lachancea thermotolerans in pure-culture...
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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
Maltotriose ■, maltose □ and ethanol ■ concentration after 21 days of fermentation
101 602 1020 Sac
• All three strains fermented 93-94% of maltose present in wort; unable to ferment maltotriose
Lactic acid and glycerol production
Glycerol ■ and lactic acid □, concentration after 10 and 21 days of fermentation
101 602 1020 Sac 101 602 1020 Sac
Pitching Rate
0
2
4
6
8
10
12
14
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Deg
rees
Pla
to
Days
Average Specific Gravity
4
4.2
4.4
4.6
4.8
5
5.2
5.4
5.6
5.8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
pH
Days
Average pH
Cells/mL/°Plato
Lactic acid produced during exponential growth phase (4,5)
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
101 UCD
1020
Oxygen Requirements
0
2
4
6
8
10
12
0 5 10 15
Deg
rees
Pla
to
Days
Specific Gravity
Air
Oxygen
• 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
O2 Air
Foam Stability
• Strain 101 compared to
a common US ale strain
of Saccharomyces in the
same wort
• Rudin evaluation of
foam stability in
decarbonated beer 0
10
20
30
40
50
60
70
80
90
100
Seco
nds
Average Rudin Half Life
Saccharomyces
Lachancea
VDK • Beers from foam and oxygen
requirement studies analyzed by SPME-GC-MS
Diacetyl (ppb) 2,3-pentanedione (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.
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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.
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