26. Pivovarsko Sladařské DNY 22.-23.12015 Non-Saccharomyces yeast in brewing Maximilian Michel, Fritz Jacob, Mathias Hutzler
Outline I. Short introduction II. Screening overview III. Screening methods and results Torulaspora delbrueckii IV. Future perspective
German beer market and the purity law Decreasing numbers in beer consume Increasing numbers in beer mix, cider. Innovations Huge hop varieties Special malts old beer styles Craft brewing
Yeasts used in breweries Fermentation behavior/ Flocculation behavior Beer style/ Beverage Genus/ Species Bottom fermenting Flocculating Dusty Pils, Export, Helles, Lager, Bottom fermented special beers, Bottom fermented low alcohol beer, etc. S. pastorianus (var.carlsbergensis) Brewery yeasts Top fermenting Dusty Wheatbeer, Ale, Stout, Kölsch, Alt, Belgian special beers, etc. S. cerevisiae - - Alcohol free beers Saccharomycodes ludwigii - - Berliner Weiße, Belgian special beers Dekkera bruxellensis - - African acient-beers Schizosaccharomyces pombe Source: Huztler M, Koob J, Riedls R, et al. 2015. Yeast identification and characterization. In: Brewing microbiology. Editor Hill A. London: Woodhead Publishing, 65-104.
Potential of Yeast approx. 20 species 1500 known species approx. 67000 yeast species Potential!!!
Potential of Yeast Metabolism - Carbon sources - Amino acids Fermentation - Main products - Side products - Technologies - Flocculation - SO2-forming - Glycerin production (mouthfeel) - Resistance ability - Pressure resistance - Temperature resistance - Hop resistance - Ethanol resistance
3.Seminar Hefe und Mikrobiologie Saccharomycodes ludwigii (Picture Saccharomycodes ludwigii TUM SL 17 Forschungszentrum Weihenstephan für Brau- und Lebensmittelqualität BLQ) Metabolism: Uses only Glucose, Fructose and Sucrose Usage for low or reduced alcohol beers Propagation as usual with pure yeast strains Fermentation temperature 15-20ºC Wort extract 7 ºP Ethanol 0,5 %Vol
3.Seminar Hefe und Mikrobiologie Saccharomycodes ludwigii
Torulaspora delbrueckii Yeast Abbreviation Origin Already little use in the beer industry Known from the wine industry Application to increase fruitiness TUM T6 T6 Wine, Sorghum TUM T9 T9 Brandy TUM T10 T10 Unknown TUM T11 T11 Beer TUM T13 T13 Wheat beer TUM T15 T15 Cheese sole TUM T17 T17 WYSC TUM T18 T18 CBS TUM T19 T19 DSM TUM T20 T20 CBS T = Torulaspora delbrueckii Occurrence on malt = purity law Metabolizes many different sugars CBS = Centralbuerau voor Schimmelcultures, Utrecht Niederlande DSM = Deutsche Sammlung für Mikroorganismen und Zellkulturen, Braunschweig Deutschland WYSC = Weihenstephan Culture Collection of Yeast and Mould Strains, Freising Deutschland
Wild yeast Screening Suitable for beer? Wort conditions /Propagation Genetic fingerprint, Real time PCR Phenolic off flavors (POF) Fermentation trials Sensorial analyses In case of success optimizing the fermentation towards taste and fermentation by products
Outcome of the screening by Yeast Ethanol Early strain Sugars Hops Ethanol POF content Esters Acids Higher Alcohols Gl uc os e Fructose Sucrose Maltose Malt otrios e Melibi ose 100 EBU Iso 50 EBU Iso Beta 10% 5% 2%Ferulic Cinnamic Cumaric v/v% Ethyla cetate Isoamyla cetate Butter acid ethyl ester Acetate acid- Isobutyl ester Acetate acid- 2-Phenylester Hexanoic acid ethyl ester Octanoic acid ethyl ester Decanoic acid ethyl ester Hexan acid Octan acid Decanacid 2-Phenyl-ethanol n-propanol i-butanol Amylalcohols Speed product s D i a c e Ace t tald y ehy l d Maltose pos Maltose neg T6 pos pos pos neg neg neg pos pos pos neg neg pos neg neg neg 87 3.83 0 0 01 04 0 0 01 2 5 2 12.8 4.8 3.4 28.03 31 1.5 High T9 pos pos pos pos pos neg pos pos pos neg pos pos neg neg neg 4 23.4 0 0 01 06 01 0 0 07 1 02 23.7 17 19.9 64.83 43 18.9 Middle T10 pos pos pos neg neg neg pos pos pos neg pos pos neg neg neg 83 2.2 0 0 01 02 0 0 01 1 3 6 15.8 3 2.3 25.26 13 7.3 High T11 pos pos pos neg neg neg pos pos pos neg pos pos neg neg neg 87 4.5 0 0 01 03 0 0 01 1 4 4 8.1 4.9 4.4 25.93 14 6.9 High T12 pos pos pos neg neg neg pos pos pos neg pos pos neg neg neg 85 5.2 0 0 01 02 0 0 01 01 02 2 9.1 4.1 3.3 26.1 T13 pos pos pos neg neg neg pos pos pos neg pos pos neg neg neg 89 5.3 0 0 01 03 0 0 01 2 5 1.1 13.9 4.1 3.5 29.1 1 8.1 High 1 5.6 High T14 pos pos pos neg neg neg pos pos pos neg pos pos neg neg neg 87 6.2 0 0 01 04 0 0 01 03 04 06 11 7.1 2.4 25.5 12 7.2 High T15 pos pos pos neg neg neg pos pos pos neg pos pos neg neg neg 94 2.7 0 0 01 02 0 0 0 1 3 08 11.8 5.7 2.1 24.1 15 2.2 High T16 pos pos pos neg neg neg pos pos pos neg pos pos neg neg neg 85 4.1 0 0 01 02 0 0 0 0 01 8 8.3 3 4.4 27.1 14 7.9 High T17 pos pos pos neg neg neg pos pos pos neg pos pos neg neg neg 89 5.9 0 0 01 03 0 0 01 1 4 6 8.9 3.6 3.3 28.36 12 1.3 High T18 pos pos pos neg neg neg pos pos pos neg pos pos neg neg neg 87 3.7 0 0 01 01 0 0 01 2 7 2 5.4 2.9 1.3 273 15 3.6 High T19 pos pos pos neg neg neg pos pos pos neg pos pos neg neg neg 9 3.9 0 0 01 03 0 0 01 1 3 1 5 2.7 2.8 15 2 3.2 High T20 pos pos pos neg neg neg pos pos pos neg pos pos neg neg neg 91 4.3 0 0 01 01 0 0 0 1 3 3 5.2 2.3 4 18.6 14 17.6 High All results are put together in a heat map As more yeasts will be screened a overview is convenient
Suitable for beer? Carbon sources: glucose, fructose, sucrose, maltose, maltotriose, melibiose Hop acids: ph-value: Ethanol: iso-α-acids (10-90 IBU), β-acids (100 and 200 ppm) ph 7-4 (pre- and post fermentation) 1% - 10% (+ mix with hop acids)
Results wort conditions T6 T9 T10 T11 T13 T15 T17 T18 T19 T20 Fructose + + + + + + + + + + Glucose + + + + + + + + + + Sucrose + + + + + + + + + + Maltose - + - - - - - - - - Maltotriose - + - - - - - - - - - Growth in ph range from ph 7-4 - Growth in ethanol concentrations till 6 vol % (but T6) IBU/Ethanol % [v v -1 ] T6 T9 T10 T11 T13 T15 T17 T18 T19 T20 50/ 5 - + - + - + + + + - 90/ 5 - - - + - + + + + -
POF-Tests Precursor Cumaric acid-, Ferulic acid-, Cinnamic acid- YM-Media: Decarboxylation 4-Vinylphenol 4-Vinylguajacol 4-Vinylbenzol (Styrol) YM-Media: Yeast Media No POF detected
Wort conditions/propagation Standardized optimal conditions Wort extract: For standardized conditions - 12ºP, FAN 200 mg/l - Good sugar distribution - Good amino acid distribution - No hops Temperature Tests for temperature optimum and cell size optimum (photometer) Propagation
Results propagation Mio. cells/ml. Viability [%] 250 98.7 98.8 200 97.9 97.8 96.9 97.1 96.6 150 96.3 96.3 95.3 100 50 0 T6 T9 T10 T11 T13 T15 T17 T18 T19 T20 Yeast 100 99.0 98.0 97.0 96.0 95.0 94.0 93.0 92.0 Vitality measurement AP (acidification potential)* <1,8 (low vitality) 1,8-2,4 (good vitality) >2,4 (very good vitality) Yeasts Measured Average 2,2 AP *Source: P. Gabriel, M. Dienstbier, D. Matoulková : Optimised Acidification Power Test of yeast vitality and ist use in brewing practise, Journal of the institute of brewing, 2012 CBS = Centralbuerau voor Schimmelcultures, Utrecht Niederlande DSM = Deutsche Sammlung für Mikroorganismen und Zellkulturen, Braunschweig Deutschland WYSC = Weihenstephan Culture Collection of Yeast and Mould Strains, Freising Deutschland
Fermentation trials 2 L small scale in triple determination for analyses and tastings First trial at 27ºC and 30 Mio. cells/ml Fermentation till final attenuation Daily analysis
Analyses Ethanol Extract reduction ph- development Diacetyl Amino acid metabolism and Sugar fermentation Fermentation main- and by-products Flocculation Cells in abeyance Extract [ P] 14 12 10 8 6 4 2 0 0 24 48 72 96 120 144 168 Time [h] Sensorial analyses
Results extract ph decreased from 5.2 to below 4.2 in the first two days Ethanol content between 87 and 4 % [v/v]
Analyses fermentation by-products
Results sensorial analyses fruity floral wort like T19 T18 T20 8 T6 T9 6 4 2 0 T10 T11 - T9 very fruity and floral - Strawberry, plum like flavors - T17 and T13 (maltose neg.) fruity flavor - T18 wort like T17 T13 T15
Future perspective - Optimizing T9 for flavor and taste to use it in breweries - Adding different hops that suit the fruity taste - Change wort concentrations for the production of fruity low alcohol beers fermented by T13 and T17 - Developing a quality control system for non-saccharomyces yeasts suitable for brewerys ( Cross contaminations ) - Screening more non-sacch. for brewing
Thank you for your attention MBAA Annual Conference Dipl.-Ing. Maximilian Michel Alte Akademie 3 85354 Freising-Weihenstephan Telefon: +49 (0) 8161 / 71-3384 Telefax: +49 (0) 8161 / 71-4181 E-Mail: m.michel@wzw.tum.de www.blq-weihenstephan.de