Analysis of 100-Year-Old Beer Originated from the Czech Republic

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Analysis of 100-years-old beer originated from the Czech Republic Jana Olšovská, Dagmar Matoulková, Martin Dusek, Jurgen Felsberg, Markéta Jelínková, Pavel #ejka, and Karel Št#rba J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05706 • Publication Date (Web): 05 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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Journal of Agricultural and Food Chemistry

Analysis of 100-years-old beer originated from the Czech Republic Jana Olšovská1*, Dagmar Matoulková1, Martin Dušek1, Jürgen Felsberg2, Markéta Jelínková2, Pavel Čejka1, Karel Štěrba1

1

Research Institute for Brewing and Malting, PLC, Lípová 15, CZ – 120 44 Prague, Czech

Republic 2

Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., CZ – 142 20

Prague, Vídeňská 1038, Czech Republic

*Corresponding author, e-mail [email protected], telephone +420 224 900 150

1 ACS Paragon Plus Environment


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ABSTRACT

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Three bottles of different beers were found in 2015 during a reconstruction of the brewery of

3

the Raven Trading s.r.o. company in Záhlinice, Czech Republic. Thanks to good storage

4

conditions it was possible to analyze their original characteristics. All three bottles contained

5

most probably lager type beer. One beer had sulfuric and fecal off-flavors; it was bright with

6

the original extract of 10.3 ° Plato. The second beer, with an original extract of 7.6 ° Plato,

7

was dark and very acidic, resembling Lambic. DNA analysis proved the presence of Dekkera

8

bruxellensis which corresponded to its chemical profile (total acidity, FAN, ethyl acetate,

9

total esters). The third beer contained traces of carbon dioxide bubbles, was light brown and

10

slightly bitter with original extract 10.4 ° Plato. Since it obviously underwent a natural aging

11

process, sweetness, honey and fruity off-flavors were detected and transformation products of

12

iso-alpha acids were found.

13 14 15 16

Key words

17

beer aging, carbonyl compounds, transformation of isohumulones, Dekkera bruxellensis,

18

Saccharomyces

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Introduction

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In the spring of 2015, three bottles of beer were found during a reconstruction of the

22

brewery of the Raven Trading s.r.o. company in Záhlinice, Czech Republic. Based on the

23

history of the Záhlinice brewery, which had been working from 1896 to 1925, the age of the

24

beer has been estimated at about 100 years. Therefore, we assume that the beer originated

25

from the period around the World War I and it was, perhaps accidentally, immured in the

26

lager cellar of the brewery. Based on preserved historical recipes, which document, that only

27

bottom-fermented beer (lager) type was brewed in Zahlinice brewery, we expected that all

28

three bottles contained most probably lager type of beer. Thanks to good storage conditions

29

the beer was preserved in such a way that it was possible to describe its original

30

characteristics using chemical analyses. Naturally, we don’t know the initial chemical

31

composition of this century-old beer but, on the assumption that 100 years ago our ancestors

32

already used similar technology and raw materials, we can approximate the original beer

33

composition and compare it to the current lager. We thus have had a unique opportunity for

34

studying and potentially characterizing chemical processes taking place in beer spontaneously

35

aging over a very long time period.

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The character and authenticity of Czech beer are based on specific technology and

37

usage of unique raw materials. The excellent two-rowed spring barley and the malt produced

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from it, along with the high-quality Saaz hops were the basic raw materials that, together with

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the decoction mashing, bottom fermentation and long cold maturation produced a unique type

40

of lager beer, called Pilsner (Pils) according to the place of its origin in 1842.1

41

Thanks to the unique discovery of the such old beer, we had the possibility to explore

42

a traditional product of our ancestors and verify and deepen the current knowledge about the

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chemical processes during beer aging. Recently, two articles regarding similar topics were

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published. Londesborough et al. described chemical parameters of beers from a 1840s 3 ACS Paragon Plus Environment


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shipwreck; two bottles found on the bottom of the ocean were 170-year-old. Based on

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analysis of hop components and their degradation products they found that each bottle

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contained a different type of beer.2 Walther et al. analyzed the composition of original lager

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beer samples from the 1880s, 1890s and 1900s with emphasis on the carbohydrate content

49

and composition. Using their results, they proved a gradual improvement of bottled beer

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handling from 1880s to the 1900s, demonstrated on a stable profile of carbohydrate

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oligomers. Only dormant cells of S. carlsbergensis yeast and Sporobolomyces roseus (a beer

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spoilage yeast) were detected in some bottles using rDNA sequencing.3 The DNA from our

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100-year-old beer was also successfully isolated and the presence of DNA of yeasts such as

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Dekkera bruxellensis and Saccharomyces pastorianus, S. cerevisiae x S. eubayanus x S.

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uvarum and Debaryomyces was proved.

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The aim of our study was to provide a sensorial and detailed chemical description of

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the century-old beers and, based on these analyses, explain the process of long-term lager beer

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aging in more detail. For this purpose, important parameters of century-old beers were

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compared with the chemical profile of 11- and 16-year-old as well as fresh lager beers. The

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maximum possible number of analyses was performed with respect to the limited volume of

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the samples.

62 63

Materials and Methods

64 65

Chemicals

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The following chemicals commercially obtained were used: methanol, acetonitrile, ethanol

67

(Merck, Darmstadt, Germany), formic acid, isooctane, 2,4,6-trinitrobenzenesulfonic acid

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(Sigma Aldrich, Steinheim, Germany), potassium hydroxide, sulfuric acid, hydrochloric acid,

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potassium dihydrogen phosphate, natrium tetraboricum, natrium sulphate, fosforic acid, 4 ACS Paragon Plus Environment

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aceton (Lach-Ner, Neratovice, Czech Republic). Gases for GC (nitrogen for ECD, helium 5.0,

71

compressed air) and AAS (nitrous oxide, acetylene) were obtained from Messer (Prague,

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Czech Republic). Deionized water used for chromatography was purified by means of a Milli-

73

Q® Integral system (Millipore, Billerica, MA, USA).

74

Certified standards of metals Certipur® (Ca, Mg, Na, K, Cu, Fe, Zn, Mn, Zn, Pb, Ni, Al, Cr,

75

Si, and Sn), and standards of carbohydrates with purity > 99% (glucose, fructose, maltose)

76

were obtained from Merck (Darmstadt, Germany). The standards of carbonyl compounds

77

with purity > 99% (2-furfural, 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, and

78

phenylacetaldehyde), internal standard 3-fluorobenzaldehyde and the derivatization reagent

79

O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride were purchased from Sigma-

80

Aldrich (Steinheim, Germany). Standards of fatty acids with purity > 99% (isobutanoic,

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butanoic, isopentanoic, pentanoic (valeric), hexanoic (caproic), heptanoic, octanoic (caprylic),

82

nonanoic (pelargonic), decanoic (capric), undecanoic and dodecanoic (lauric), tridecanoic,

83

tetradecanoic

84

octadecanoic (stearic), octadecadienoic (linoleic), eicosatrienoic (linolenic), octadecenoic

85

(oleic) acids were obtained from Sigma-Aldrich (Steinheim, Germany). Standard of DMS was

86

purchased from Merck (Darmstadt, Germany); standards of volatile compounds (diacetyl,

87

pentandion, alcohols and esters) were purchased from Sigma-Aldrich (Steinheim, Germany).

88

The following kits were used: Nucleospin Tissue Kit (Macherey Nagel, Düren, Germany),

89

DNA Clean and Concentrator TM-5 kit (Zymo Research, Irvine, California, USA), PPP

90

Master Mix (Top-Bio, Praha, Czech Republic), MinElute™ PCR Purification Kit (Qiagen,

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Hilden, Germany), BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems,

92

Foster City, California, USA).

(myristic),

pentadecanoic,

hexadecanoic

93 94

Beer samples


(palmitic),

heptadecanoic,


95

Century-old beers were in dark glass bottles covered with cork, somewhat dilapidated

96

but well-sealed. The beers were marked A, B, and C. Beer A and C was in brown bottles

97

while beer B was in a green bottle. Based on the place of beer finding, we assume that the

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bottles were stored in the dark at a quite stable temperature around 10 °C. During the

99

experiment, we also decided to analyze lager beers from 1999 (beer D, stabilized, pasteurized)

100

and 2004 (beer E, stabilized, pasteurized), both of which were stored in our lab in the dark at

101

20 °C.

102

Finally, fresh beers of traditional Czech commercial lagers (beers F and G, stabilized,

103

pasteurized) and a fresh lager produced at our institute (beer H, non-stabilized, non-

104

pasteurized) were analyzed and used as reference samples to provide an overview of the

105

current composition of Czech lagers. We chose purposely lager beers F and G that are very

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well known, are produced in large quantities and are very different in their chemical and also

107

sensorial parameters.

108 109

Beer analysis

110

Sensory analysis

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Because of the small volume of the century-old beer samples, sensory analysis was

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carried out by only 5 members of our sensory panel. A descriptive analysis of flavor and taste

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was performed immediately after opening of the bottles.

114 115

Chemical analysis

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Chemical analyses such as determination of original extract (EBC 9.4), alcohol (EBC

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9.2.6), total acidity (ASBC Beer 8), pH (EBC 9.35), color (EBC 9.6), content of bitter

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substances (EBC 9.8), total nitrogen (EBC 9.9.1), FAN (EBC 9.10), vicinal diketones


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(EBC 9.24.2) and volatile compounds (EBC 9.39) were performed according to EBC4 and

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ASBC5 methods.

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Minerals and heavy metals were analyzed using AAS on Varian SpectrAA 240FS

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using the following EBC methods: Ca (EBC 9.19), Mg (EBC 9.18), Na (EBC 9.16), K (EBC

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9.17), Cu (EBC 9.14.3), Fe (EBC 9.13.3), Zn (EBC 9.20), Mn (similar as Zn at 279.5 nm), Pb

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(similar as Zn at 217.0 nm), Ni (similar as Zn at 232.0 nm). Al, Cr, Si, and Sn were

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determined in acetylene–nitrous oxide flame at 309.3, 357.9, 251.6, 235.5 nm, respectively.

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As was measured using Graphite Tube Atomizer GTA 120 module of Varian SpectrAA

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240FS at 193.7 nm. Hg was determined by Advanced Mercury Analyzer AMA – 254 (Altec,

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Czech Republic).

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The profiles of residual α-acids, iso-α α-acids and their cis/trans isomers were

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determined using HPLC-UV on Dionex UltiMate 3000 UHPLC system consisting of a pump,

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a degasser, a column oven, an auto sampler (Dionex, Sunnyvale, CA, USA), and UltiMate

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3000 RS Diode Array Detector. The residual acids were separated on Alltima C18 5 U

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chromatography column (150 x 4.6 mm, 5 µm, Alltech, Grace, USA) according to a

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previously described method.6 Unlike in the original work, the sample was cleaned using

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solid phase extraction on SPE C18 columns (Strata-X 33µ 60 mg/3mL, Phenomenex,

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Torrance, CA,USA).

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The profiles of transformation products of alpha acids were determined using liquid

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chromatography coupled with high resolution mass spectrometry (HPLC-HR/AM-MS).

139

Analyses were performed on a hybrid quadrupole-orbitrap (Q-Exactive) mass spectrometer

140

(Thermo Fisher Scientific, San Jose, CA, USA) coupled with Dionex UltiMate 3000 UHPLC

141

system, consisting of a pump, a degasser, a column oven, and an autosampler (Dionex,

142

Sunnyvale, CA, USA). Alternatively, the same chromatographic system was coupled with

143

UltiMate 3000 RS Diode Array Detector (Dionex, Sunnyvale, CA, USA). Separations were



144

performed on XSelect HSS T3 C18 column (100 × 2.1 mm i.d., 3 µm, Waters, Milford, MA,

145

USA) with a mobile phase flow rate of 0.4 mL/min and column temperature 40 °C. The

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mobile phase consisted of (A) aqueous solution containing 0.1% (v/v) formic acid and (B)

147

acetonitrile solution containing 0.1% (v/v) formic acid with the gradient elution (min/% A):

148

0/95, 1.0/95, 13/70, 24/5, and 30/5, followed by a 6-min equilibration. The samples were kept

149

at 10 °C and the injection volume was 2 µL. The electrospray operated in negative mode

150

using the following parameters: electrospray voltage, 2.5 kV; sheath gas, 48 arbitrary units;

151

auxiliary gas, 11 arbitrary units; sweep gas, 2 arbitrary units. The heater in the source was set

152

to 295 °C, and heated capillary in the mass spectrometer was operated at 295 °C. Full Scan

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data were collected across a mass range of 120-900 m/z at resolution 70,000 FWHM. A

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maximum inject time of 250 ms for the Full Scan was applied.

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Iso-octanol extracts of century-old beer samples were prepared in accordance with

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EBC 9.8 method. For determining the profile of bitter compounds, 2.5 mL of iso-octanol

157

extract was evaporated under vacuum at 40 °C and the residue re-dissolved in 1 mL of

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acetonitrile.

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Carbohydrates were determined using HPLC-RI on a system equipped with a high-

160

pressure pump with degasser, column thermostat (SISw, Czech Republic) and autosampler

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Midas (Spark, Holland) connected with a high-sensitivity RI detector Shodex RI 101 (Japan).

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Chromatographic data were collected and processed by the DataApex Clarity data system,

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version 3.0.5.505. Separations were performed on Rezex RSO-Oligosaccharide ion exchange

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column in Ag+ mode, (200 x 10 mm, Phenomenex, U.S.A.) with deionized water as mobile

165

phase (Millipore S.A., France). The flow rate was 0.3 mL/min and column temperature was

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80 °C. Injection volume was 10 µL.7

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Fatty acids were determined using GC-FID; analyses of both short- and long-chain

168

fatty acids were carried out using a Chrompack CP 9001 gas chromatograph with a


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split/splitless injector, DB-WAX 20 m x 0.18 mm x 0.18 µm column (Thermo Scientific,

170

Runcorn, Cheshire,

171

United Kingdom), and a flame ionization detector (FID) under the same chromatographic

172

conditions. The gas chromatograph was equipped with Labio ASG 40 autosampler. The

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chromatographic column was maintained at 120 °C and after sample injection this

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temperature was kept for 0.7 min. Then the column oven was ramped at a rate of 30 °C/min to

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200 °C, and held isothermally for 6 min. The split mode with split ratio 1:10 was used. The

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injector and detector temperature was 220 °C. The carrier gas was helium of 5.0 quality with a

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column head pressure of 200 kPa at 120 °C. Prior to the analysis, free fatty acids were

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extracted from beer on SPE column LiChrolut EN 200 mg (Merck, Darmstad, Germany).

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Firstly, the column was conditioned with 2.5 ml of methanol and 5.0 ml of water.

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Subsequently, 20 ml of degassed beer sample acidified by 1 ml of 1 M HCl was spiked with

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10 µl of internal standard (C9, C13, C15 acid) and loaded on the column. Finally, the column

182

was rinsed by 5.0 ml of water, dried under gently nitrogen stream and purified sample was

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eluted with 2 x 0.5 ml of chloroform. During the SPE procedure, the flow of mobile and

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temperature was 3 ml/min and 20 °C, respectively.

185

Then the short-chain fatty acids were immediately determined using GC-FID. Long-chain

186

fatty acids were at first derivatized to their methyl esters and then analyzed by gas

187

chromatography.8

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Carbonyl compounds were determined using GS-MS analysis on the TRACE GC

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Ultra gas chromatograph coupled with DSQ II quadrupole mass spectrometer (Thermo

190

Electron Corporation). The polar column TR-WAX MS, 30 m x 0.25 mm i.d., 0.25 µm film

191

thickness (Agilent Technologies, Inc., Santa Clara, Ca, USA) was used. The carrier gas was

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set at 1.2 mL/min, one microliter of extract was injected in a splitless mode and the splitless

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time was 1 min. The injection temperature was 250 °C. The chromatographic oven was held



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at 60 °C for 2 min, then was increased to 235 °C at 6 °C/min. The carbonyl compounds were

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derivatized prior to the GC-MS analysis using PFBOA (derivatization reagent O-(2,3,4,5,6-

196

pentafluorobenzyl) hydroxylamine hydrochloride).9

197 198

Microbiology/genetic analysis

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Isolation of genomic DNA

200

Chromosomal DNA was extracted from the sediment of beer liquid by using

201

NucleoSpin Tissue Kit (Macherey Nagel, Germany) according to the manufacturer´s

202

instructions. Because of the extremely low concentration of DNA isolated that way, a

203

concentration step with further purification was added by application of the DNA Clean &

204

Concentrator™-5 kit (Zymo Research, USA). Concentration and integrity of all isolated DNA

205

samples were controlled by electrophoresis.

206 207

PCR and sequencing

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To identify the microflora parts of genes coding for ribosomal DNA, the ITS region

209

for yeast and 16S rDNA for bacteria were amplified. The PCR reactions were performed

210

using the PPP Master Mix (Top-Bio, Czech Republic). The ITS regions of yeast were

211

amplified using primer sets ITS1F + ITS4 and NS7 + ITS4, according to White et al.10 The

212

following PCR regime was used: denaturation 95.0 °C/1 min; 40 cycles (denaturation 95.0

213

°C/45 s, annealing 56.0 °C/30 s, extension 72.0 °C/60 s); final extension 72.0 °C/5 min. The

214

primer set 0028F + 1521R11 was used for the amplification of bacterial 16S rDNA. The PCR

215

conditions were the same as for yeast ITS amplification, but the extension time was prolonged

216

to 90 s. The PCR products were controlled by electrophoresis. A reamplification step with the

217

same primer set was employed in cases of very low amounts of amplified DNA fragments.


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Finally, the fragments were purified with the MinElute™ PCR Purification Kit

219

(Qiagen, Germany) and sequenced by the dideoxy chain termination method according to

220

Sanger and Coulson12 using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied

221

Biosystems, Foster City, California, USA). The sequencing products were analyzed with an

222

ABI Prism 3130xl DNA analyzer (Applied Biosystems, Foster City, California, USA). The

223

edited sequences were compared with GenBank nucleotide sequences using the BLAST

224

algorithm.13

225 226

Results and discussion

227

The opening the original bottles, which were covered by a cork, was immediately

228

followed by the basic sensory analysis. Subsequently, the chemical analyses such as

229

determination of original extract, alcohol, acidity, pH, color, variety of polyphenols, content

230

of bitter substances, amino nitrogen, volatile and carbonyl compounds, carbohydrates, fatty

231

acids, minerals and heavy metals were performed.

232

The DNA was also analyzed in order to identify microorganisms present in the beer.

233

PCR reamplification was used because the DNA isolation was done directly from the beer

234

sediment. As target regions for reamplification, we used internal transcribed spacer regions

235

ITS1 and ITS2, and 16S rRNA (or 16S rDNA) for identification of yeast and bacteria,

236

respectively. However, because of the time and conditions of beer storage we can assume that

237

the DNA was to a large extent degraded. The results of DNA analysis are demonstrated in

238

Table 1. The sequences of PCR products were compared with the GenBank using the BLAST

239

algorithm. The homology is expressed as the percentage of the coincident sequences. The

240

presence of DNA of bacteria and soil fungi (e.g. Coniochaeta, Lecythophora, Hypocreales) in

241

samples was probably caused by the cork-cup leakage during the storage of beer sample that

242

led to the contamination. 11 ACS Paragon Plus Environment


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Beer A was sensorially the least acceptable. It was light, hazy with very intensive

244

sulphuric and fecal off-flavor. No Saccharomyces yeast DNA was detected, but DNA of

245

bacteria Staphylococcus and Streptomyces was found.14

246

Beer B resembled Lambic. It was dark, very sour with madeira and nicely fruity off-

247

flavors. DNA of yeast Dekkera bruxellensis and Saccharomyces bayanus / S. pastorianus and

248

S. cerevisiae x S. eubayanus x S. uvarum was identified. No bacterial DNA was detected.14

249

Beer C was light brown and contained traces of carbon dioxide bubbles. While the

250

beer was oxidized, with typical sweetness and papery off-flavors, it was very slightly bitter

251

and it really appeared as beer. The slightly higher color of beer C comparing with A is

252

probably caused by oxidation. DNA of yeast belonging to the genus Debaryomyces was

253

identified. DNA of Bacillus and Streptomyces bacteria was proved.14

254 255

The good agreement between sensory, microbiological, and chemical analysis of these beers is discussed below.

256

Basic parameters: extract, alcohol, color, pH, total acidity, total nitrogen, FAN,

257

and total polyphenols. The basic parameters of studied beers are summarized in Table 2. In

258

comparison with the current beers, all three century-old beers A, B, and C had lower original

259

extract (10.30, 7.62, and 10.40 °Plato, respectively). Probably because of a secondary

260

fermentation or even some kind of contamination of century-old beers, higher content of

261

alcohol and very low real extract were detected. The other parameters, such as low pH and

262

very high value of total acidity, low concentration of FAN total nitrogen and polyphenols

263

(mainly in beer B), are also very probably caused by infection, which was subsequently

264

proved.

265

Minerals and metals. Century-old beers A, B, C had a higher content of Fe, Cu, Mn

266

and Zn (see Table S1); especially the concentration of Fe ranging from 0.25 to 0.46 mg/L is

267

very high compared to current beers. We assume that the higher concentration of these metals


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relates to metal composition of the brewhouse equipment 100 years ago. The concentration of

269

Mg, and K were very similar in beers A and C (about 95 and 580 mg/L, respectively),

270

whereas the concentration of Mg,

271

respectively). The concentration of Na in beer A, B, and C was 91, 17, and 51 mg/L,

272

respectively. The bconcentration of toxic metals (As, Al, Cd, Hg, Sn and Pb,) were under

273

LOQs (0.05, 0.2, 0.005, 0.0005, 0.5 and 0.03 mg/L, respectively), except sample B, where the

274

concentration of Pb was 0.182 mg/L. The different profile of mineral ions could be in direct

275

connection to malt composition (sample B was dark beer type). The concentration of ions and

276

namely high amount of Pb cations is also affected by the composition of glass, which the

277

green bottle was made from, due to elution of these metal ions from glass wall during such a

278

long time period.

and K in beer B was lower (51, , and 316 mg/L,

279

Finally, significantly lower concentrations of silicon were determined in the century-

280

old beers A, B, C compared with current Czech beer. Silicon in beer originates from barley,

281

namely from palea (grain without palea does not contain silicon). Barley malt is therefore a

282

rich silicon source. The content of silicon in barley malt depends on the barley variety and

283

growing region. The silicon concentration in Czech beer in 2013 ranged from 16 mg/L to 113

284

mg/L.15 We assume that a lower concentration of Si could be caused primarily by barley

285

variety and secondarily by some type of adjuncts. Because of a lack of barley during World

286

War I and also after it, a ministerial decree imposed barley rationing for breweries, which

287

covered only 22.4 % of normal need16. Therefore, a relatively high percentage of adjunct

288

could be supposed.

289

α-Acids, iso-α α-acids and their cis/trans isomers. The most interesting findings were

290

obtained during bitterness and iso-α-acids analysis. The data on residual α-acids and their

291

isomers, which were actually zero, are given in Table S2,. These results clearly show a trend

292

of degradation of iso-α-acids during beer aging. While the concentration of cis-iso-α-acids is



293

still noticeable after 16 and 11 years, the concentration of trans-iso-α-acids after one hundred

294

years’ period is near zero. This fact is in accordance with a number of previous studies on

295

different sensitivities of cis- and trans-isomers to degradation; the trans-isomers are described

296

as the less stable of the two.6, 17, 18 After 100 years, all monitored iso-α-acids are degraded.

297

This result strongly correlates with the sensory analysis, which did not detect bitterness in the

298

century-old samples.

299

However, the determined values of BU units of bitterness of these three century-old

300

beers A, B, and C (15, 8, and 22, respectively) are in contradiction to the sensory evaluation

301

which showed no or only minimal bitter taste of these samples. Thus, in this case, the

302

determination of bitterness according to EBC method gives false positive results. Therefore,

303

the non-specific measurement of absorbance of iso-octanol extract was altered by high-

304

performance liquid chromatography (HPLC) on reversed phase in combination with UV

305

detection at a wavelength of 275 nm. The HPLC chromatograms show a group of peaks

306

eluting between 15 to 25 min that should represent, in accordance with EBC method,

307

compounds responsible for bitter taste present in the beer sample (see Figure 1a). To obtain

308

more detailed information, the samples were analyzed again under the same chromatographic

309

conditions using high-resolution mass spectrometer operated in negative ionization mode.

310

Figure 1b shows total ion current chromatogram (TIC) of the iso-octanol extract of century-

311

old beer C, which nicely corresponds with the UV chromatogram of the same sample shown

312

in Figure 1a. Two of the most intense chromatographic peaks eluted at retention times 18.5

313

and 19.7 minutes in TIC chromatogram (see Figure 1b) correspond to compounds of at m/z

314

347.1860 and m/z 361.2018. These exact masses could be assigned to the deprotonated

315

molecular ions [M-H]− with elemental composition corresponding to the summary formulas

316

C20H27O5 (2.0 ppm) and C21H29O5 (2.2 ppm), respectively. Four extracted ion chromatograms

317

(XIC) marked a - d, using a 5 ppm window around the monoisotopic mass (m/z) of the target


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Page 15 of 34


318

compounds are shown in Figure 2 and Figure 3, respectively. Figure 2a and Figure 3a show

319

the profile of these two compounds in one sample of century-old beers (sample C) and

320

Figure 2b and Figure 3b show a sample of beer F (Bitterness unit (BU) = 20) analyzed under

321

the same LC/MS conditions and used as a reference sample for comparison of the profile of

322

individual “bitter” compounds. The peaks at retention time of 21.6 min (Figure 2b) and 22.1

323

(Figure 3b) were identified as those corresponding to cohumulone and humulone, which are

324

prevalent members of the class of compounds known as alpha acids. In terms of structure,

325

alpha acids are phloroglucinol derivatives with two side-chains formed by prenyl groups and

326

one side chain that could be isobutyryl for cohumulone (C20H28O5), isovaleryl for humulone

327

(C21H30O5) and methyl-ethyl-acetyl for adhumulone (C21H30O5). In contrast with traces of

328

these compounds present in sample of Beer F, the most intense chromatographic peaks

329

represent cis- and trans-isocohumulone eluted at 19.7 and 19.9 min (Figure 2b) and cis- and

330

trans-isohumulone co-eluted together with cis- and trans-isoadhumulone from 20.5 to 21.0

331

min (Figure 3b). The correct identification of iso-alpha acids was proved by analyzing the

332

standard mixture of iso-alpha acids (Figure 2d and Figure 3d), where also the peak of

333

adhumulone at retention time 22.3 min could be observed. It is well known that alpha acids

334

get isomerized to form iso-alpha acids by the application of heat in the slightly acidic wort

335

and collectively these compounds give hopped beer its characteristic bitter flavor. The profile

336

of alpha and iso-alpha acids in sample of Beer F is therefore in accordance with expectations.

337

In samples of century-old beer, the profile of alpha and iso-alpha acids is completely

338

different. Figure 2a shows one major peak at m/z 347.1860 eluted at 18.5 min and Figure 3a

339

shows two peaks at m/z 361.2018 eluted at 19.7 and 20.0 min, respectively. Surprisingly,

340

these can be most probably attributed to spontaneous isomerization of iso-alpha acids that has

341

never before been reported in the literature, while some studies using isomerization of

342

hulupone in alkaline methanol describe laboratory preparation of these compounds that have a



343

spiro structure and are isomeric with isohumulones. The authors isolated four

344

diastereoisomers of compounds formed from humulone by an internal cyclization reaction

345

(Figure 4, structures 6-9). These compounds have been consequently designed as spiro-

346

isohumulones and their chemical properties, like the comparable pKa values and the almost

347

identical UV spectra, follow from the similarity with isohumulones.20 We confirmed the

348

presence of these compounds, tentatively identified as spiro-isohumulones, by preparing these

349

compounds using a modified procedure described by Verzele and De Keukeleire20 and by

350

comparing their structure with compounds from century-old-beer using high resolution MS.

351

Figure 2c and Figure 3c show the profile of humulones (alpha acids), isohumulones and

352

most probably spiro-isohumulones extracted by iso-octane from a sample of lyophilized beer

353

re-suspended in ethanolic solution of potassium hydroxide (0.8 M) and heated under reflux

354

for 30 minutes. Two groups of peaks eluted from 18.5 to 19.8 min and from 19.2 to 19.6 min,

355

respectively, nicely corresponded to the peaks that were found in samples of century-old-beer.

356

The retention times of these compounds and their fragmentation patterns were the same and,

357

therefore, confirmed the presence of identical compounds in both samples (compare Figure

358

2a and 2c). The mechanism of isomerization of isohumulones during natural aging of beer is

359

still unclear but on the basis of our set of experiments we were able to prove that identical

360

compounds arise by boiling of beer under alkaline conditions and thus could be identified as

361

spiro-isohumulones in accordance with a literature source20 at a high level of confidence. The

362

presence of these compounds in samples of century-old beers could explain the similarity

363

between values of BU units of century-old and contemporary beers, while both the sensory

364

bitterness and concentration of iso-alpha-acids are nearly zero. It is obvious that the false

365

positive results of bitterness of the century-old beers are caused by a wide range of other

366

compounds eluted from 14 to 23 min (see Figure 1); however, the limited space of this article

367

did not allow us to list all these compounds that have emerged during almost 100 years.


Page 16 of 34

Page 17 of 34


368

Volatile compounds. The concentration of diacetyl and pentandione in all analyzed century-

369

old samples was negligible (from 8 to 45 µg/L) compared to fresh beers G and H (105 µg/L);

370

the higher concentration of diacetyl is specific for beer G. Unfortunately, we cannot discuss

371

diacetyl concentration in beers A-C because we haven’t any information about the original

372

concentration of this compound.

373

A relatively high concentration of DMS (66 µg/L, see Table S3) was found in beer A,

374

which is in agreement with sensory analysis (the flavor threshold in beer is 30 – 50 µg/L).21 It

375

was evident, that also other sulfuric compounds (hydrogen sulfide and mercaptanes were

376

present in sample A but we were not able to determine all these components because of the

377

limited amount of the sample.

378

Further, the century-old samples A, B, and C contained a high concentration of esters

379

(data not shown) of acetic acid; in particular, beer B, which contained the highest

380

concentration of acetic acid (nearly 9000 mg/L), contained also a high level of ethyl acetate

381

(624 mg/L). Also, the concentration of ethyl lactate in samples A, B and C was high, namely

382

0.64, 0.55, and 0.92 mg/L, respectively. The concentration of lactic acid in these samples was

383

also very high and ranged between 2500 and 3500 mg/L. Finally, the high content of ethyl

384

myristate determined in beer C correlated with the higher concentration of myristic acid

385

(1.405 mg/L, Table S2) in the same sample. The fermentation of wort by Dekkera is

386

accompanied by the production of considerable amounts of ethyl caprylate, ethyl caprate,

387

ethyl lactate and ethyl acetate. Conversely, the amount of acetic acid esters (e.g. isoamyl

388

acetate) is lower than during the fermentation by Saccharomyces. Cellular esterases of

389

Dekkera hydrolyze these esters during fermentation.22

390

To sum up, the high content of esters evident in century-old beers A, B, and C is most

391

likely caused by infection; the ratio of the sum of esters to the sum of alcohol is ranging from

392

0.15 to 0.98 whereas in fresh beers the ratio is ranging from 4.89 to 6.27.23



393

Carbohydrates. The profile of simple sugars and maltose oligomers, and the sum of

394

carbohydrates with DP ≤ 10 (DP = degree of polymerization = number of glucose units) are

395

given in Table S4. During the storage, simple fermentable sugars such as glucose, fructose

396

and maltose were completely fermented by yeasts and other microorganisms (see below) and

397

they are completely missing in A, B, C beers; the sum of carbohydrates with DP ≤ 10 in these

398

samples is 0.19, 0.08, and 0.21 g/100 mL. Unlike Walther et al3, who analyzed the

399

maltooligosaccharide profile in nearly two centuries-old beer samples using HILIC-1H-NMR,

400

we therefore cannot assess the raw material quality of the samples.

401

Fatty acids. Fifteen basic fatty acids (short-chain saturated, long-chain saturated and

402

three unsaturated) were determined and compared in century-old and current beer; the results

403

are given in Table S2. Higher concentration of isobutyric, butyric, and isovaleric acids was

404

found in all three century-old beers. These fatty acids with unpleasant odor most probably

405

come from microbial infection and may appear also as a result of oxidation of hop

406

compounds. Although their levels (ranging from 0.7 to 4.4. mg/L) are near the sensory

407

threshold (1 - 2.5 mg/L and 3 mg/L for isovaleric and butyric acid, respectively)Error!

408

Bookmark not defined., the off-flavor was not really recognized during the sensory analysis

409

except for beer B, where the concentrations of isovaleric and isobutyric acid were 4.4 and 2.2

410

mg/L, respectively. This result correlates with the finding of Dekkera bruxellensis, which

411

breaks down leucine and valine present in beer into isovaleric and isobutyric acid,

412

respectively.24

413

Further, a very interesting result has been the four-fold higher concentration of

414

myristic acid in century-old beers A, B, and C compared with the current beers. This saturated

415

fatty acid is neither a product of contaminating bacteria nor a product of yeasts. The higher

416

concentration of myristic acid may probably be caused by two factors. One of them is the

417

possible use of surrogates, which can increase the concentration of this fatty acid25 and


Page 18 of 34

Page 19 of 34


418

corresponds also with the lower concentration of silicon. The other factor may be the

419

influence of barley variety; according to our long-time research, the profile of fatty acids is

420

variety dependent, especially the myristic acid concentration being a characteristic feature of

421

each Czech barley variety (study in preparation, note of the author).

422

Finally, the concentration of saturated fatty acids with longer chain (see Table S2)

423

from hexanoic to decanoic acid) was determined to be very similar in century-old and current

424

beers, so it seems that these compounds are chemically stable.

425

Carbonyl compounds. Carbonyl compounds belong to the main compounds arising

426

during beer aging. Some of these compounds are sensorially active and the increase of their

427

concentration therefore causes (mostly adverse) beer taste changes.26 Some of these

428

compounds are sensorially inactive and were referred to as “aging indicators”.26,27 Some

429

studies found a statistically significant correlation between the formation of stale flavor and

430

concentration of aging indicators (mainly 2-furfural, 2-methylpropanal, 2-methylbutanal, 3-

431

methylbutanal).8 Figure 5 demonstrates a huge increase of 2-furfural, 2-methylbutanal, 3-

432

methylbutanal, and phenylacetaldehyde during the 100 years of beer storage. Whereas the

433

concentration of 2-furfural in fresh beers is about 50 µg/L, in 10 years-old beer it is about

434

2000 µg/L and in the 100-year-old beer C it was more than 6500 µg/L. It is very interesting

435

that the authors who analyzed beer from the 1840s shipwreck (Londesborough 2015) found a

436

much lower amount of 2-furfural (644 µg/L)

437

samples, it was below the threshold value (150 mg/L). This fact can be probably caused by

438

the low and stable temperature (we assume 4 °C) on the ocean floor. 2-Methylpropanal and 3-

439

methylbutanal (fruity off-flavor) were found in comparable concentrations in our 10-year-old

440

beer (from 51.67 to 170.44 µg/L) and in the 100-year-old beer (5.29 to 118.77 µg/L), whereas

441

the concentrations of these sensory active compounds in fresh beers are below 20 µg/L. An

442

increased concentration of phenyl acetaldehyde (flowery, rose, hyacinth) was also proved in

Error! Bookmark not defined.


; however, like in our


443

100-year-old beer (mostly in beer C, 894 µg/L) and also in 10-year-old beer (119 – 190 µg/L).

444

Finally, on comparing century-old beers A, B, and C, beer C is seen to contain the highest

445

concentration of carbonyl compounds, similar to that in beers D and E from 1999 and 2004,

446

respectively. As we know that beer C was well-sealed, because it contained rests of carbon

447

dioxide bubbles resulting in unspoiled sensory profile relative to today´s beer, this beer could

448

be considered as a unique example of a naturally aged beer. A different situation was with

449

beers A and B (sulfuric and acidic flavor, respectively) in which the aging reaction

450

mechanisms seem to be different due to noticeable contamination.

451

To sum up, all three beers can be concluded to be of the lager type. Moreover, one

452

can assume that a century ago our ancestors produced beer from similar raw materials and in a

453

similar way as today. The chemical changes in beers A and B were caused mainly by

454

microbial contamination. The chemical profile of beer B (total acidity, FAN, ethyl acetate,

455

total esters) nicely corresponds to the finding of Dekkera bruxellensis yeast DNA. The

456

relevant genetic study is being published.

457

knowledge about a 100-year-old beer which, due to undamaged sealing cork plug and most

458

probably constant temperature in the cellar, underwent a “natural” aging process unmarred by

459

microbial contamination, which resulted in an unspoiled sensory profile but included the

460

production of carbonyl compounds and a very high concentration of aging indicators 2-

461

methylpropanal, 3-methyl-propanal, phenylacetaldehyde and namely furfural that was found

462

in this beer. The beer aging during the whole 100-year period including carbonyl compound

463

production thus seems to be a stable and ongoing process. Beer C can also be used for

464

studying the progression of natural transformation of iso-alpha bitter compounds. During the

465

lengthy beer storage, iso-alpha acids were transformed to their subsequent “iso” products,

466

which are determined by the routine EBC 9.8 method along with iso-alpha acids, but are not

467

sensorially bitter and their polarity is different, as documented by their retention time in

14

The beer C enabled us to acquire deeper


Page 20 of 34

Page 21 of 34


468

chromatographic separation which is different from that of iso-alpha acids. They may include

469

spiro-isohumulones, which have been as yet reported only as products of alkaline hydrolysis

470

of alpha acids.20 This finding is so interesting, that we plan to continue our studies of this

471

topic.

472 473 474 475 476

Acknowledgement

477

This study was supported by the project Ministry of Agriculture of the Czech Republic

478

No. RO1915 and Ministry of Education Youth and Sports of the Czech Republic No. LO

479

1312 and the project RVO61388971. The authors thank Mr. Aleš Přinosil from the Raven

480

Trading s.r.o. for providing beer samples and his cooperation.

481 482 483 484

References

485 486

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487

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488

2

489

Seppänen-Laakso, T.; Viljanen, K.; Virtanen, H.; Wilpola, A.; Hofmann, T.; Wilhelmson, A.

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Analysis of Beers from an 1840s’ Shipwreck. J. Agric. Food Chem. 2015, 63, 2525-2536.

Olšovská, J.; Čejka, P.; Sigler, K.; Hönigová, V. The Phenomenon of Czech Beer: a review.

Londesborough, J.; Dresel, M.; Gibson, B.; Juvonen, R.; Holopainen, U.; Mikkelson, A.;



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in (and since) the late 19th century: Molecular profiles of 110–130 year old beers. Food

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Chem. 2015, 183, 227-234.

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Analytica EBC, EBC Analysis Commitee-Nürnberg: Carl Getranke-Fachverlag. 2012

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ASBC methods of analysis. American Society of Brewing Chemists, 14th edition. 2016.

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ISBN 978-1-881696-21-6.

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6

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Beer Ageing, Marked Instability of Trans-Iso-oc-Acids and Implications for Beer Bitterness

499

Consistency in Relation to Tetrahydroiso-oc-Acids. J. Inst. Brew. 2000, 106(3), 169-178.

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in beer using its pre-column enzymatic cleavage and HPLC-RI. Food Anal Met, 2014, 7(8),

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1677-1686.

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Beer by Fast Routine Analyse. Kvasny Prum 2013, 59, 58-62.

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Aging for Ex-post Checking of Storage Conditions and Prediction of the Sensory Stability of

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Beer. J. Agric. Food Chem. 2013, 61, 12670-12675.

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sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to

510

Methods and Applications; Innis MA., Gelfand D.H., Sninsky JJ., White TJ., eds.; Academic

511

Press: New York, NY, 1990, 315-322.

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11

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nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal

514

DNA. Nucleic Acids Res. 1989, 17(19), 7843-7853.

Walther, A.; Ravasio, D.; Qin, F.; Wendland, J.; Meier, S. Development of brewing science

De Cooman, L.; Aerts, G.; Overmeire, H. Alterations of the Profiles of Iso-a-Acids During

Jurková, M.; Čejka, P.; Štěrba, K.; Olšovská, J. Determination of total carbohydrate content

Horák, T.; Čulík, J.; Jurková, M.; Čejka, P.; Olšovská, J. Determination of Fatty Acids in

Čejka, P.; Čulík, J.; Horák, T.; Jurková, M.; Olšovská, J. Use of Chemical Indicators of Beer

White, T. J.; Bruns, T. D.; Lee, S. B.; Taylor J. W. Chapter 38. Amplification and direct

Edwards, U., Rogall, T., Blöcker, H., Emde, M., Böttger E. Isolation and direct complete


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synthesis with DNA polymerase. J. Mol. Biol. 1975, 94(3), 441–448.

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Search Tool. J. Molecular Biology 1990, 215(3), 403-410.

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Analysis of century-old beer – Chemical, sensorial and genetic profile of 100-year-old beer

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from Zahlinice. Kvasny Prum. 2016, 62 (11-12), DOI:10.18833/kp2016032.

522

15

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During the Brewing Process. Czech J. Food Sci. 2013, 31,166–171.

524

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Institute of Brewing and Malting: Praha, Czech Republic, 2005. ISBN 8086576167.

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17

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staling in beer. J. Am. Soc. Brew. Chem. 2002, 60(1), 26-30.

528

18

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G. Flavour stability of pale lager beers: Determination of Analytical markers in relation to

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sensory ageing. J. Inst. Brew. 2008, 114(2), 180-192.

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Bitter Acids, 1st Edition; Elsevier Sience Publishers B.V., Amsterdam, Netherlands, 1991, pp.

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114-115. ISBN 9780444881656

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http://www.aroxa.com/beer/beer-flavour-standard/

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22

Steensels, J.; Daenen, L.; Malcorps, P.; Derdelinckx, G.; Verachtert, H.; Verstrepen, K.J.,

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2015: Brettanomyces yeasts - From spoilage organisms to valuable contributors to industrial

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fermentations. Int. J. Food Microbiol. 2015, 206, 24-38.

Sanger, F.; Coulson, A. R. A rapid method for determining sequences in DNA by primed

Altschul, S. F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D. J. Basic Local Alignment

Olšovská, J.; Matoulková, D.; Felsberg, J.; Jelínková, M.; Dušek, M.; Čejka, P.; Štěrba, K.

Cejnar, R.; Mestek, O.; Dostálek, P. Determination of Silicon in Czech beer and its Balance

Kratochvíle, A. Pivovarství v českých zemích v proměnách 20. století, 1st edition. Research

Araki, S.; Tahashio, M.; Shinotsuka, K. A new parameter for determination of the extent of

Malfliet, S.; VanOpstaele, F.; De Cllippeller J.; Syryn, E.; Goiris, K.; De Cooman, L.; Aerts,

Maes, L.; Antenuis, M.; Verzele, M. Spiro-isohumulones. J. Inst. Brew. 1970, 76(3), 250-

Verzele, M.; De Keukeleire, D. Volume 27. Chemistry and Analysis of Hop and Beer



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technologische Faktoren. Brauwelt 1995, 135, 2286-2301.

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winemaking: a synoptic review. S. Afr. J. Enol. Vitic. 2008, 29, 128–144.

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545

Woodhead Publishing Limited, Cambridge, United Kingdom, 2004, 313 – 315. ISBN

546

0849325471.

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München, Weihenstephanm, 1991.

549

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Narziss, L. Über den Biergesmack und seine Beeinflussung durch Rohstoffe und

Oelofse, A.; Pretorius, I.S.; du Toit, M. Significance of Brettanomyces and Dekkera during

Briggs, D. E.; Boulton, Ch. A.; Brookes, P. A.; Stevens, R. Brewing. Science and practice.

Eichhorn, P. Die Erhaltung der Gesmackstabilität von Bier. Disertation Arbeit, TU

Meilgaard, M. Stale flavour carbonyls in Brewing. Brew. Dig. 1972, 47, 48–57.

550


Page 24 of 34

Page 25 of 34


551

Figure 1. LC–DAD chromatogram at 275 nm (a) and LC–ESI-MS (negative ion mode) total

552

ion current (TIC) chromatogram (b) of the iso-octanol extract of century-old beer A

553

evaporated and re-dissolved in methanol.

554 555

Figure 2. Extracted ion chromatogram (EIC) for m/z 347.1864 of sample of century-old beer

556

C (a), beer F (b), a standard mixture of iso-alpha acid (c) and a standard of alpha acids (d).

557 558

Figure 3. Extracted ion chromatogram (EIC) for m/z 361.2020 of sample of century-old beer

559

C (a), beer F (b), standard mixture of iso-alpha acid (c), standard mixture of alpha acids (d).

560 561

Figure 4. Structural formulae of cohumulone (1), humulone (2), adhumulone (3), trans-

562

isohumulone(4), cis-isohumulone (5), exo trans-spiro-isohumulones (6), endo trans-spiro-

563

isohumulones (7), exo cis-spiro-isohumulones (8), and endo cis-spiro-isohumulones (9).

564 565

Figure 5. The concentration profile of 2-furfural, 2-methylbutanal, 3-methylbutanal, and

566

phenylacetaldehyde in fresh, 11-, 16-, and 100-year-old beers.

567



Page 26 of 34

Table 1. Results of DNA analysis

Beer Amplified

Primers

NCBI Blast*

DNA homology

ITS1F + ITS4

Uncultured Coniochaeta clone AEW4_82 18S ribosomal RNA gene

98 %

Lecythophora sp. olrim15 internal transcribed spacer

99 %

Hypocreales sp. PSU-ES23 internal transcribed spacer 1

99 %

NS7+ITS4

Uncultured Dikarya clone PA2009D1 18S ribosomal RNA gene

96 %

0028F + 1521R

Staphylococcus hominis 16S rDNA gene

100 %

Streptomyces paucisporeus 16S rDNA gene

99 %

Uncultured Coniochaeta clone AEW4_82 18S ribosomal RNA gene

96 %

Lecythophora sp. olrim15 internal transcribed spacer

99 %

Simplicillium sp. M-27 18S ribosomal RNA gene

100 %

1

Saccharomyces bayanus strain DBMY771 internal transcribed spacer

99 %

2

Saccharomyces cerevisiae x Saccharomyces eubayanus x Saccharomyces

99 %

region 1

ITS

16S rDNA

2

ITS

ITS1F + ITS4

uvarum strain CBS1503 internal transcribed spacer 1 3

Saccharomyces pastorianus internal transcribed spacer 1

99 %

4

Saccharomyces uvarum strain CBS 377 18S ribosomal RNA gene

99 %

5

Dekkera bruxellensis voucher Bb_Tou_Fr2 18S ribosomal RNA gene

98 %

26

ACS Paragon Plus Environment

Page 27 of 34


6

NS7 + ITS4

Dekkera bruxellensis 18S rRNA gene

96 %

Uncultured Dikarya clone PA2009D1 18S ribosomal RNA gene

98 %

Debaryomyces hansenii 18S ribosomal RNA gene

98 %

Uncultured Coniochaeta clone AEW418S ribosomal RNA gene

95 %

Simplicillium sp. M-27 18S ribosomal RNA gene

99 %

Uncultured Dikarya 18S ribosomal RNA gene

96 %

NS7 + ITS4

Uncultured Ascomycota clone

98 %

0028F + 1521R

Bacillus simplex 16S rDNA gene

100 %

Streptomyces sp. 16S rDNA gene

99 %

7 3

ITS

ITS1F + ITS4

8 9

16S

rDNA 10

* NCBI Blast - National Center for Biotechnology Information Basic Local Alignment Search Tool

27



Page 28 of 34

Table 2. Basic chemical parameters of century-old and current beers Analyte

Units

Uncertainty

Beer A

Beer B

Beer C

Beer D

Beer E

Beer F

Beer G

Beer H

1900?*

1900?*

1900?*

1999*

2004*

2015*

2015*

2015*

Extract real

%

0.02

1.87

1.58

2.09

5.04

4.09

3.66

5.1

4.61

Alcohol (v/v)

%

0.05

5.45

3.85

5.38

4.41

4.79

5.2

4.3

5.16

Alcohol (w/v)

%

0.04

4.31

3.04

4.25

3.24

3.75

4.08

3.35

4.04

Original extract

%

0.06 10.30

7.62

10.40

11.71

11.39

11.59

11.61

12.42

Plato° Bitterness

IBU

1

15

8

22

15

20

31

32

25

EBC u.

0.5

10.0

60.2

17.2

24.0

22.4

8.4

12.0

11.3

-

0.05

3.89

3.7

3.86

4.67

4.43

4.53

4.6

4.6

mL 1M NaOH/100

3 % (rel.) 5.08

12

4.34

2.6

2.86

2.16

2.4

3.63

0.47

0.47

0.37

0.45

0.48

Color pH

Total acidity Total nitrogen

mL (g/100 mL)

20

0.36

0.13

0.48

FAN

mg/L

6

149

12

180

99

198

180

257

Total polyphenols

mg/L

8

105

5

21

116

100

150

125

*year of beer production

28


Page 29 of 34


Figure 1



Figure 2

30


Page 30 of 34

Page 31 of 34


Figure 3

31



Figure 4

32


Page 32 of 34

Page 33 of 34


Figure 5

33



TOC

34


Page 34 of 34

Analysis of 100-Year-Old Beer Originated from the Czech Republic

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