Lachancea Thermotolerans As a Novel, Single-Strain Brewing Yeast Claire Svendsen, M.S

Investigation of Lachancea thermotolerans as a novel, single-strain brewing yeast Claire Svendsen, M.S. NC State University Raleigh, NC Investigating Wild Yeast for Brewing Beer • Collaborative project creating local beer • Dr. Rob Dunn • NCSU Biology • Isolate wild yeasts • Dr. John Sheppard • NCSU Food Science • NCSU Brewery • Ferment wild yeasts

Lachancea thermotolerans

• Isolated from a bumble bee • Identified by PCR and phenotypic analysis • Confirmed GRAS status Experimental Goals

1) Differentiate L. thermotolerans NCSU from type strain: • L. thermotolerans NRRL Y-8284 (ATCC® #56472) 2) Demonstrate L. thermotolerans NCSU can be used as a pure culture brewing strain

Lachancea thermotolerans

• Previously Kluyveromyces thermotolerans

• Mixed evaluations as as a wine yeast: • Useful for bioacidification and aroma enhancement • Poor fermenter, needs to be co-fermented with Saccharomyces for wine production

Lachance & Kurtzman, 2011; Moreno-Arribas, 2009; Jolly, 2013 Fermentation Conditions

• Laboratory scale NCSU • Fermented in triplicate • Pilsner wort at 18 °C • OG: 13 °P

• Sampled daily ATCC

Wort Attenuation

1.060

1.050

1.040 Gravity

1.030 NCSU Specific NRRL Y-8284

1.020

1.010

1.000 0 1 2 3 4 5 6 7 Fermentation Time (Day) Maltose Utilization and Ethanol Production

NCSU NRRL Y-8284 80.0 80.0

70.0 70.0

60.0 60.0

50.0 50.0

40.0 40.0 Maltose Maltose

30.0 Ethanol 30.0 Ethanol

Concentration(g/L) Concentration(g/L) 20.0 20.0

10.0 10.0

0.0 0.0 -1 1 3 5 7 -1 1 3 5 7 Fermentation Time (Day) Fermentation Time (Day) Monosaccharide Utilization and Glycerol Production

NCSU NRRL Y-8284

18 18

16 16

14 14

12 12

10 10 Glucose Glucose 8 Fructose 8 Fructose

6 Glycerol 6 Glycerol

Concentration(g/L) Concentration(g/L) 4 4

2 2

0 0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Fermentation Time (Day) Fermentation Time (Day) Wort Acidification

6.00

5.50

5.00

4.50 pH NCSU NRRL Y-8284 4.00

3.50

3.00 0 1 2 3 4 5 6 7 Fermentation Time (Day) L. thermotolerans Comparison

• Strain NCSU compared to NRRL Y-8284 • Increased wort attenuation • Increased wort acidification • Increased ethanol production • Increased glycerol production “Lambic Style” Fermentation

• Lambic wort (barley and wheat, lightly hopped)

• Pure culture of L. thermotolerans NCSU

• Pilot-plant scale (300 L) at 18 °C

• Pitched 4 L of active yeast

Wort Attenuation in Lambic Fermentation

1.07

1.06

1.05

1.04

1.03 Specific Gravity Specific

1.02 1.011 1.005 1.01

1 0 5 10 15 20 25 30 35 Fermentation Time (Day) Wort Acidification in Lambic Fermentation 5.50

5.00

4.50

4.00

3.50

3.00 0 5 10 15 20 25 30 35 Fermentation Time (Day) “Lambic” Sugar Utilization

Fermentation Time Maltotriose Maltose Glucose Fructose (Day) (g/L) (g/L) (g/L) (g/L)

0 29.0 67.0 18.7 6.8

25 4.8 3.5 0.8 n.d. Metabolite Production

Fermentation Time Lactic Acid Glycerol Ethanol ABV% (Day) (g/L) (g/L) (g/L)

0 n.d. n.d. n.d. n.d.

25 7.3 2.4 54 6.8% Aromatic Analysis

EtOH 6 5

13 7

10 17 3 4 8 18 16 19 2 9 11 12 14 15 20 Detected Esters

Peak Peak Compound Flavor Descriptor # Ratio 1 Ethyl acetate 3.56 fruity, sweet 2 Isobutyl acetate 0.08 fruity, banana 3 Ethyl butyrate 0.41 fruity, papaya 5 Isoamyl acetate 6.57 fruity, banana 7 Ethyl hexanoate 4.28 fruity, sour apple 9 Acetic acid, hexyl ester 0.08 fruity, green apple 10* 2-Heptanol 1.00 n.a. 11 Ethyl lactate 0.04 fruity, raspberry 13 Ethyl octanoate 6.67 fruity, apple 17 Ethyl decanoate 1.04 fruity, apple 18 Phenylethyl acetate 0.47 roses, honey 19 Ethyl laurate 0.17 caprylic, estery Other Detected Aromatics

Peak Peak Compound Flavor Descriptor # Ratio 4 Isobutanol 0.58 alcoholic, solvent-like 6 Isoamyl alcohol 9.77 alcohol, sweet 8 Styrene 0.35 sweet, floral 10* 2-Heptanol 1.00 n.a. 12 1-Hexanol 0.04 coconut, unpleasant 14 2-Ethylhexanol 0.07 citrus, floral 15 2,3-Butanediol 0.06 warming, butterscotch 16 Linalool 0.15 fruity, flowerly 20 Caproic acid 0.38 sour, fatty “Lambic” Fermentation

• Successful fermentation in brewhouse environment

• Reduces pH through lactic acid production

• Final ABV of 6.8%

Conclusions for L. thermotolerans

• L. thermotolerans NCSU is: • Metabolically different from the type strain, L. thermotolerans NRRL Y-8284 • A yeast brewers may use for pure culture, sour beer fermentations • Patent pending for fermenting various beverages (beer, cider, etc.)

Questions? Contact Information

• Dr. John Sheppard: [email protected]

• Claire Svendsen: [email protected]