When Beer

Goes Sour:

An NMR

InvestigationADAM DICAPRIO

PROCESS NMR ASSOCIATES

87A SAND PIT ROAD DANBURY, CT 06810

What is Sour Beer?

Wikipedia says:

“Sour beer is a beer style characterized by an intentionally acidic, tart,

sour taste.”

“Wild Brews: Beer beyond the Influence of Brewer’s Yeast”

Category 17 of the Beer Judge Certification Program

Encompasses: Berliner Weisse, Flanders Red Ale, Flanders Brown Ale,

Lambic, Fruit Lambic, Gueuze

What goes in to a sour beer? Grain

Malted Barley, Unmalted Wheat, Specialty Malts

Dextrins, Dextrins, Dextrins

Sikaru beer (3000 B.C.) – 62.5% Barley Malt + 37.5% Raw Wheat2

Modern Lambic – Brasserie Cantillon recipe – 65% Barley Malt + 35% Raw Wheat2

Hops

Aged & Oxidized

Aging Vessels – A sour beers home for up to a full century

Oak Barrels (French & American)

Oak Foudre

Stainless Steel Tank

http://www.newbelgium.com/Community/Blog/12-03-23/Who-wants-more-sour-beer.aspx

http://www.belgianbeermagazine.com/oud-beersel-brewery/

Who goes in to a sour beer? Dozens of organisms2,3

Bacteria

Enterobacteriaceae

Citrobacter spp., Enterobacter spp., Klebsiella spp., Hafnia spp.

Lactobacillaceae

Pediococcus spp., Lactobacillus spp.,

Acetobacter spp.

Klebsiella spp.

Yeasts

Kloeckera apiculata

Saccharomyces spp.

Brettanomyces spp.

Pichia spp.

Candida spp.

Hansenula spp.

Cryptococcus spp.

Why is Sour Beer Sour?

Straight Lambic, Flanders Ales, Gueuze, Berliner Weisse

Lactic, Acetic, Succinic Acid1

85% - 10% - 5%

Fruit Lambics1

Cherries, Grapes – Malic Acid

Raspberries – Citric Acid

Chemistry of Sour Beers

Application of Quantitative NMR to Biologically Acidified Mashes

Quantitative NMR and Descriptive Chemistry of American Wild Ales

and genuine Belgian Lambic

Berliner Weisse & Biological

Acidification

Reinheitsgebot of 1516

Beer can contain only malt, hops & water

Unmalted wheat and yeast added in the Provisional Law of 19964

Artificial alteration of pH is illegal5

Development of Biological Acidification/Sour Mashing

Utilization of native microbes for pH adjustment6

Perform Starch

Conversion

Cool to ~120°F

InnoculateHold

Temperature (120°F)

Temperature Dependence of the

Sour Mash Technique Goal:

Does the “magic” temperature of 120°F have a chemical significance?

Record and quantify sour metabolites & contaminant products as a function of sour mash

temperature

Determine wt% of metabolites using Maleic Acid internal standard & manual integration

Lactic Acid

Acetic Acid (contaminant)

Succinic Acid

Ethanol

γ-Amino Butyric Acid (contaminant)

Straight Run of 95°F Sour mash; Assignments from Rodrigues et al. 2010

Malt-040-H.esp

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

Chemical Shift (ppm)

0

0.0005

0.0010

0.0015

Nor

mal

ized

Inte

nsity

HOD

Pyruvic Acid

Acetic Acid

Succinic Acid

2,3-butanediol

Lactic Acid

B-Glucose

a-Glucose

Ethanol

Maleic Acid

Lactic Acid

a(1-4) Dextrins

Sugar Anomeric Region

Freeze Dried Run of 95°F sour Mash; Assignments from Rodrigues et al. 2010

Malt-025-H.esp

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

Chemical Shift (ppm)

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

Nor

mal

ized

Inte

nsity

Acetic Acid

2,3-Butanediol

Succinic Acid TMSP

Maleic Acid

Ethanol

Lactic Acid

B-Glucose

a-Glucose

Lactic Acid

a(1-4) Dextrins

HOD

Sugar Anomeric Region

Absolute Metabolite Proportions

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

90 95 100 105 110 115 120 125 130 135 140

Metabolite Weight % vs. Temperature

Lactic Acid Acetic Acid Succinic Acid Poly. (Lactic Acid) Poly. (Acetic Acid) Poly. (Succinic Acid)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

90 100 110 120 130 140

Pe

rce

nt

by

Ma

ss

Temperature (°F)

Weight % Ethanol

Relative Proportions

R² = 0.9676

0.0

50.0

100.0

150.0

200.0

250.0

300.0

95 105 115 125 135W

T %

LA

CTI

C /

WT

% S

UC

CIN

IC

TEMPERATURE

Lactic:Succinic

R² = 0.9941

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

90 100 110 120 130 140

WT

% L

AC

TIC

/ W

T %

AC

ETI

C

TEMPERATURE (°F)

Lactic:Acetic

Conclusions

120°F is sub-optimal for acid production

Around 120°F Lactic acid reaches a relative maximum

Lactic good, Acetic bad

Aim is pH adjustment, not flavor adjustment

Lambics of Belgium & Lambic-Styles

of the USA What’s the difference?

Lambic – From the Pajottenland / Senne River

Valley Region of Belgium

American likenesses styled as “American Wild Ale

(AWA)” or “American Coolship Ale (ACA)”

Different Microbial Community

Follow same general succession

ACA involves a more diverse community of Lactic

Acid Bacteria and Minority Yeasts3

The Chemistry of Sour Beers

Goals

Comparative analysis of organic acids using quantitative NMR

Manually integrated against a known mass of Maleic Acid

Lactic Acid, Acetic Acid, Succinic Acid, Citric Acid, Malic Acid

Analyze linear and branched dextrin ratios among multiple styles

Utilize multivariate analysis to discriminate multiple styles of sour beer

Drie Fonteinen Oude Gueuze Straight Run; Assignments from Rodrigues et al. 2010

Allagash-014-H.esp

6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

Chemical Shift (ppm)

0

0.0005

0.0010

Nor

mal

ized

Inte

nsity Acetic Acid

Succinic Acid

Lactic Acid

Maleic Acid EthanolEthanol

Lactic AcidCitric Acid & Malic Acid (if present)

HOD

Freeze dried Run of Drie Fonteinen Oude Gueuze; Assignments from Rodrigues et al. 2010, Rodrigues & Gil 2011, & Nord et al. 2004

Allagash-014-H_FD.esp

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

Chemical Shift (ppm)

0.001

0.002

0.003

0.004

0.005

0.006

0.007

Nor

mal

ized

Inte

nsity

Pyruvic Acid

2,3-butanediol

B-Glucose

Alaninea-Glucose

Ethanol

a(1-6) Maltodextrin

Lactic Acid

Residual Ethanol

Lactic Acid

Maleic Acid

a(1-4) Maltodextrin

Arginine

Citric Acid & Malic Acid (if present)

Asparagine

Proline

Proline

Valine, Leucine, Isoleucine

Aromatic Amino Acids

Residual Phenols

Benzoic Acids

Sugar Anomeric Region

Aromatic Amino Acids

Residual Phenols

Benzoic Acids

Allagash-006-H_FD.esp

3.0 2.5 2.0 1.5

Chemical Shift (ppm)

0.005

0.010

0.015

0.020

0.025

0.030

Norm

alize

d In

tensi

ty

Acetic Acid

Succinic Acid

Lactic Acid

Malic Acid

Allagash-005-H_FD.esp

3.0 2.5 2.0 1.5

Chemical Shift (ppm)

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

Norm

alize

d In

tensi

ty

Acetic Acid

Succinic Acid

Lactic Acid

Citric Acid

Allagash-004-H_FD.esp

3.0 2.5 2.0 1.5

Chemical Shift (ppm)

0.005

0.010

0.015

0.020

0.025

0.030

Nor

mal

ized

Inte

nsity

Succinic Acid

Acetic Acid

Lactic Acid

No Malic Acid

Little to No Citric Acid

Framboise-Style Kriek-Style

Gueuze-Style

Acid Differences

77%

20%

3%

American Geuze-Style

Lactic Acetic Succinic

85%

11%4%

Belgian Geuze

Lactic Acetic Succinic

43%

18%

4%

35%

American Kriek-Style

Lactic Acetic Succinic Malic

35%

27%

3%

35%

American Framboise-

Style

Lactic Acetic Succinic Citric

Beer LA (mg/L) AA (mg/L) SA (mg/L) CA (mg/L) MA (mg/L)

American

Geuze-

Style

5386.0 1410.0 238.5 0 0

American

Framboise

-Style

3896.7 2972.1 394.6 3890.7 0

American

Kriek-Style4682.8 1965.7 423.4 0 3777.8

Boone

Mariage

Parfait

2009

4506.5 488.1 217.0 0 0

Oude

Geuze

Vieille

3497.8 454.1 175.2 0 0

Geuze

Fond

Tradition

6807.8 698.6 218.8 0 0

Drie

Fonteinen

A

5137.6 865.9 234.6 0 0

Drie

Fonteinen

B

5389.9 917.7 228.9 0 0

Dextrin Ratios

0

1

2

3

4

5

6

7

Α(1

-4)

MA

LTO

DEX

TRIN

/ Α

(1-6

)

MA

LTO

DEX

TRIN

STYLE

Ratio of α(1-4) Maltodextrin to α(1-6)

Maltodextrin by Style

“Degree of Fermentation”

Dependent on:

Strain

Style

Ingredients

Multivariate Analysis – Principal Component Analysis

Segregate based

largely on sugar

detail

Independent of

ingredients

Ameri

can

Cools

hip

Beers

Belgi

an

Geuz

es

Saiso

ns Belgi

an

Foudr

e

Beer

IPA +

Lager

s

100%

Brett

Beers

Ameri

can

Foudr

e

Beer

Hybri

d

Beer

Saisons

Lambic

ACA

IPA + Lager

Bel. Foudre

100% Brett.

Am. Foudre

Conclusions

ACAs & Belgian Lambics have differing acid profiles

Ratio of linear and branched maltodextrins can be used to

differentiate styles

Multivariate analysis can differentiate between ACAs, Belgian

Lambics and 6 other styles

References1. Sparrow, J. (2005). Wild brews: Beer Beyond the Influence of Brewer’s Yeast, (Strong, G.,

Ed.), Brewers Publications, Boulder, CO.

2. Guinard, J. X. (1990). Lambic, (Thomas, V., Ed.), Brewers Publications, Boulder, CO.

3. Bokulich, N. A., Bamforth, C. W., Mills, D. A. (2012). Brewhouse-Resident Microbiota Are

Responsible for Multi-Stage Fermentation of American Coolship Ale, PLoS One. 7:4, 1-11.

4. Dornbusch, H., Heyse, K. U. (2012) Reinheitsgebot, in the Oxford Companion to Beer, (Oliver,

G., Ed.), pp. 691-693, Oxford University Press, Oxford.

5. Narziss, L. (1984) The German Beer Law, J. Inst. Brew. 90, 351-358.

6. Sour Mashing: Techniques, Brew Your Own Magazine. http://byo.com/all-grain-

brewing/item/1691-sour-mashing-techniques (last accessed July 2013).

7. Rodrigues, J. E., Erny, G. L., Barros, A. S., et al. (2010). Quantification of organic acids in beer by nuclear magnetic resonance (NMR)-based methods, Anal. Chim. Acta. 674(2):166-

175.

8. Rodrigues, J.E., Gil, A. M. (2012). NMR methods for beer characterization and quality

control, Magn. Reson. Chem. 49: S37-S45.

9. Nord, L. I., Vaag, P., Duus, J. O. (2004). Quantification of Organic and Amino Acids in Beer

by 1H NMR Spectroscopy, Anal. Chem. 76(16):4790-4798.