The science of malting, brewing, and fermenting beer Oct 6, 2010 Protein: a chain made up of 20 different amino acids from a few to as many as 34,350 residues α-amylase DNA transcription translation RNA Temperature effects on biology and chemistry Water is the universal solvent of life : The Structure and Properties of Water D. Eisenberg and W. Kauzmann 308 pages Oxford Press 1969 + Overview of metabolism • Most efficient way of getting energy is by combining reduced carbon with oxygen. The more reduced the carbon, the more energy it has, the more oxygen added, the more energy released. • In the absence of oxygen, fermentation is a suitable alternative. glucose and other sugars fatty acid (-2880 kJ/mol) pyruvate lactic acid (-198 kJ/mol) ethanol + CO2 (-235 kJ/mol) Overview of metabolism = = = = = α β Overview of metabolism R = some chemical group Overview of metabolism Barley: A member of the grass family. It is a self-pollinating, diploid species with 14 chromosomes. The wild ancestor of domesticated barley, Hordeum vulgare subsp. spontaneum, is abundant in grasslands and woodlands throughout the Fertile Crescent and has been cultivated for millennia. Malting barley is usually lower protein which leads to more uniform germination, with shorter steeping. The lower protein content also reduces the haze that results from precipitated protein. Two-row barley generally has a lower protein content compared to six-row. -A single mutation is responsible for the difference between two-row and six-row barley. -Two mutations of wild barley prevent the spike from shattering. Traditionally barley was classified by morphological differences and were considered to be different species. -Two-rowed barley with shattering spikes (wild barley) is classified as Hordeum spontaneum K.Koch. -Two-rowed barley with non-shattering spikes is classified as H. distichum L. -Six-rowed barley with non-shattering spikes as H. vulgare L. (or H. hexastichum L.). -Six-rowed with shattering spikes as H. agriocrithon Åberg. Recent cytological and molecular evidence has led most recent classifications to consider all forms as a single species, H. vulgare L. Maris Otter is a 2-row, "winter" variety bred by researchers at Cambridge and introduced in 1966 possessing low nitrogen (protein) and superior malting characteristics. It is a cross of Proctor and Pioneer. Germinating/malting breaking down chains of stuff http://plantphys.info/plant_physiology/gibberellin.shtml Drying and Kilning During drying and or kilning, some enzymes become denatured. Generally darker grains are roasted longer and at higher temperatures and thus have less active enzymes then pale malt. Crystal malt is kilned without drying which denatures all enzymes. Importantly, during the drying phase, most lipase and lipoxygenase enzymes are destroyed. These enzymes are implicated in the formation of off flavors in beer as it ages. Kilning also roasts the grain. During the roasting process a glorious reaction called the Maillard reaction occurs. Modification: the degree of breakdown of the starch-protein matrix during the malting, drying, and kilning process The Maillard reaction A general reaction between an amino acid and a reducing sugar and contributes to the color and flavor of browned bread, chocolate, seared meat, caramel and deep fried death. sugar amino acid Strecker degradation melanoidins (dark color and toasty aroma) pyrazines thiophenes pyrroles furans isobutyraldehyde (wet cereal/straw) The modern history of enzymes began in 1833 when French chemists described the isolation of an amylase complex from germinating barley and named it diastase. Sugar enzymology I http://blog.targethealth.com/?p=9586 Mashing and resting breaking down more chains of stuff Temp °C Temp °F Enzyme Breaks down 40 °C 104.0 °F β-Glucanase Cellulose (β-Glucan) 50 °C 122.0 °F Protease Protein 62 °C 143.6 °F β-Amylase Starch 72 °C 161.6 °F α-Amylase Starch Generally only needed when using >25% unmalted wheat or barley, corn, or rye β-1,4 glycosidic linkage cellulose Temp °C Temp °F Enzyme Breaks down 40 °C 104.0 °F β-Glucanase Cellulose (β-Glucan) 50 °C 122.0 °F Protease Protein 62 °C 143.6 °F β-Amylase Starch 72 °C 161.6 °F α-Amylase Starch ~122 °F Protein rest: lower temperatures (122ºF (50ºC) ) yield shorter peptides and single amino acids (free amino nitrogen aka FAN) which aren’t as good for head retention. ~133 °F Temperatures closer to 133ºF (55ºC) leave longer peptide chains and more available amino acids for yeast. Some recent research suggests that most proteases are destroyed during kilning and that there is no significant reduction in the molecular weight spectrum of the mash. Generally only needed when using minimally modified malt or a lot (>25%) of adjuncts Sugar enzymology II amylopectin amylose limit dextrinase (debranching enzyme) O β O O… O O O O O O O O O O O O O Sugar enzymology II α β Temp °C Temp °F Enzyme Breaks down 40 °C 104.0 °F β-Glucanase Cellulose (β-Glucan) 50 °C 122.0 °F Protease Protein 62 °C 143.6 °F β-Amylase Starch 72 °C 161.6 °F α-Amylase Starch General enzymology Enzyme activity is affected mainly by temperature, but also pH, presence of metals or cofactors, substrate concentration, viscosity, etc The thicker the mash, the more active the enzymes. reaction rate ([producet]/second) Maximum rate possible KM [Substrate] Humulus lupulus ΔT humulone isohumulone Lupulin16% Soft Resins 13% Alpha Acids 8% Beta Acids 4% Other Soft Resins 1% Hard Resins 2% Essential Oils 1% Hydrocarbons 0.75% Oxidation Products 0.2% Sulphur containing compounds 0.05% Vegative Matter 84% lupulones Linalool (spicy) Geraniol Myrcene Caryophylene Faresene Selinene trans-isohumulone cis-isohumulone Further adventures in stereochemistry carvone thalidomide D-form amino acids tend to taste sweet, L-form amino acids are generally tasteless. Proteins use L-amino acids Most sugars we digest and incorporate are D (R) spearmint (S) caraway Antibiotic properties of hops Hop compounds act as ionophores that exchange protons for cellular divalent cations. This decreases the intracellular pH and dissipates the transmembrane proton gradient (ΔpH) and the proton motive force (pmf). Bacteria have evolved a number of ways to resist killing by hops. HorA (a) and probably also by a pmf-dependent transporter (b) overexpressed H+-ATPase increases the pumping of protons released from the hop compounds (c) Galactosylated glycerol teichoic acid in the cell wall and a changed lipid composition of the cytoplasmic membrane of beer spoilage lactic acid bacteria may increase the barrier to hop compounds. + H+ antibacterial form trans-isohumulone Saccharomyces cerevisiaea -Single-celled fungus from the phylum Ascomycota -One of the most well characterized organisms -Genome sequenced in 1996 -Capable of sexual and asexual reproduction -Found in wild on fruit surfaces Lager yeast is more complex. First called S. carlsbergensis or S. pastorianus, then considered to be S. cerevisiae, are now recognized as a hybrid of S. cerevisiae and S. bayanus S. cerevisiae life cycle Gene expression in lag phase and early log phase Time Brejning et al. J Appl Microbiol. 2005. If fermentation is anaerobic why is so much oxygen needed when pitching? S. cerevisiae life cycle Fermentation profiles with various sugar supplements Piddocke et al. Applied Microbiology and Biotechnology 2009 S. cerevisiae life cycle Stationary phase is more complex than it seems Extending healthy life span--from yeast to humans. Fontana L, Partridge L, Longo VD. Science. 2010 Apr 16;328(5976):321-6. Review. Insulin/IGF-I and related signaling pathways regulate aging in nondividing cells: from yeast to the mammalian brain. Parrella E, Longo VD. ScientificWorldJournal. 2010 Jan 21;10:161-77. Review. Genetic links between diet and lifespan: shared mechanisms from yeast to humans. Bishop NA, Guarente L. Nat Rev Genet. 2007 Nov;8(11):835-44. Review. Gray et al. Microbiology and Molecular Biology Reviews. 2004. Yeast metabolism Why is S. cerevisiae so good at making beer? “Make-accumulate-consume” Yeast can suppress respiration in the presence of glucose and oxygen Yeast settling to the bottom is not a passive process What goes wrong when beer goes bad? diacetyl rest: yeast convert acetolactic acid into valine instead of diacetyl (butanedione) and converts any butanedione into butanediol which is neutral as far as beer flavoring ethanol valine butanediol pyruvate Lagering: beer stored at 34-40 F for a few weeks. levels of diacetyl, acetaldehyde and sulfur compounds decrease. acetaldehyde acetolactate acetoin The dynamics of the Saccharomyces carlsbergensis brewing yeast transcriptome during a productionscale lager beer fermentation. Olesen K, Felding T, Gjermansen C, Hansen J. FEMS Yeast Res. 2002 Dec;2(4):563-73. diacetyl Two-dimensional gel analysis of the proteome of lager brewing yeasts. Joubert R, Brignon P, Lehmann C, Monribot C, Gendre F, Boucherie H. Yeast. 2000 Apr;16(6):511-22. Flavor in beer Organoleptic threshold (ppm) Concentration in Japanese beer (ppm) Alcohol 800 8–15 Alcohol 200 7–14 65 46–71 Higher alcohols Propan-1-ol (npropanol) 2-Methyl propanol (isobutyl alcohol) 2-Methyl butanol (active amyl alcohol) 3-Methyl butanol (isoamyl alcohol) 2-Phenyl ethanol Alcohol, banana, medicinal, solvent Alcohol, banana, sweetish, aromatic Roses, sweetish, perfumed 70 125 20–27 30 10–20 1.2 1.3–2.5 3.8 0.4–1.3 Esters Solvent, fruity, sweetish Banana, apple, solvent, Isoamyl acetate estery Roses, honey, apple, 2-Phenylethyl acetate sweetish Ethyl caproate Sour apple Ethyl caprylate Sour apple Ethyl acetate 0.21 0.9 Carbonyl compounds Acetaldehyde 2,3-Butanedione (diacetyl) Green leaves, fruity 25 2.9–3.4 Butter-scotch 0.15 <0.01–0.06 Kobayashi et al. J. Biosci. and Bioengr. 2008. Kobayashi et al. J. Biosci. and Bioengr. 2008. Parameter Amino acid Metal Lees oil EDTA Fatty acid Gravity Temperature Top pressure Oxygen Production of higher alcohols and esters Addition promoted (amyl alcohols and esters) and no effect on isobutyl alcohol Addition promoted (only isobutyl alcohol Val production) Ile Addition promoted (only amyl alcohol production) Addition promoted (ethyl acetate and n-propanol) Asp and repressed (isobutyl alcohol) Addition promoted (higher fermentation rates was Zn obtained.) Addition promoted (isoamyl acetate) and no effect on ethyl acetate Addition did not significantly promote C18:2 Addition repressed (only acetate esters) Promoted (from 12 to 20° Plato media) and repressed in higher-gravity media Promoted as temperature increased Repressed as top pressure increased Repressed during aeration prior to pitching Leu pH (4.9–8.5) Promoted (isoamyl alcohol and isoamyl acetate) and repressed (ethyl acetate) Serial repitching Promoted as the number of repitchings increased Kobayashi et al. J. Biosci. and Bioengr. 2008. Compounds Flavor note Odor threshold in beer Probable precursor (ppb)a Concentration in finished beer (ppb) Sulfur dioxide (SO2) Burnt matches 25 ppm Sulfate/sulfite 200 Pungent, rotten eggs Cooked cabbage; putrid 5–10 2 Sulfate, cysteine Methionine 0.5–20 Nd 3-Methyl-2-butene-1-thiol Onion, leek, skunky flavor 1–100 ppt 2-Mercaptoethanol rotten eggs Thiols Hydrogen sulfide (H2S) Methanethiol (MTL) Polyfunctional thiols 3-Mercaptopropanol Hop (isohumulone) + cysteine + ri Nd boflavine + light Cysteine Nd Homocysteine Nd 30 SMM, dimethylsulfoxide 5–90 3–50 0.3–1.5 1.2 MTL MTL, H2S, 3-MTP, Smethylcysteinesulfoxide Unknown Cheese, cooked vegetables Ripened cheese, cabbage >100 0.8–3.5 MTL and acetyl–CoA Unknown 3–8 40 Soap, potato Cauliflower 250 < 0.1b 2,000 Methionine Methionine Nd Nd Cardboard, musty 200 hop 10–9,300 ppm Sulfides Dimethyl sulfide Dimethyl disulfide Dimethyl trisulfide Dimethyl tetrasulfide Cabbage, corn, onion, blackcurrant Cooked cabbage, onion Fresh onion, cooked vegetables Onion, cooked vegetables 0.1 0.1–1.8 0.2 Thioesters S-Methylthioacetate S-Ethylthioacetate Alkyl thio derivatives Methional Methionol Sulfured terpens 1,2-Epithiohumulene nd Not determined, SMM S-methyl methionine, and 3-MTP 3-methylthiopropionaldehyde aUnless stated otherwise, odor threshold values were determined in beer. bIn alcohol-free beer Landaud et al. App. Microbiol. Biotechnol. 2008 Landaud et al. App. Microbiol. Biotechnol. 2008 How can we make even better beer? Breeding an Amylolytic Yeast Strain for Alcoholic Beverage Production. Cheng MC, Chang RC, Dent DF, Hsieh PC. Appl Biochem Biotechnol. 2010 Sep 5. [Epub ahead of print] Improvement of Saccharomyces yeast strains used in brewing, wine making and baking. Donalies UE, Nguyen HT, Stahl U, Nevoigt E. Adv Biochem Eng Biotechnol. 2008;111:67-98. Review. Multiobjective optimization and multivariable control of the beer fermentation process with the use of evolutionary algorithms. Andrés-Toro B, Girón-Sierra JM, FernándezBlanco P, López-Orozco JA, Besada-Portas E. J Zhejiang Univ Sci. 2004 Apr;5(4):378-89. The potential of genetic engineering for improving brewing, wine-making and baking yeasts. Dequin S. Appl Microbiol Biotechnol. 2001 Sep;56(56):577-88. Review. Genetic improvement of brewer's yeast: current state, perspectives and limits. Saerens SM, Duong CT, Nevoigt E. Appl Microbiol Biotechnol. 2010 May;86(5):1195-212. Epub 2010 Mar 2. Review. Use of a modified alcohol dehydrogenase, ADH1, promoter in construction of diacetyl non-producing brewer's yeast. Onnela ML, Suihko ML, Penttilä M, Keränen S. J Biotechnol. 1996 Aug 20;49(1-3):101-9. Study Blieck et al. (2007) Olesen et al. (2002) Gibson et al. (2008) James et al. (2003) Higgins et al (2003) Mizuno et al. (2006) Bond et al. (2004) Pope et al. (2007) Joubert et al. (2001) Joubert et al. (2000) Brejning et al. (2005) Kobi et al. (2004) Minato et al. (2009) Yoshida et al. (2008) Strains and conditions investigated Purpose of investigation Level of global analysis (method applied) Lager yeast strain (CMBS33) and an UV-induced mutant of this strain showing improved fermentation performance in high-gravity wort (23 °P, 2 Strain improvement via inverse engineering Transcriptome (microarray, S.c. gene probeset) l scale, tall tube vessels) Industrial lager yeast strain in 5,000 hl 14 °P wort in cylindroconical Dynamics of brewing fermentation Transcriptome (microarray, S.c. gene probeset) fermentation tanks Study of the response of lager brewing yeast to Lager yeast strain (CB11) in cylindroconical fermentation tanks (3,275 hl changes in wort fermentable carbohydrate Transcriptome (microarray, S.c. gene probeset) scale, 17 °P wort) concentration and composition Two bottom-fermenting lager strains (Guinness 6701 and 7012) in 2 l 15 °P wort in European Brewery Convention (EBC) tall cylindroconical Dynamics of brewing fermentation Transcriptome (microarray, S.c. gene probeset) fermentation vessels Study of the stress response during an industrial lager Industrial lager yeast in 20 l 12 °P wort in industrial fermentation vessels Transcriptome (microarray, S.c. gene probeset) fermentation Top-fermenting brewer’s yeast strain (NCYC1245) and a 2-deoxyglucoseIdentification of the genes involved in the low acetic resistant mutant of this strain with low acetic acid and high ethanol Transcriptome (microarray, S.c. gene probeset) acid/high ethanol phenotype productivities (100 ml scale, 13 °P wort) Two bottom-fermenting lager yeast strains (CMBS33 and Guinness 6701) in Aneuploidy and copy number breakpoints in lager Genome (CGH, S.c. gene probeset) comparison with the haploid laboratory strain S-150 yeast strains Genome (CGH, S.c. gene probeset) Two ale brewer’s strains, six lager brewer’s strains and one type strain of S. Differentiation between industrially used brewer’s cerevisiae in complex medium (YM) strains Exometabolome (DIMS, GC-TOF-MS) Identification of proteins which do not co-migrate with Proteome (2D gel electrophoresis, MALDI–MS and Lager brewer’s yeast strain (K11) in minimal medium (YNB, 2% glucose) the known proteins of S.c. MS/MS) Seven lager brewer’s yeast strains, type strains of S. cerevisiae, S. bayanus, Obtain information about the identity of the ancestors Proteome (2D gel electrophoresis, gas–liquid phase S. carlsbergensis, S. monascensis, S. pastorianus and S. uvarum in minimal of lager brewer’s yeast sequencing) medium (YNB, 2% glucose) Identify proteins whose expression is induced in lager Lager brewer’s yeast strain (KVL001) in minimal medium (YNB, 0.5% Proteome (2D gel electrophoresis, MALDI–MS and brewing during lag phase and early exponential glucose) MS/MS) growth Ale yeast strain (A38) in complex medium (YPD) and brewer’s wort Comparison of an ale, a lager and a laboratory yeast Lager brewers’ yeast strain (K11) in YPD medium Proteome (2D gel electrophoresis, MALDI–MS) strain Laboratory yeast (S288c) in complex medium (YPD) Lager brewers’ yeast strain (KBY011), S. cerevisiae laboratory strain and S. Expression of S.c-type and non-S.c.-type genes in a Transcriptome (microarray, S.c. gene and non-S.c. EST pastorianus in complex medium (YPD) lager brewer’s yeast probes) Transcriptome (microarray with S.c. gene and non-S.c. One lager brewer’s yeast (KBY011) and one baker’s yeast (S288c) showing Strain improvement via inverse engineering (increase EST probes) significant differences in sulphite production (SD10 medium lacking amino of sulphite production) acids, 2 l scale, anaerobic conditions) Endometabolome (CE–ESI–MS) Genome (CGH, “two-species array” with probes for genes from S.c. and S. bayanus var. uvarum) Genome (whole-genome array CGH with S.c. gene and non-S.c. probesets) Three lager brewer’s yeast strains which show significant differences in Strain improvement via inverse engineering (reduction Duong Cam et al. (in preparation) Transcriptome (whole-genome array with S.c. gene diacetyl production analysed in wort under conditions relevant in brewing of diacetyl formation) and non-S.c. probesets) Proteome (2D gel electrophoresis, MALDI–MS) Identify the complete genomic sequence of a Nakao et al. (2009) Lager brewer’s yeast strain (Weihenstephan 34/70) Genome (whole genome sequencing) commonly used lager yeast strain Dunn and Sherlock (2008) 17 lager brewers strains and 3 ale strains Differentiation between brewer’s yeast strains and identification of the ancestors of S. Pastorianus Saerens et al. Appl Microbiol Biotechnol. 2010. Yeast and human health Tumor cell energy metabolism and its common features with yeast metabolism. Diaz-Ruiz R, Uribe-Carvajal S, Devin A, Rigoulet M. Biochim Biophys Acta. 2009 Dec;1796(2):25265. Epub 2009 Aug 12. Review. Yeast cell wall polysaccharides as antioxidants and antimutagens: can they fight cancer? Kogan G, Pajtinka M, Babincova M, Miadokova E, Rauko P, Slamenova D, Korolenko TA. Neoplasma. 2008;55(5):387-93. Review. Saccharomyces cerevisiae: a useful model host to study fundamental biology of viral replication. Alves-Rodrigues I, Galão RP, Meyerhans A, Díez J. Virus Res. 2006 Sep;120(1-2):49-56. Epub 2006 May 15. Review. Winderickx J, Delay C, De Vos A, Klinger H, Pellens K, Vanhelmont T, Van Leuven F, Zabrocki P. Biochim Biophys Acta. 2008 Jul;1783(7):138195. Epub 2008 Feb 11. Review. Brewer's/baker's yeast (Saccharomyces Combined yeast-derived beta-glucan with anti- cerevisiae) and preventive medicine: Part II. Moyad MA. tumor monoclonal antibody for cancer Urol Nurs. 2008 Feb;28(1):73-5. Review. immunotherapy. Liu J, Gunn L, Hansen R, Yan J. Protein folding diseases and Exp Mol Pathol. 2009 Jun;86(3):208-14. Epub neurodegeneration: lessons learned from 2009 Jan 21. Review. yeast. References and further reading National Institutes of Health digital archive of biomedical and life sciences journal literature http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed http://www.wikipedia.org http://homebrewandchemistry.blogspot.com/ Site of Brew Your Own magazine http://www.byo.com/ http://forum.northernbrewer.com/ http://www.brewingtechniques.com/ Site of UC-Davis Anheuser-Busch Endowed Professor of Brewing Science Charles Bamforth http://foodscience.ucdavis.edu/bamforth/ Rensselaer Polytechnic Institute brewing class http://www.rpi.edu/dept/chem-eng/Biotech-Environ/beer/index1.htm Tools of the trade High performance liquid chromatograph (HPLC) Gas chromatograph (GC) Fermenter Electrospray ionization m mass spectrometer (ESI-MS)