Newly-created hybrid lager yeast strains (S. cerevisiae x S. eubayanus) outperform both parents during brewery fermentation

Transcription

1 Newly-created hybrid lager yeast strains (S. cerevisiae x S. eubayanus) outperform both parents during brewery fermentation th Congress EBC Porto Kristoffer Krogerus, Frederico Magalhães, Virve Vidgren, Brian Gibson

2 Background Pale lager is the most popular beer style worldwide Clean flavour profile Lack of yeast-derived aroma compounds Joel Olives 28/04/2015 2

3 Background S. pastorianus is a natural hybrid between S. cerevisiae and S. eubayanus: Saccharomyces cerevisiae (cold-sensitive, good fermentation) Saccharomyces eubayanus (cold-tolerant, poor fermentation) Saccharomyces pastorianus (Good fermentation performance at low temperature, lager brewing) 28/04/2015 3

4 Background Poor diversity among the traditional lager yeasts Belong to one of two distinct lineages: Saaz Frohberg Group 1 (Saaz) Group 2 (Frohberg) Weak fermentation Very cold-tolerant S. eubayanus genome dominates Strong fermentation Cold-tolerant S. cerevisiae genome dominates 28/04/2015 4

5 Background Making new lager yeast hybrids by mating S. eubayanus with selected S. cerevisiae parents? S. cerevisiae S. eubayanus Lager yeast hybrid 28/04/2015 5

6 Aim Generate artificial lager yeast hybrids by mating S. eubayanus with selected S. cerevisiae parents Assess fermentation performance of hybrids Low temperature fermentation Sugar utilization Production of aroma compounds Flocculation 28/04/2015 6

7 Parent strains Saccharomyces cerevisiae VTT-A81062 Origin: Brewery in the United Kingdom Strongly flocculent Maltotriose utilization Saccharomyces eubayanus VTT-C12902 (CBS12357) Type strain No maltotriose utilization Good cold tolerance 28/04/2015 7

8 Selection of hybrids Auxotrophic markers (ura-, lys-, leu-, ade-, etc.) Inability to grow with NH 4 + as sole nitrogen source S. cerevisiae S. eubayanus ura-, lys+ ura+, lys- Lager yeast hybrid ura+, lys+ 28/04/2015 8

9 Hybrid generation mass mating Yeast cultures of parent strains Purification Incubation Selection 28/04/2015 9

10 Confirmation of hybrid status ITS-PCR and RFLP: Hybrids H1-H4 Sc A62 Se C902 28/04/

11 Confirmation of hybrid status Amplification with FSY1- and MEX67-specific primers: Hybrids H1-H4 Sc A62 Se C902 FSY1 MEX67 28/04/

12 DNA content of hybrids Estimated with SYTOX Green-staining and flow cytometry. 1n 2n 4n 28/04/

13 Growth at various temperatures Strain 8 C 25 C 37 C S. cerevisiae A62 S. eubayanus C902 Hybrid H1 Hybrid H2 Hybrid H3 Hybrid H4 28/04/

14 Fermentation The fermentation performance of 4 hybrids was compared in 2L all-malt wort fermentations at 12 P and 12 C. 28/04/

15 Alcohol (%) Fermentation performance (12 C, 12 P) Hybrids H1H1-H4 A81062 S. cerevisiae (Sc) A62 C12902 S. eubayanus (Se) C Fermentation time (hours) 28/04/

16 Maltotriose utilization Wort Original wort Hybrids H2H1-H4 A62 S. cerevisiae ura3- A62 C902 lys- S. eubayanus C902 0 Maltotriose g/l 28/04/

17 µmol/min/g DY µmol/min/g DY Maltotriose transport Maltose transport activity 0 Ale S. eub Hyb1 Hyb2 Hyb3 Hyb Maltotriose transport activity Ale S. eub Hyb1 Hyb2 Hyb3 Hyb4 Strain 28/04/

18 Flocculation potential Hybrid H1 Hybrid H2 Hybrid H3 Hybrid H4 S. cerevisiae A62 S. eubayanus C902 H1 H2 H3 H4 A81062 ura3- C12902 lys- 28/04/

19 ppm Vicinal diketones Diacetyl is unwanted in lager beers Concentrations of diacetyl in beers fermented with hybrid strains in between those of the parent strains Diacetyl 0,3 0,2 0,1 0,0 Hybrid H1 Hybrid H2 Hybrid H3 Hybrid H4 A81062 (Sc) C12902 (Se) 28/04/

20 Aroma compounds - higher alcohols Methylpropanol Hybrid H1 Hybrid H2 Hybrid H3 Hybrid H4 A81062 (Sc) 2-Methylbutanol C12902 (Se) ppm Hybrid H1 Hybrid H2 Hybrid H3 Hybrid H4 A81062 C12902 Hybrid H1 Hybrid H2 Hybrid H3 Hybrid H4 A81062 C12902 (Sc) (Se) (Sc) (Se) 28/04/ ,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 3-Methylbutanol Hybrid H1 Hybrid H2 Hybrid H3 Hybrid H4 A81062 (Sc) 3-Methylbutyl acetate C12902 (Se)

21 Aroma compounds ethyl esters Ethyl acetate ppm 0,6 0,5 0,4 0,3 0,2 0,1 Ethyl hexanoate 0 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 Hybrid H1 Hybrid H2 Hybrid H3 Hybrid H4 A81062 (Sc) Ethyl octanoate Hybrid H1 Hybrid H2 Hybrid H3 Hybrid H4 A81062 (Sc) C12902 (Se) C12902 (Se) 28/04/ ,0 0,4 0,3 0,2 0,1 0,0 Hybrid H1 Hybrid H2 Hybrid H3 Hybrid H4 A81062 (Sc) Ethyl decanoate Hybrid H1 Hybrid H2 Hybrid H3 Hybrid H4 A81062 (Sc) C12902 (Se) C12902 (Se)

22 Aroma compounds The beers fermented with the hybrid strains: All had similar aroma profiles Concentrations of higher alcohols and their esters in between those of the parent strains Concentrations of ethyl esters higher than those of the parents strains 28/04/

23 Mass loss (%) Mass loss (%) Fermentation at various temperatures 6% 5% 4% A62 C902 A15 (Froh) A62 C902 hybrid 20 C 3% 2% 1% 0% Fermentation time (hours) 6% 5% A62 C C 4% 3% A15 (Froh) A62 C902 hybrid 2% 1% 0% Fermentation time (hours) 28/04/

24 ppm ppm Aroma compounds at various temperatures Mary Siebert 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 A62 C902 A15 (Froh) Isoamyl acetate A62 C902 hybrids 20 C 15 C 10 C 0,4 Ethyl caproate 0,3 0,2 arsheffield 0,1 0,0 A62 C902 A15 28/04/2015 A62 C902 hybrids (Froh) 24

25 Conclusions Successful generation of de novo lager yeast hybrids by interspecific hybridization Non-GM Hybrid strains inherited beneficial properties from both parents, and showed apparent hybrid vigour: Cryotolerance Flocculation ability Maltotriose utilization Faster fermentation Higher aroma production 28/04/

26 Acknowledgements Alfred Kordelin Foundation PBL Brewing Laboratory Academy of Finland FP7 Marie-Curie ITN YEASTCELL Sirpa Jylhä Annika Wilhelmson Aila Siltala Arvi Wilpola Eero Mattila 28/04/