Post on 21-Aug-2020
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.