Lachancea thermotolerans in pure-culture fermentations

Transcription

1 Lachancea thermotolerans in pure-culture fermentations Jen House UC Davis

2 Lachancea Saccharomycetaceae family Formerly Kluyveromyces (6,7) Reclassified by Kurtzman in 2003 Named after Dr. Marc-André Lachance Unique in its ability to produce lactic acid (4,5) Souring potential in beer? Maltose fermentation capacity is strain-variable (7) Found naturally on insects and plants (10,11,13) Apian microbiota

3 Oenological use Found in natural fermentations of Majorcan wines (9) Glycerol production for improved mouthfeel (1) Pitched pre-saccharomyces to assist with ph drop in low-acidity wines (1-3,8,9) Chr. Hansen Concerto strain Trials suggest not optimal for beer Highly phenolic

4 Experimental Design Initially investigated two strains from UC Davis and one strain from yeast culture from the Polytechnic University of Marche SAIFET (Ancona, Italy) Selected Italian strain (Strain 101) for organoleptic character, flocculation, and ability to produce higher levels of lactic acid Most studies performed in benchtop trials with 300 ml wort in 500 ml flasks

5 Performance in wine fermentation Previous study of Strain 101 (1) 8% alcohol by volume (ABV) Positive for β-glucosidase activity Negative for α-glycosidase, protease, and esterase activity Resistant to up to 20 ppm SO 2 Negative for killer factor

6 Performance in beer fermentation (Strain 101) Sugar metabolism: maltose and maltotriose Lactic acid and glycerol production Pitching rate Re-pitching capacity Flocculation characteristics Oxygen requirements Foam stability Vicinal diketone (VDK) production Tolerance of hop iso-alpha-acids

7 Sugar metabolism Sac Maltotriose, maltose and ethanol concentration after 21 days of fermentation All three strains fermented 93-94% of maltose present in wort; unable to ferment maltotriose

8 Lactic acid and glycerol production Sac Sac Glycerol and lactic acid, concentration after 10 and 21 days of fermentation

9 Pitching Rate Average Specific Gravity Cells/mL/ Plato Degrees Plato Average ph Days Lactic acid produced during exponential growth phase (4,5) ph Days

10 Pitching Rate Rate (Cells/mL/ P) ph Plato % ABV % Viability 1 (2.5x10 5 ) 4.28± ± ± (5.0x10 5 ) 4.23± ± ± (1.0x10 6 ) 4.20± ± ± (2.0x10 6 ) 4.21± ± ± (3.0x10 6 ) 4.19± ± ±

11 Re-pitching Capacity Generation ph Plato % ABV % Viability ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.6 Potential for improved alcohol production High viability after 5 generations

12 Flocculation Helm assay (calcium sulfate) used to evaluate flocculation capacity Classified non-flocculent Settles well 24 hours postfermentation in benchtop and larger-scale fermentations Easy yeast maintenance 101 UCD 1020

13 Oxygen Requirements Wort bubbled to saturation with air (~7.8 ppm) or pure oxygen (~37 ppm) No significant difference observed; both fermenting vigorously 24 hours post-pitch Paola suggests that higher O2 is better, although ~7.8 ppm was sufficient for successful fermentation Too much O2 may increase VDK production 12 Specific Gravity 10 Degrees Plato Air Oxygen O 2 Air Days

14 Foam Stability Seconds Average Rudin Half Life Saccharomyces Lachancea Strain 101 compared to a common US ale strain of Saccharomyces in the same wort Rudin evaluation of foam stability in decarbonated beer

15 VDK Beers from foam and oxygen requirement studies analyzed by SPME-GC-MS Diacetyl (ppb) 2,3-pentanedione (ppb) Foam Study: Mid-fermentation Saccharomyces Mid-fermentation Lachancea Final Saccharomyces Final Lachancea Oxygen Study: O 2 Lachancea Air Lachancea

16 Iso-alpha-acid tolerance BU ph Plato % ABV % Viability ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.3 Study utilized isomerized kettle extract (Hopsteiner) Other studies utilizing various pellet additions to the kettle suggest likewise

17 UC Davis Strains Viticulture & Enology Wine Yeast and Bacteria Collection UCD Strain Species Source 602 thermotolerans Wine or must 1020 thermotolerans Unknown, Madrid, Spain 2820 fermentati Zinfandel must, CA foothills 2989 thermotolerans Alder tree, Spenceville, CA 2996 thermotolerans Oak tree, Big Sur, CA 2997 thermotolerans Oak tree, Big Sur, CA 3826 thermotolerans Oak tree, Cedar Roughs Wilderness, CA 3829 thermotolerans California bay laurel, Cedar Roughs Wilderness, CA 3830 thermotolerans Oak tree, Cedar Roughs Wilderness, CA 3834 thermotolerans California poppy flower, Cedar Roughs Wilderness, CA

18 UC Davis Strains UCD Strain Days ph Plato % ABV

19 Summary Most strains of Lachancea are capable of wort fermentation Strain of interest was robust and viable after several generations and demonstrated no major flaws Practicality of Lachancea will depend on purpose Flavor, enzyme activity, lactic acid production

20 Acknowledgements Charlie Bamforth Paola Domizio Linda Bisson Lucy Joseph Joe Williams Cary Doyle Double Mountain

21 References 1. Comitini, F., Gobbi, M., Domizio, P., Romani, C., Lencioni, L., Mannazzu, I., et al. Selected non-saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiol. (online) /j.fm , Cordero-Bueso, G., Esteve-Zarzoso, B., Cabellos, J. M., Gil-Díaz, M., and Arroyo, T. Biotechnological potential of non-saccharomyces yeasts isolated during spontaneous fermentations of Malvar (Vitis vinifera cv. L.). Eur. Food Res. Technol. (online) /s , Gobbi, M., Comitini, F., Domizio, P., Romani, C., Lencioni, L., Mannazzu, I., et al. Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: a strategy to enhance acidity and improve the overall quality of wine. Food Microbiol. (online) /j.fm , Kapsopoulou, K., Kapaklis, A., and Spyropoulos, H. Growth and fermentation characteristics of a strain of the wine yeast Kluyveromyces thermotolerans isolated in Greece. World J. Microbiol. Biotechnol. (online) /s , Kapsopoulou, K., Mourtzini, A., Anthoulas, M., and Nerantzis, E. Biological acidification during grape must fermentation using mixed cultures of Kluyveromyces thermotolerans and Saccharomyces cerevisiae. World J. Microbiol. Biotechnol. (online) /s , Kurtzman, C. Phylogenetic circumscription of Saccharomyces, Kluyveromyces and other members of the Saccharomycetaceae, and the proposal of the new genera Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma, and Zygotorulaspora. FEMS Yeast Res. (online) /S (03) , Kurtzman, C., and Fell, J. W. The Yeasts - A Taxonomic Study. Elsevier Mora, J., Barbas, J. I., and Mulet, A. Growth of yeast species during the fermentation of musts inoculated with Kluyveromyces thermotolerans and Saccharomyces cerevisiae. 41 (2) : Mora, J., Barbas, J. I., Ramis, B., and Mulet, A. Yeast microflora associated with some Majorcan musts and wines. 39 (4) : Naumova, E. S., Serpova, E. V., and Naumov, G. I. Molecular systematics of Lachancea yeasts. Biochem. Mosc. (online) /S , Nguyen, N. H., Suh, S.-O., and Blackwell, M. Five novel Candida species in insect-associated yeast clades isolated from Neuroptera and other insects. Mycologia (online) /mycologia , Rudin, A. D. Measurement of the foam stability of beers. 63 : Sláviková, E., Vadkertiová, R., and Vránová, D. Yeasts colonizing the leaf surfaces. J. Basic Microbiol. (online) /jobm , 2007.

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