Thesis Title: “Metabolism of nitrogenous wort components by brewers’ yeast” Christoforos Lekkas Submitted for the degree of Doctor of Philosophy Heriot-Watt University School of Life Sciences/ICBD June 2010 The copyright in this thesis is owned by the author. Any quotation from the thesis or use of any of the information contained in it must acknowledge this thesis as the source of the quotation or information. ABSTRACT A quantitative and qualitative identification of the nitrogenous constituents of unfermented and final fermented wort has been performed for five industrial yeast strains. Results highlight the importance of three key amino acids in yeast fermentation performance. Supplementation of wort using individual amino acids and ammonia clearly demonstrate lysine and arginine to be marker nitrogen wort constituents. Synergistic effects of individual wort free amino acids and ammonia on yeast fermentation efficiency were also examined. Further analysis of nitrogeneous wort constituents included examination of oligopeptides with a molecular weight of less than 500 Daltons and the influence of yeast proteases on the availability of small peptides during fermentation. Both lager and ale yeast can simultaneously use amino acids and small peptides as sources of assimilable nitrogen. Laboratory malt and mash analysis of 28 malt types revealed that 80-95% of total free amino nitrogen originates during malting and the remaining 5-20% during mashing. ACKNOWLEDGEMENT I would like to express my gratitude to all those who gave me the possibility to complete this thesis. I want to thank Scottish Courage Ltd for partially funding this thesis and especially my industrial supervisors Drs. Behnam Taidi and Jeff Hodgson for their scientific assistance on various project research related issues. I am deeply indebted to my academic supervisors Prof. Graham G. Stewart and Dr. Annie Hill from ICBD whose help, stimulating suggestions and encouragement helped me in all the time of research and writing of this thesis. I am also obliged to the department technicians James MacKinlay for his valuable assistance with HPLC and GC analyses, Graham McKernan for his great help in the pilot brewery and Vicky Goodfellow for providing with all the requested laboratory equipment. Especially, I would like to thank my parents and my brother who believed in me and for their continuous support for the completion of this thesis. Finally, I would like to give my special thanks to my wife Maria whose patient love enabled me to complete this work. TABLE OF CONTENTS Chapter 1: Introduction 1 1.1 The brewing process; an ancient biotechnology 1 1.2 Outline of malting and brewing 2 1.3 Brewing yeast growth and fermentation performance 5 1.4 Brewing yeast nutrition 5 1.4.1 Carbon 6 1.4.2 Oxygen 6 1.4.3 Inorganic ions 6 1.4.4 Vitamins 7 1.4.5 Nitrogen 7 1.5 Free amino nitrogen (FAN) 7 1.5.1 FAN production during malting 9 1.5.2 FAN production during mashing 11 1.5.3 FAN in wheat 11 1.5.4 FAN in sorghum 12 1.6 Amino acids 13 1.6.1 Amino acids and other beer characteristics 16 1.6.2 The general amino acid permease (GAP) system 17 1.6.3 Amino acid biosynthesis 20 1.6.3.1 Glutamate family 23 1.6.3.2 Proline biosynthesis 25 1.6.3.3 Arginine biosynthesis 25 1.6.3.4 Lysine biosynthesis 28 1.6.3.5 Aromatic amino acid biosynthesis (phenylalanine, tryptophan, tyrosine) 30 1.6.3.6 Serine family biosynthesis (serine, glycine, cysteine) 33 1.6.3.7 Aspartate family biosynthesis (aspartate, asparagine, threonine, methionine, isoleucine) 36 1.6.3.8 Pyruvate family biosynthesis (valine, leucine, alanine) 42 1.6.3.9 Histidine biosynthesis 42 i 1.7 Oligopeptides 45 1.7.1 Oligopeptide utilization 45 1.7.2 The peptide transport system (PTR) 49 1.7.3 Oligopeptides and other beer characteristics 51 1.8 Ammonia 51 1.9 Yeast proteases 54 1.10 Flavour compounds 55 1.10.1 Higher alcohols 57 1.10.2 Esters 58 1.10.3 Vicinal diketones 59 1.10.4 Aldehydes 60 Chapter 2: Materials and Methods 62 2.1 Shake flask fermentations 62 2.1.1 Maintenance of stock cultures 62 2.1.2 Yeast identification 62 2.1.3 Preparation of inoculum cultures 62 2.1.4 Wort preparation 63 2.1.5 Shaken fermentations 64 2.2 Static fermentations 64 2.2.1 Yeast strains 64 2.2.2 Yeast identification 64 2.2.3 Maintenance of stock cultures 65 2.2.4 Propagation of yeasts 65 2.2.5 Determination of yeast pitching volume 66 2.2.6 Wort preparation 68 2.2.7 Fermentations 68 2.3 Experimental wort and beer analysis 70 2.3.1 Determination of cell number and viability 70 2.3.2 Biomass determination 70 2.3.3 Determination of pH 70 2.3.4 Determination of specific gravity 71 2.3.5 Determination of total wet and dry yeast biomass 71 ii 2.3.6 Determination of free amino nitrogen (FAN) 71 2.3.7 Determination of individual wort amino acids 72 2.3.8 Determination of ammonia 76 2.3.9 Oligopeptide determination 76 2.3.9.1 Sample preparation 76 2.3.9.2 Vacuum filtration 77 2.3.9.3 Acid hydrolysis 77 2.3.9.4 Alkaline hydrolysis 77 2.3.9.5 HPLC analysis 78 2.3.10 Determination of protease activity 78 2.3.11 Determination of carbohydrates by HPLC 79 2.3.12 Determination of ethanol 81 2.3.13 Determination of volatile compounds 82 2.4 Re-pitching of static fermentations 82 2.5 Amino acid supplementations 83 2.6 Mashing trials 88 2.6.1 Mashing at 65oC 88 2.6.2 Mashing at 4oC 90 Chapter 3: Results 91 3.1 Shake flask fermentations 93 3.1.1 12oPlato wort 93 3.1.1.1 Fermentation profile and sugar utilization (12oPlato) 93 3.1.1.2 Amino acid and ammonia utilization (12oPlato) 96 3.1.1.3 Wort nitrogen distribution and nitrogen source utilization (12oPlato) 98 3.1.2 20oPlato wort 100 3.1.2.1 Fermentation profile and sugar utilization (20oPlato) 100 3.1.2.2. Amino acid and ammonia utilization (20oPlato) 102 3.1.2.3 Wort nitrogen distribution and nitrogen source utilization (20oPlato) 104 3.1.3 20oPlato wort + 30% Glucose 106 3.1.3.1 Fermentation profile and sugar utilization 106 iii 3.1.3.2 Amino acid and ammonia utilization (20oPlato + 30% Glucose) 108 3.1.3.3 Wort nitrogen distribution and nitrogen source utilization (20oPlato + 30% Glucose) 110 3.2 Static fermentations 112 3.2.1 Lager fermentations 112 3.2.1.1 SC3 lager fermentations 112 3.2.1.1.1 Fermentation profile and sugar utilization 112 3.2.1.1.2 Amino acid and ammonia utilization 114 3.2.1.1.3 Wort nitrogen distribution and nitrogen source utilization 116 3.2.1.1.4 Final fermentation measurements 118 3.2.1.2 SC3 lager fermentations (Repitching) 119 3.2.1.2.1 Fermentation profile and sugar utilization 119 3.2.1.2.2 Amino acid and ammonia utilization 121 3.2.1.2.3 Wort nitrogen distribution and nitrogen source utilization 123 3.2.1.2.4 Final fermentation measurements 125 3.2.1.3 SC4 lager fermentations 126 3.2.1.3.1 Fermentation profile and sugar utilization 126 3.2.1.3.2 Amino acid and ammonia utilization 128 3.2.1.3.3 Wort nitrogen distribution and nitrogen source utilization 130 3.2.1.3.4 Final fermentation measurements 132 3.2.1.4 SC4 lager fermentations (Repitching) 133 3.2.1.4.1 Fermentation profile and sugar utilization 133 3.2.1.4.2 Amino acid and ammonia utilization 135 3.2.1.4.3 Wort nitrogen distribution and nitrogen source utilization 137 3.2.1.4.4 Final fermentation measurements 139 3.2.2 Ale fermentations 140 3.2.2.1 SC5 ale fermentations 140 3.2.2.1.1 Fermentation profile and sugar utilization 140 iv 3.2.2.1.2 Amino acid and ammonia utilization 143 3.2.2.1.3 Wort nitrogen distribution and nitrogen source utilization 145 3.2.2.1.4 Final fermentation measurements 147 3.2.2.2 SC5 ale fermentations (Repitching) 148 3.2.2.2.1 Fermentation profile and sugar utilization 148 3.2.2.2.2 Amino acid and ammonia utilization 151 3.2.2.2.3 Wort nitrogen distribution and nitrogen source utilization 153 3.2.2.2.4 Final fermentation measurements 155 3.2.2.3 SC8 ale fermentations 156 3.2.2.3.1 Fermentation profile and sugar utilization 156 3.2.2.3.2 Amino acid and ammonia utilization 158 3.2.2.3.3 Wort nitrogen distribution and nitrogen source utilization 160 3.2.2.3.4 Final fermentation measurements 162 3.2.2.4 SC8 ale fermentations (Repitching) 163 3.2.2.4.1 Fermentation profile and sugar utilization 163 3.2.2.4.2 Amino acid and ammonia utilization 165 3.2.2.4.3 Wort nitrogen distribution and nitrogen source utilization 167 3.2.2.4.4 Final fermentation measurements 169 3.3 Fermentation supplementations 170 3.3.1 Control fermentations 171 3.3.1.1 Fermentation profile and sugar utilization 171 3.3.1.2 Amino acid and ammonia utilization 173 3.3.1.3 Wort nitrogen distribution and nitrogen source utilization 175 3.3.1.4 Final fermentation measurements 177 3.3.2 Lysine supplemented fermentations (five times) 178 3.3.2.1 Fermentation profile and sugar utilization 178 3.3.2.2 Amino acid and ammonia utilization 180 3.3.2.3 Wort nitrogen distribution and nitrogen source utilization 182 3.3.2.4 Final fermentation measurements 184 v 3.3.3 Arginine supplemented fermentations (five times) 185 3.3.3.1 Fermentation profile and sugar utilization 185 3.3.3.2 Amino acid and ammonia utilization 187 3.3.3.3 Wort nitrogen distribution and nitrogen source utilization 189 3.3.3.4 Final fermentation measurements 191 3.3.4 Methionine supplemented fermentations (five times) 192 3.3.4.1 Fermentation profile and sugar utilization 192 3.3.4.2 Amino acid and ammonia utilization 194 3.3.4.3 Wort nitrogen distribution and nitrogen source utilization 196 3.3.4.4 Final fermentation measurements 198 3.3.5 Ammonia supplemented fermentations (twice) 199 3.3.5.1 Fermentation profile and sugar utilization 199 3.3.5.2 Amino acid and ammonia utilization