2 Comments to the FITC Conjugation Procedure . . . . . . 423.2.3 Conjugation with Other Fluorochromes 44

3.3 Biotinylation 453.3.1 Protocol for Conjugation with Biotin 453.3.2 Comments to the Biotin Conjugation 45

3.4 Fixation 463.4.1 Protocol for PLP Fixation of Seeds to be Sectioned . . . . 463.4.2 Surface Fixation of Sanded Seeds for Retention

of Germination Capacity 473.4.3 Comments to the Fixation 47

3.5 Preparation of Tissue 483.5.1 Thin Sections 483.5.2 Sanded Seeds 48

3.6 Staining Procedures 483.6.1 General Considerations 483.6.2 Direct Staining of Thin Sections 493.6.3 Indirect Staining of Thin Sections 493.6.4 Use of Biotin-Labelled Primary Antibodies 503.6.5 Use of Biotin-Labelled Secondary Antibodies 503.6.6 Two-Colour Staining of Thin Section 503.6.7 Direct Staining of Sanded Seeds 52

4 Concluding Remarks 52References 53

Fluorimetric Methods for the Analysis of Malt Modificationand Pre-Harvest SproutingS. AASTRUP and K. G. JBRGENSEN (With 6 Figures)

1 Introduction 562 Modification and Homogeneity of Barley Malt 57

Contents XIII

2.1 Field of Application 572.2 Sample Size . . . . 572.3 Reagents and Materials 582.4 Apparatus 582.5 Procedure 59

2.5.1 Fixation and Sanding 592.5.2 Staining 592.5.3 Examination 59

2.6 Expression of Results 592.7 Use of Malt Modification Analysis 61

3 Pre-Sprouting in Cereal Seeds 633.1 Field of Application 633.2 Sample Size 633.3 Reagents and Materials 633.4 Apparatus 633.5 Procedure 64

3.5.1 Fixation and Sanding 643.5.2 Staining 643.5.3 Examination 64

3.6 Expression of Results 643.7 Use of Pre-Sprouting Test 64

4 Further Applications of the Carlsberg Seed Fixation System 65References 65

Hop AnalysisL. C. VERHAGEN (With 4 Figures)

1 Introduction 672 Chemical Composition of Hops 673 Analysis 70

3.1 Resin Analysis of Hops : . . 703.2 The Conductometric Lead Value of Hops . 733.3 Verzele Conductometric Analysis of a-Acids 733.4 Beer Bitterness Analysis 753.5 High Performance Liquid Chromatography (HPLC) 75

4 Essential Oil of Hops 825 Polyphenols 846 Prospect 84References 84

Determination of /J-Glucan in Barley, Malt, Wort and BeerK. G. J0RGENSEN and S. AASTRUP (With 6 Figures)

1 Introduction 882 The Nature of J?-Glucan 89

XIV Contents

3 Quantification of Barley j3-Glucan 893.1 Methods Based on Extraction 90

3.1.1 Viscosity Methods 903.1.2 Difference Methods 913.1.3 Selective Precipitation Methods 92

3.2 Non-Extraction Methods 923.2.1 Near Infrared Reflectance 923.2.2 Calcofluor Staining Used in Flour Suspensions 93

3.3 Enzymatic Methods 933.3.1 Methods Using Bacterial /J-Glucanase 933.3.2 Methods Based on Fungal Cellulases 95

4 Quantification of /?-Glucan in Malt, Wort and Beer 974.1 Total /J-Glucan Content of Malt 98

4.1.1 Non-Enzymatic Methods 984.1.2 Enzymatic Methods 98

4.2 )8-Glucan Content of Wort and Beer 984.2.1 Non-Enzymatic Methods 984.2.2 Enzymatic Methods 99

5 An Automatic /?-Glucan Analyzer System 1005.1 Principle 1005.2 Apparatus and Conditions 1015.3 Quantification of j?-Glucan in Barley, Malt, Wort and Beer . . .102

5.3.1 Sample Preparation for Wort and Beer 1025.3.2 Sample Preparation for Barley and Malt 103

6 Evaluation of Methods 1046.1 Specificity 104

References 105

Analysis of Proanthocyanidins and Phenolic Acids inBarley, Malt, Hops and BeerB. JENDE-STRID (With 8 Figures)

1 Introduction 1092 Choice of Analytical Methods 1123 Chromatographic Methods 115

3.1 High Performance Liquid Chromatography (HPLC) 1153.1.1 Preparation of Barley and Malt Samples 1153.1.2 Preparation of Hop Samples 1173.1.3 Preparation of Beer Samples 1173.1.4 Choice of HPLC Columns 1173.1.5 Mobile Phase 1183.1.6 Standards 1183.1.7 Quantitation 120

3.2 Thin Layer Chromatography (TLC) 1223.3 Column Chromatography 122

4 Colourimetric Methods 1234.1 Determination of Proanthocyanidins in Barley, Malt, Wort and Beer. 123

Contents XV

4.2 Measurement of Total Polyphenols in Beer and Wort 1234.3 Analysis of Proanthocyanidins and Catechins Using

Acidified Vanillin 1234.4 Determination of Proanthocyanidins and Catechins in Beer with

p-Dimethylaminocinnamaldehyde as a Reagent 1245 Vanillin Test of Barley Grains 124References 126

Yeast

Breeding and Characterization of Brewer's Yeast IncludingSingle Chromosome TransferC. GJERMANSEN (With 5 Figures)

1 Introduction 1312 Sporulation, Isolation of Meiotic Progeny and Construction of Hybrids . 132

2.1 Mass Spore Isolation 1322.2 Isolation of Colonies and Spore Staining 1322.3 Determination of Mating Type 1332.4 Construction of Hybrids by Mating of Meiotic Segregants . . . .1332.5 Random Mating 134

3 Genetics of Diacetyl Formation 1343.1 Isolation of Spontaneous Mutants Resistant to Sulfometuron Methyl 136

4 Transformation 1375 Single Chromosome Transfer 137

5.1 Transfer of Chromosome V 1385.2 Characterization of Chromosome V by Restriction

Endonuclease Analysis 140References 142

Genetic Analysis and Transformation of Distiller's YeastA. K. KEIDING (With 6 Figures)

1 Introduction 1452 Genetic Analysis 145

2.1 Sporulation and Mating Ability .1452.1.1 Sporulation Test 1462.1.2 Spore Isolation 1462.1.3 Mating Type Tests 146

2.1.3.1 Induction of Respiratory-Deficient Mutations . . . 1462.1.3.2 Determination of Mating Type 1462.1.3.3 Rare Mating on Solid Medium 1472.1.3.4 Rare Mating in Liquid Medium 147

2.1.4 Characterization of Auxotrophic Spore Clones 1472.1.5 Sporulation and Mating Ability of Strain A 147

XVI Contents

2.2 Mutagenesis 1482.2.1 Procedure 1492.2.2 Mutants Obtained from Spore Clones of Strain A 149

2.3 Crossing and Tetrad Analysis 1503 Molecular Characterization 150

3.1 Procedure .1513.2 Analysis of Chromosomes III of Strain A 151

4 Genetic Transformation of an Industrial Yeast Strain 1544.1 Selection of Transformants by Acquired Prototrophy 155

4.1.1 Transformation Procedure 1564.1.2 Screening for Amylase Activity 1564.1.3 Stability Test of Transformants 1564.1.4 Integration of a Self-Replicating Plasmid 1574.1.5 Breeding Through Transformation of Spore-Derived Strains . 158

4.2 Integration Vector with Drug Resistance as Selective Marker . . .1584.2.1 Transformation Procedure Using a Non-Replicating Plasmid . 1604.2.2 Screening for /?-Galactosidase Activity 160

References 161

Automated Testing of Brewer's Yeast Strains for FermentationCharacteristics (Multiferm, System Carlsberg)P. SIGSGAARD (With 12 Figures)

1 Introduction 1642 Testing Techniques 165

2.1 Small-Scale Fermentations 1652.2 MULTIFERM Fermentations 165

2.2.1 Description of the MULTIFERM System 1662.2.2 Working Procedures 169

2.2.2.1 Propagation of Yeast 1692.2.2.2 Wort Collection and Handling 1702.2.2.3 Start of Fermentations 1702.2.2.4 Sampling During Fermentations 1712.2.2.5 End of Fermentations 1722.2.2.6 Cleaning in Place of the MULTIFERM 1722.2.2.7 Re-Use of Yeast 173

2.2.3 Analyses Performed 1732.2.3.1 Yeast Cell Size and Yeast Cell Number 1732.2.3.2 Pitching Rate 1732.2.3.3 Extract Determination 1742.2.3.4 Yeast Yield 1742.2.3.5 Vicinal Diketones and Ester/Alcohol (Aroma

Profile) Analyses 1742.2.4 Data 175

2.2.4.1 Data Handling and Storage 1752.2.4.2 Data Evaluation 175

3 Conclusion 177References 178

Contents XVII

The Use of Nucleotide Sequence Polymorphisms and DNA Karyotyping in theIdentification of Brewer's Yeast Strains and in Microbiological ControlM. BOHL PEDERSEN (With 8 Figures)

1 Introduction 1801.1 Restriction Fragment Length Polymorphisms . . . • 1811.2 DNA Karyotypes 185

2 Storage and Propagation of Yeast Strains 1883 Restriction Site Analysis of Yeast Genomic DNA 188

3.1 Isolation of Yeast Genomic DNA for Restriction Site Analysis . . 1883.2 Analysis of Restriction Fragment Length Polymorphisms (RFLP) . 189

3.2.1 RFLP Level A 1893.2.2 RFLP Level B 190

4 Southern Transfer and Molecular Hybridization of RadioactivelyLabelled Probes 1904.1 Transfer of DNA Molecules from Agarose Gels onto Nitrocellulose

Membranes by Southern Transfer 1904.2 Molecular Hybridization 191

4.2.1 Hybridization Buffers 1914.2.2 Nick Translation 1914.2.3 Post Hybridization Wash 192

5 Isolation of Yeast Genomic DNA for Chromosome Separation . . . . 1925.1 Orthogonal Field Alternation Gel Electrophoresis 1925.2 Preparation of Chromosomal DNA for OFAGE Karyotypes . . . 193

References 193

Screening and Testing New Distillers' Yeasts for Their Potential inMolasses Ethanol FermentationsB. E. CHRISTENSEN and H. ERIKSEN (With 3 Figures)

1 Introduction . . . 1952 Screening and Testing Techniques . 196

2.1 Small-Scale Batch Fermentations 1962.1.1 Description of Apparatus 1962.1.2 Propagation of Yeast 1962.1.3 Fermentation and Analyses 197

2.2 Small-Scale Continous Fermentations 1972.2.1 Propagation of Yeast 1982.2.2 Fermentation and Analysis 198

2.3 Full-Scale-Fermentation 1982.3.1 Propagation of Yeast 1982.3.2 Fermentation Equipment 1992.3.3 Analyses Performed 200

2.3.3.1 Oxidation Method of Ethanol Determination . . . . 2002.3.3.2 Sugar Concentration Determined by Fermentation . 201

2.3.4 Regulation of Fermentation 203

XVIII Contents

3 Evaluation of Results 2033.1 Small-Scale Fermentations 2033.2 Production-Scale Fermentations 204

References 205

Genetic Analysis and Manipulation of Mucor Species by DNA-MediatedTransformationR. VAN HEESWIJCK, M. I. G. RONCERO and L. P. JEPSEN (With 1 Figure)

1 Introduction 2071.1 Taxonomy of Mucor 2071.2 Industrial Use of Mucor 2071.3 Genetic Analysis and Manipulation 207

2 General Methods 2082.1 Media 2082.2 Growth Conditions 2082.3 Harvesting, Storage and Germination of Sporangiospores . . . . 2092.4 Replica Plating of Colonies 209

3 DNA-Mediated Transformation of Mucor 2103.1 Plasmids pMCL006 and pMCL1302 2103.2 Method for Transformation of Mucor with Plasmid DNA . . . . 2103.3 Properties of Transformants 211

4 Isolation, Regeneration and Fusion of Protoplasts from Mucor . . . . 2124.1 Formation of Protoplasts from Mucor 2124.2 Preparation of a Lytic Enzyme from Streptomyces sp. no. 6

(Streptozyme) 2144.3 Regeneration of Protoplasts 2144.4 Fusion of Protoplasts 214

5 Isolation of Marker Genes for Use in DNA-Mediated Transformation . 2155.1 Selectable Marker Genes Coding for Resistance to Antibiotics . . 2155.2 Selectable Marker Genes Coding for Metabolic Enzymes 2155.3 Isolation and Enrichment of Auxotrophic Mutants of Mucor . . . 217

6 Analysis of Transformants 2186.1 Isolation of RNA 2186.2 Isolation of DNA 2196.3 Recovery of Plasmid DNA 219

7 Genetic Manipulation of Mucor by DNA-Mediated Transformation.Concluding Remarks 220

References 220

Final Product

Malt and Hop Flavanoids in Pilsner BeerJ. A. DELCOUR (With 5 Figures)

1 Importance 2252 Identification of Flavanoids in Barley, Malt, Hops and Beer 226

2.1 Barley and Malt Flavanoids 226

Contents XIX

2.2 Hop Flavanoids 2272.3 Beer Flavanoids . 227

3 Analysis of Beer Flavanoids 2283.1 Analysis of Beer Anthocyanogens 229

3.1.1 Introduction 2293.1.2 Method 230

3.2 Analysis of Total Polyphenolics 2313.2.1 Introduction 2313.2.2 Method 231

3.3 Analysis of Flavanoids 2323.3.1 Introduction 2323.3.2 Method 234

3.4 Comparison of the Methods 2344 Significance of the Analytical Results 2365 Analysis of Beer Flavanoids by High-Performance Liquid

Chromatography 2365.1 Gas Chromatography 2375.2 High Performance Liquid Chromatography 237

5.2.1 Isolation of Proanthocyanidins from Beer 2375.2.2 High Performance Liquid Chromatography 238

References 238

Analytical Measurement of Volatile Sulphur Compounds in BeerT. PEPPARD (With 2 Figures)

1 Introduction 2412 Volatile Sulphur Compounds in Beer 2413 Analytical Methods for Determining Volatile Sulphur Compounds in Beer 242

3.1 General Remarks 2423.2 Non-Chromatographic Methods 243

3.2.1 Sulphur Dioxide 2433.2.1.1 The Monier-Williams Distillation/Titrimetric

Procedure 2433.2.1.2 The Para-Rosaniline Colorimetric Procedure . . . . 2443.2.1.3 The DTNB Method 244

3.2.2 Hydrogen Sulphide 2453.3 Chromatographic Methods 245

3.3.1 Analyte Detection 2463.3.2 Chromatographic Separation 2463.3.3 Sample Preparation/Introduction into Gas Chromatograph . 247

3.3.3.1 Static Headspace Sampling 2473.3.3.2 Dynamic Headspace Sampling (Purge and

Trap Methods) 2483.3.3.3 Methods for Concentrating Sulphur Compounds of

Lower Volatility 2514 Conclusions 252References 252

XX Contents

Short Introduction to Headspace AnalysisJ. STRATING

1 Introduction 2542 The Static Headspace Method 2553 The Dynamic Headspace Method 2564 Sampling Methods 2575 Factors Affecting Choice of Equilibrium Conditions 2586 Quantitative Headspace Analysis 2607 Final Remarks 261References 262

The Use of Principal Components Analysis in Monitoring theQuality of BeerT. PEPPARD (With 5 Figures)

1 Introduction 2642 The Principles of PCA 265

2.1 The Objectives 2652.2 Conceptualizing the Multivariate Problem 2652.3 A Stepwise Explanation of PCA 266

3 The Use of PCA in Brewing Science 2693.1 A Detailed Example of the Use of PCA in Brewing Science . . . 2703.2 Other Examples of the Use of PCA in Brewing Science 274

4 Conclusions 278References 278

German Beer Purity Law and its Influences on the Propertiesand Analysis of BeerS. DONHAUSER (With 14 Figures)

1 The German Beer Purity Law 2802 Complaint of the EEC Commission at the European Court of Law

Against the Federal Republic of Germany 2813 Arguments for Retaining the Beer Purity Law 2814 Beers with Adjuncts 2825 Analytical Analysis of Additives and Harmful Substances 283

5.1 Immunological and Immunochemical Methods 2845.1.1 Qualitative and Half-Quantitative Indication Methods . . . 284

5.1.1.1 Double Diffusion in Agar 2845.1.1.2 Immunoelectrophoresis 2855.1.1.3 Counter Current Electrophoresis 287

5.1.2 Quantitative Identification Methods 2875.1.2.1 Basic Radial Immunodiffusion 2875.1.2.2 Electroimmunodiffusion 2875.1.2.3 Two-Dimensional Immunoelectrophoresis 288

5.1.3 Highly Sensitive Immunochemical Methods 2905.1.3.1 Radio Immuno Assay 2905.1.3.2 ELISA of EIA (Enzymelinked Immunosorbent Assay) 291

Contents XXI

5.2 Gas Chromatography and High Performance Liquid Chromatography(HPLC) 293

5.3 Electrophoresis 2935.4 Isotachophoresis 2945.5 Atomic Absorption Spectrometry 294

6 Summary 294References 295

Subject Index 297