Presentation

Combining Lactic Acid Bacteria and Active Dry Yeasts for Sour Beer Production

Kevin Lane Technical Sales Support Manager – The Americas BA Collab Hour Webinar

Sour Beers Characterization Souring Processes 1. Alcohol fermentation performance 1 4 2. Sensory analysis 3. Aromatic expression

2 Lactic Acid Bacteria metabolism

Conclusions Lactic Fermentation Parameters 5 1. Pitching rate 3 2. Wort density 3. Temperature 4. Aeration Condition 1 Beer Production Process

Milling Mashing Filtration Boiling Cooling Fermentation Maturation Bottling

Lambic/Gueuze/Gose beers Mixed primary fermentation in Bottling Blend of Pale oak barrel ale and colored Berliner Weissebier malts < 6 IBU beers Yeast/Lactobacill Malt and Wheat us co-inoculation

Milling Mashing Filtration Boiling Cooling Fermentation Maturation Bottling

Malt and Mixed secondary Wheat Fermentation/ Maturation in oak foeders

Flanders Red ale beers Bottling

Milling Mashing Filtration Boiling Cooling Fermentation Maturation Bottling

Non-hopped Lactic wort boiling Fermentation

Kettlesour

Lambic/Gueuze/Gose beers Mixed primary fermentation in Bottling Blend of Pale oak barrel ale and colored Berliner Weissebier malts < 6 IBU beers Yeast/Lactobacill Malt and Wheat us co-inoculation

Milling Mashing Filtration Boiling Cooling Fermentation Maturation Bottling

Malt and Mixed secondary Wheat Fermentation/ Maturation in Non-hopped Lactic oak foeders wort boiling Fermentation

HOMOfermentative bacteria HETEROfermentative bacteria

Glucose Glucose

CO2

O 2 + O 2 2 Lactate Lactat Ethano Acetate e l Lactiplantibacillus plantarum Lp652 Levilactobacillus brevis Lb1

0 1 g/hL 8 P

1 0 g / hL Pitching 120 P Rate °P 100 g/hL 160 P

450 C A n a e r o b i c 400 C °C 300 C O 2 A e r o b i c Lactiplantibacillus plantarum Lp652 370 C Levilactobacillus brevis Lb1

Acidification kinetics Analysis Organic acids

0 1 g/hL 8 P

1 0 g / hL Pitching 120 P Rate °P 100 g/hL 160 P

450 C A n a e r o b i c 400 C °C 300 C O 2 A e r o b i c Lactiplantibacillus plantarum Lp652 370 C Levilactobacillus brevis Lb1

Acidification kinetics Analysis Organic acids

5,2 5,2 5 5 4,8 4,8 4,6 4,6 4,4 4,4 4,2 4,2 1 g/hL 1 g/hL 10 g/hL

4 4 10 g/hL pH 100 g/hL pH 3,8 3,8 100 g/hL 3,6 3,6 3,4 3,4 3,2 3,2 3 3 0 8 16 24 32 40 48 0 8 16 24 32 40 48 Time (h) Time (h) Final pH are lower for Lp652 than for Lb1 at equivalent pitching rate © Fermentis communication – All rights reserved 1 g/ hL 1 0 g/ hL Pitching 3 Organic acids production Rate 100 g/ hL 12°P (non-hopped) – 30°C – Anaerobic Lp652 Lb1

8 8 24h 24h 7 7 48h 48h 6 6 5 5 4 4 3 3 2 2

Organic Acids (g/L) Acids Organic 1 Organic Acids (g/L) Acids Organic 1 0 0 1 g/hL 10 g/hL 100 g/hL 1 g/hL 10 g/hL 100 g/hL 1 g/hL 10 g/hL 100 g/hL 1 g/hL 10 g/hL 100 g/hL Lactic acid Acetic acid Lactic acid Acetic acid

0 1 g/hL 8 P

1 0 g / hL Pitching 120 P Rate °P 100 g/hL 160 P

450 C A n a e r o b i c 400 C °C 300 C O 2 A e r o b i c Lactiplantibacillus plantarum Lp652 370 C Levilactobacillus brevis Lb1

Acidification kinetics Analysis Organic acids

10 g/hL – 30°C – Anaerobic Lp652 Lb1

5,2 5,2 5 5 4,8 4,8 4,6 4,6 4,4 4,4 4,2 8°P 4,2 8°P

4 12°P 4 pH

pH 12°P 16°P 3,8 3,8 16°P 3,6 3,6 3,4 3,4 3,2 3,2 3 3 0 4 8 12 16 20 24 28 32 36 40 44 48 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (h) Time (h)

10 g/hL – 30°C – Anaerobic Lp652 Lb1

5 5 24h 24h 4 4 48h 48h 3 3 2 2

1 1

Organic acids (g/L) acids Organic Organic acids (g/L) acids Organic 0 0 Lactic Acetic Lactic Acetic Lactic Acetic Lactic Acetic Lactic Acetic Lactic Acetic 8°P 12°P 16°P 8°P 12°P 16°P

0 1 g/hL 8 P

1 0 g / hL Pitching 120 P Rate °P 100 g/hL 160 P

450 C A n a e r o b i c 400 C °C 300 C O 2 A e r o b i c Lactiplantibacillus plantarum Lp652 370 C Levilactobacillus brevis Lb1

Acidification kinetics Analysis Organic acids

5,2 5,2 5 5 4,8 4,8 4,6 4,6 4,4 4,4 45°C 4,2 4,2 45°C

40°C pH

4 4 40°C pH 3,8 37°C 3,8 37°C 3,6 30°C 3,6 30°C 3,4 3,4 3,2 3,2 3 3 0 4 8 12 16 20 24 28 32 36 40 44 48 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (h) Time (h) Temperatures higher than 37°C impact lactic fermentation performance for both strains!

5 5

24h 24h 4 4 48h 48h 3 3

2 2 Organic acids (g/L) acids Organic

Organic acids (g/L) acids Organic 1 1

0 0 Lactic Acetic Lactic Acetic Lactic Acetic Lactic Acetic Lactic Acetic Lactic Acetic Lactic Acetic Lactic Acetic 30°C 37°C 40°C 45°C 30°C 37°C 40°C 45°C

Temperatures higher than 37°C impact lactic acid production of both strains!

0 1 g/hL 8 P

1 0 g / hL Pitching 120 P Rate °P 100 g/hL 160 P

450 C A n a e r o b i c 400 C °C 300 C O 2 A e r o b i c Lactiplantibacillus plantarum Lp652 370 C Levilactobacillus brevis Lb1

Acidification kinetics Analysis Organic acids

O2 3 Acidification kinetics Aer obi c

10 g/hL – 12°P (non-hopped) – 30°C Lp652 Lb1

5,2 5,2 Wo O2 Wo O2 5 5 O2 O2 4,8 4,8 4,6 4,6 4,4 4,4

4,2 4,2 pH

4 pH 4 3,8 3,8 3,6 3,6 3,4 3,4 3,2 3,2 3 3 0 4 8 12 16 20 24 28 32 36 40 44 48 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (h) Time (h)

Lactic fermentation is slightly improved in anaerobic conditions

O2 3 Organic acids production Aer obi c

10 g/hL – 12°P (non-hopped) – 30°C

4 3,5 3 2,5 2

1,5 Lp 652 wo O2

Organic acid (g/L) acid Organic 1 Lp 652 O2 0,5 Lb1 wo O2 0 Lactic acid Lb1 O2 Acetic acid Ratio L/A Acetic acid quantity produced by Lb1 is promoted by aeration

1) Common features

• Acidification kinetics are relatively equivalent • No significative impact of wort density on lactic fermentation • Optimal temperature (30°C – 37°C)

2) Significant Differences • Lp652 produces more lactic acid • Lb1 produces acetic acid • Oxygen presence improves strongly acetic acid production by Lb1

Pitching Rate °P 20 IBU 1 0 g/ hL 120 P Pitching 100% Pilsen malt °C Rate

°C O2 230 C 5 0 g/ hL 300 C Anaer obi c

pH 3 , 5

Fermentation Performance

time (h) Sensory Analysis

Aromatic components © Fermentis communication – All rights reserved 4 Sour beers characterization 1. Typical fermentation performance K-97 6 5 4 Ale yeasts i.e. 3 POF -: K-97, S-33, S-04, BE-256 2 Control

Ethanol [%v/v] Ethanol Lp652 POF +: BE-134, T-58, WB-06 1 Lb1 0 0 2 4 6 8 10 Time [day] 90 Control US-05 85 6 Lp652 80 5 Lb1 4 75 3 Ale yeasts i.e. 70

2 Control ADF [%] ADF

Ethanol [%v/v] Ethanol 65 1 Lp652 POF -: US-05 Lb1 0 60 0 2 4 6 8 10

Time [day] 55

T-58

S-33 S-23 S-04

S-23 K-97

S-189 US-05

7 WB-06

BE-134 BE-256 6 W-34/70 5 4 3 Control 2 Lager yeasts i.e. S-23, W-34/70, S-189 Lp652 Ethanol [%v/v] Ethanol 1 Lb1 0 0 2 4 6 8 10 12 © Fermentis communication – All rights reserved Time [day] 4 Sour beers characterization Typical sensory profiles S-33 BEBE--134134 Lp 652 Lp 652 Fruity_O Lb 1 Fruity_O Lb 1 Fruity_O Lb 1 10 1010 8 88 Alcohols_F Phenolic_O Alcohols_F Phenolic_OPhenolic_O 6 66 4 44 2 22 Phenolic_F 0 Alcohols_O Phenolic_F 00 Alcohols_OAlcohols_O

Fruity_F Sweetness Fruity_F SweetnessSweetness

Acidity AcidityAcidity POF- POF+ • Low phenolic perception detected for POF+ yeasts when lactic fermentation is performed with Lp652 • Lb1 respects phenolic character of POF+ yeasts POF- • Acidity perception is higher with Lp652 than Lb1, increasing drinkability • Lb1 develops rounder profiles with more balanced organicsensory acid profiles© Fermentis profile communication – All withrights reserved Lp652 4 Sour beers characterization 3. Phenolic Aroma Expression

4-Vinyl Guaiacol Control 700 Lb 1 Lp 652 No 4-EG 600 POF+ 4-VG 4-EG detected Control + - 500 Lp652 - - 400 Lb1 + -

300 Concentration [µg/L] Concentration 200 ? 100

0 D BE-134 S-33 T-58 Fer ul i c aci d 4VG 4EG? Brettanomyces bruxellensis Lp 652 POF+ Saccharomyces cerevisiae D Fer ul i c aci d 4VG 4EG Brettanomyces bruxellensis var. diastaticus p- coumar i c aci d 4VP 4EP Lp 652 • Lp652 seems to consume ferulic acid during lactic fermentation • POF+ character is not detected when lactic fermentation is performed© FermentisPOF+ communication Saccharomycesby –Lp652All rights reserved cerevisiae var. diastaticus p- coumar i c aci d 4VP 4EP 5 Conclusions

Sour beer diversity can be created by the LAB strain choice for the lactic fermentation (kettle-souring); more specifically:

1) Quality of the acidity produced during the lactic fermentation Lactic (Dairy) and/or Acetic (Vinegar)

• Lb1 produces acetic acid and wort aeration can modulate the amount

• Lp652 produces two times more lactic acid than Lb1

2) Flavor impact of lactic fermentation

• Lb1, due to its aromatic neutrality, respects the yeast flavor/aromatic signature developed during main fermentation

• Lp652 develops citrusy, tropical fruit and peach notes during lactic fermentation

• Lp652 reveals the potential of the fruity expression of the fermentation, more specifically with POF+ yeasts strains which hinders phenolic expression