Lachancea thermotolerans in pure-culture fermentations
Jen House
UC Davis 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 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
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
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 SO2 • Negative for killer factor 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 Sugar metabolism
101 602 1020 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 Lactic acid and glycerol production
101 602 1020 Sac 101 602 1020 Sac
Glycerol ■ and lactic acid □, concentration after 10 and 21 days of fermentation Pitching Rate
Average Specific Gravity Cells/mL/°Plato 14
12
10
8
6 Degrees Plato Degrees 4 Average pH 2 5.8
0 5.6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 5.4 Days 5.2
5
Lactic acid produced during pH 4.8 exponential growth phase 4.6 (4,5) 4.4 4.2
4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Days Pitching Rate
Rate (Cells/mL/°P) pH °Plato % ABV % Viability
1 (2.5x105) 4.28±0.01 1.60±0.03 5.65±0.03 93.4
2 (5.0x105) 4.23±0.03 1.57±0.00 5.66±0.02 91.5
3 (1.0x106) 4.20±0.00 1.52±0.00 5.67±0.01 92.0
4 (2.0x106) 4.21±0.01 1.46±0.01 5.72±0.02 89.5
5 (3.0x106) 4.19±0.02 1.50±0.01 5.69±0.03 88.8 Re-pitching Capacity
Generation pH °Plato % ABV % Viability
1 4.21±0.01 1.46±0.01 5.72±0.02 89.5
2 4.17±0.10 1.70±0.09 5.82±0.08 93.0±1.4
3 4.24±0.02 1.78±0.00 5.99±0.04 94.7±0.5
4 4.30±0.02 1.66±0.01 5.90±0.13 90.7±0.3
5 4.23±0.00 1.66±0.02 6.08±0.02 89.1±0.6
• Potential for improved alcohol production • High viability after 5 generations Flocculation
• Helm assay (calcium sulfate) used to evaluate flocculation capacity – Classified “non-flocculent” • Settles well 24 hours post- fermentation in benchtop and larger -scale fermentations • Easy yeast maintenance UCD 101 1020 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 Specific Gravity 12
10
8
6 Air
Oxygen Degrees Plato Degrees 4
2 O2 Air 0 0 5 10 15 Days Foam Stability
Average Rudin Half Life 100 • Strain 101 compared to 90 a common US ale strain 80
70 of Saccharomyces in the
60
Saccharomyces same wort 50 Lachancea Seconds 40 • Rudin evaluation of 30
20 foam stability in 10 decarbonated beer 0 • Beers from foam and oxygen VDK requirement studies analyzed by SPME-GC-MS
2,3-pentanedione
Diacetyl (ppb) (ppb)
Foam Study: Mid-fermentation
Saccharomyces 46.7 13.2
Mid-fermentation Lachancea 30.8 33.8
Final Saccharomyces 47.3 15.7
Final Lachancea 29.29 4.5
Oxygen Study: O2 Lachancea 31.3 7.5
Air Lachancea 22.8 6.0 Iso-alpha-acid tolerance
BU pH °Plato % ABV % Viability
30 4.18±0.03 1.72±0.01 5.88±0.10 94.9±0.8
40 4.22±0.03 1.78±0.02 5.92±0.03 96.2±0.2
50 4.22±0.01 1.73±0.01 5.93±0.00 95.4±0.5
60 4.22±0.02 1.78±0.01 5.97±0.02 94.5±0.3
• Study utilized isomerized kettle extract (Hopsteiner) • Other studies utilizing various pellet additions to the kettle suggest likewise 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 UC Davis Strains
UCD Strain Days pH °Plato % ABV
602 12 4.24 3.71 4.25
1020 12 4.20 3.76 4.15
2820 12 4.03 3.69 4.15
2989 6 4.49 11.22 0.24
2996 12 4.10 3.85 4.11
2997 12 3.84 3.98 4.17
3826 8 3.52 4.42 2.66
3829 8 3.69 5.82 1.94
3830 8 3.57 4.16 2.64
3834 8 3.58 4.10 2.82 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 Acknowledgements
Charlie Bamforth Paola Domizio Linda Bisson Lucy Joseph Joe Williams Cary Doyle Double Mountain 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). 10.1016/j.fm.2010.12.001, 2011.
2. 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). 10.1007/s00217-012-1874-9, 2012.
3. 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). 10.1016/j.fm.2012.10.004, 2013.
4. 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). 10.1007/s11274-005-8220-3, 2005.
5. 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). 10.1007/s11274-006-9283-5, 2007.
6. 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). 10.1016/S1567-1356(03)00175-2, 2003.
7. Kurtzman, C., and Fell, J. W. The Yeasts - A Taxonomic Study. Elsevier. 1998.
8. 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) :156–159 1990.
9. Mora, J., Barbas, J. I., Ramis, B., and Mulet, A. Yeast microflora associated with some Majorcan musts and wines. 39 (4) :344–346 1988.
10. Naumova, E. S., Serpova, E. V., and Naumov, G. I. Molecular systematics of Lachancea yeasts. Biochem. Mosc. (online). 10.1134/S0006297907120097, 2007.
11. 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). 10.3852/mycologia.99.6.842, 2007.
12. Rudin, A. D. Measurement of the foam stability of beers. 63 :506–509 1957.
13. Sláviková, E., Vadkertiová, R., and Vránová, D. Yeasts colonizing the leaf surfaces. J. Basic Microbiol. (online). 10.1002/jobm.200710310, 2007.