SCREENING FOR THE BREWING ABILITY OF NON-SACCHAROMYCES YEASTS

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1 Young Scientists Symposium on Malting, Brewing and Distilling April 2016 SCREENING FOR THE BREWING ABILITY OF NON-SACCHAROMYCES YEASTS Dipl.-Ing. Maximilian Michel

2 Overview Little introduction How does yeast screening work? My results Future perspective Schizosaccharomyces pombe Dipl.-Ing. Maximilian Michel Slide 2

3 Yeasts in use Fermentation behavior/ Flocculation behavior Beer style/ Beverage Genus/ Species Brewery yeasts Bottom fermenting Top fermenting Flocculating Dusty Dusty Pils, Export, Helles, Lager, untergäriges Spezialbier, untergäriges Leichtbier, etc. Wheatbeer, Ale, Stout, Kölsch, Alt, Belgian special beers, etc. - - Low alcohol beers Berliner Weiße, Belgian special beers African acientbeers S. pastorianus (ssp.carlsbergensis) S. cerevisiae Saccharomycodes ludwigii Dekkera bruxellensis Schizosaccharomyces pombe Dipl.-Ing. Maximilian Michel Folie 3

4 Potential approx. 20 species 1500 known species approx yeast species Potential!!! Dipl.-Ing. Maximilian Michel Folie 4

5 Screening yeasts Torulaspora delbrueckii Used in wine fermentation Adds fruitiness Schizosaccharomyces pombe Known from ancient beers pombe refers to Swahili language = beer Kluyveromyces lactis Occurs mostly in milk products Referred to as generally save Hansenisaspora uvarum Used in wine fermentation Spontaneous fermentations of banana wine Kluyveromyces marxianus Fermentation of Cacao beans Also found in ancient beers Dipl.-Ing. Maximilian Michel Folie 5 Pichia anomala Phenolic off flavor pos. Found as spoilage organism in many foods

6 Screening Wild yeast Screening Genetic fingerprint, Real time PCR Suitable for beer? Off flavors - Tests on Characteristics of beer - Propagation ability Fermentation trials Tastings In case of success optimizing the fermentation towards taste and fermentation by products Dipl.-Ing. Maximilian Michel Folie 6

7 Screening Suitable for beer? Carbone sources: Hop acids: ph-value: glucose, fructose, sucrose, maltose, maltotriose, melibiose iso-α-acids ( IBU), β-acids (0.2% - 0.4%) ph 7-4 (pre- and post fermentation) Ethanol: 1 10 Vol. % Dipl.-Ing. Maximilian Michel Folie 7

8 Screening POF-Tests: Already been used in the wine industry Precursor Cumaric acid-, Ferulic acid-, Cinnamic acid- YM-Media: Decarboxylation 4-Vinylphenol 4-Vinylguajacol 4-Vinylbenzol (Styrol) Dipl.-Ing. Maximilian Michel Folie 8

9 Screening and propagation preparation 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 Dipl.-Ing. Maximilian Michel Folie 9

10 Screening Small scale fermentations: - Only if prior tests were positive - Analysis of fermentation byproducts - Analysis of extract, final attenuation, ph Dipl.-Ing. Maximilian Michel Folie 10 - If promising results, strain is used in larger scale fermentations

11 Screening Preparation: Fermentation Vesels 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 Dipl.-Ing. Maximilian Michel Folie 11

12 Screening Analyses: Fermentation main- and by products Amino acid metabolism and Sugar fermentation Extract reduction ph- development Higher alcohols Ethanol Diacetyl Tastings Flocculation Cells in abeyance Beer color In case of promising results Optimization: - cell count - starting temperature - storage Dipl.-Ing. Maximilian Michel Folie 12

13 Results T: Torulaspora delbrueckii S: Schizzosaccharomyces pombe Pa: Pichia anomala H: Hanseniaspora uvarum K: Kluyveromyces lactis Km: Kluyveromyces marxianus T6 T9 Pa2 Pa5 Pa6 Pa9 Pa13 Pa16 Pa18 Pa19 Pa21 Pa23 Pa24 Pa25 Pa26 Pa27 Pa28 S5 S6 S7 S11 Pa1 Pa3 Pa10 Pa12 Pa14 Pa15 Pa8 Pa22 K2 K4 K7 S4 T13 T15 T17 T18 T19 T20 K3 KM3 KM6 KM7 KM8 KM9 KM10 KM11 S2 S10 Pa20 T12 KM1 KM2 K5 K8 KM5 SM1 KM4 H1 H2 H3 H4 H5 H6 H8 H9 H10 H7 Pa4 K1 K6 K9 S3 Pa7 S8 S9 Pa17 T Glucose Fructose 1% Ethan 2% Ethan 200ppm b ph 6 5% Ethan 50 IBU 100 IBU 100ppm b 50 IBU u ph4 ph IBU ph 7 ph 5 Saccharo 8% Ethan 10% Etha Maltose Maltotri Melobios Dipl.-Ing. Maximilian Michel Folie 13

14 Screening Torulaspora delbrueckii - Known from the wine industry - Application for more fruitiness - Growth on malt = purity law - Already little use in the beer industry - Metabolizes many different sugars Yeast T6 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 Origin Wine, Sorghum Brandy Unknown Beer Beer Weatbeer Feta production Chees sole WYSC WYSC CBS DSM CBS T = Torulaspora delbrueckii CBS DSM WYSC = Centralbuerau voor Schimmelcultures, Utrecht Niederlande = Deutsche Sammlung für Mikroorganismen und Zellkulturen, Braunschweig Deutschland = Weihenstephan Culture Collection of Yeast and Mould Strains, Freising Deutschland Dipl.-Ing. Maximilian Michel Folie 14

15 Results Torulaspora delbrueckii T6 T9 T10 T11 T13 T15 T17 T18 T19 T20 Fructose Glucose Sucrose Maltose Maltotriose Yeast T6 T9 T10 T11 T13 T15 T17 T18 T19 T20 Ethanol (%. v/v) 0.87 ±0.01 a 4.00 ±0.01 a 0.83 ±0.01 a 0.87 ±0.02 a 0.89 ±0.01 a 0.94 ±0.01 a 0.89 ±0.01 a 0.87 ±0.02 a 0.9 ±0.02 a 0.91 ±0.01 a - T9 maltose positive strain - All strains POF negative - T9 produced 4 v/v% alcohol Dipl.-Ing. Maximilian Michel Folie 15

16 Results Torulaspora delbrueckii fruity floral wort like Yeast Origin Flocculation T6 Wine, Sorghum Little dust T19 T20 8 T6 T T10 T9 Brandy Very dusty T10 Unknown Little dust T11 Beer Little dust T13 Wheat beer Little dust T15 Chees sole Curdy T18 T11 T17 WYSC Little dust T18 CBS Little dust T17 T15 T13 T19 DSM Little dust T20 CBS Little dust Dipl.-Ing. Maximilian Michel Folie 16

17 Results Torulaspora delbrueckii Optimization of the fermentation of T9 with the two parameters Pitching rate 1-70 Mio. cells/ml Temperature ºC Design Expert (Response Surface Method) 32ºC emperature Responses: Fermentation-by-products Tastings Attenuation, ph-drop 15ºC 1 Pitching rate Mio. cells/ml 70 Dipl.-Ing. Maximilian Michel Folie 17

18 Results Torulaspora delbrueckii 10 trained panelists 5 high intensity 0 no intensity Honey Caramel Grapefruit Berry Currant Plum Lemon Maracuja Malty Wine Gummy Bear 60 Mio. cells/ml pitching rate 30 C 60 Mio. cells/ml pitching rate 18 C Dipl.-Ing. Maximilian Michel Folie 18

19 Results Torulaspora delbrueckii Alcohol content averaged % Vol. ph-value Average of 7-10 days of fermentation 60 Mio. cells/ml pitching rate 18 ⁰C 30 ⁰C 2-phenyl ethanol ethylacetate amyl-alcohol Dipl.-Ing. Maximilian Michel Folie 19

20 Future perspective Screen more species Move up to bigger batches with T9 Optimize wort composition Find well fitting hops to the currant/fruity flavor of T9 Dipl.-Ing. Maximilian Michel Folie 20

21 Thank you for your attention Dipl.-Ing. Maximilian Michel Alte Akademie Freising-Weihenstephan Telefon: +49 (0) 8161 / Telefax: +49 (0) 8161 / m.michel@wzw.tum.de Dipl.-Ing. Maximilian Michel Folie 21