Studies on the Simultaneous Formation of Aroma-Active and

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Studies on the Simultaneous Formation of Aromaactive and Toxicologically Relevant Vinyl Aromatics from Free Phenolic Acids during Wheat Beer Brewing Michael Granvogl, and Daniel Langos J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05606 • Publication Date (Web): 22 Jan 2016 Downloaded from http://pubs.acs.org on January 23, 2016

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Journal of Agricultural and Food Chemistry

Studies on the Simultaneous Formation of Aroma-active and Toxicologically Relevant Vinyl Aromatics from Free Phenolic Acids during Wheat Beer Brewing Daniel Langos# and Michael Granvogl§*

# Deutsche Forschungsanstalt für Lebensmittelchemie, Lise-Meitner-Straße 34, D-85354 Freising, Germany § Lehrstuhl für Lebensmittelchemie, Technische Universität München, Department für Chemie Lise-Meitner-Straße 34, D-85354 Freising, Germany

___________________________________________________________________ *Corresponding Author Phone:

+49 8161 71 2987

Fax:

+49 8161 71 2970

E-mail:

[email protected] 1 ACS Paragon Plus Environment


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ABSTRACT: During the brewing process of wheat beer, the desired aroma-active

2

vinyl aromatics 2-methoxy-4-vinylphenol and 4-vinylphenol as well as the undesired

3

and toxicologically relevant styrene are formed from their respective precursors free

4

ferulic acid, p-coumaric acid, and cinnamic acid, deriving from the malts. Analysis of

5

eight commercial wheat beers revealed high concentrations of 2-methoxy-4-

6

vinylphenol and 4-vinylphenol always in parallel with high concentrations of styrene

7

or low concentrations of the odorants in parallel with low styrene concentrations,

8

suggesting a similar pathway. To better understand the formation of these vinyl

9

aromatics, each process step of wheat beer brewing and the use of different strains

10

of Saccharomyces cerevisiae were evaluated. During wort boiling, only a moderate

11

decarboxylation of free phenolic acids and formation of desired and undesired vinyl

12

aromatics was monitored due to the thermal treatment. In contrast, this reaction

13

mainly occurred enzymatically catalyzed during fermentation with Saccharomyces

14

cerevisiae strain W68 with normal Pof+-activity (phenolic off-flavor) resulting in a

15

wheat beer eliciting the typical aroma requested by consumers due to high

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concentrations of 2-methoxy-4-vinylphenol (1790 µg/L) and 4-vinylphenol (937 µg/L).

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Unfortunately, also a high concentration of undesired styrene (28.3 µg/L) was

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observed. Using a special Saccharomyces cerevisiae strain without Pof+-activity

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resulted in a significant styrene reduction (< LoQ), but also in low amounts of 2-

20

methoxy-4-vinylphenol (158 µg/L) and 4-vinylphenol (46.7 µg/L), resulting in a less

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pronounced wheat beer aroma.

22 23 24

KEYWORDS: Wheat beer, phenolic acids, styrene, 2-methoxy-4-vinylphenol, 4-

25

vinylphenol

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INTRODUCTION

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Wheat beer is a Bavarian specialty beer, which is brewed with at least 50% of

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wheat malt. Its outstanding feature is the flavor, mainly characterized by clove-like

29

and slightly phenolic aroma notes caused by 2-methoxy-4-vinylphenol (4-

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vinylguaiacol) and 4-vinylphenol.1-6 When these compounds occur in concentrations

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above their respective odor thresholds in other beer types, the induced aroma is

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undesired and known as “phenolic off-flavor” (Pof).7 However, in wheat beer, exactly

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this aroma impression is requested by consumers and lowered concentrations of 2-

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methoxy-4-vinylphenol and 4-vinylphenol result in a less pronounced overall wheat

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beer aroma as recently shown.8 The vinyl aromatics are formed by decarboxylation of

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the corresponding free phenolic acids, namely ferulic acid and p-coumaric acid

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(Figure 1), which can be caused either by elevated temperatures9 or by enzyme

38

activity of Pof+ strains of the top-fermenting yeast Saccharomyces cerevisiae.10

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Recent studies also reported about the presence of styrene in wheat beer, another

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vinyl aromatic formed by the same decarboxylation mechanism from free cinnamic

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acid (Figure 1).11-14 Styrene is undesired in all foods due to its toxicological

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relevance15,16 and its classification as “possibly carcinogenic to humans” (group 2B)

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by the International Agency for Research on Cancer (IARC).17

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Because phenolic acids are the precursors of the desired phenolic aroma

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compounds as well as of the undesired styrene, their control during the brewing

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process is of special interest for wheat beer breweries. Phenolic acids originate from

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barley and wheat malts, in which they occur either in free form or bound to cell wall

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arabinoxylans.18-20 During mashing, phenolic acids in free or ester bound form can be

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extracted from the malts and, hence, be solubilized in the mash, wort, and, finally, in

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the beer.21,22 Some studies already investigated the influence of the mashing 3 ACS Paragon Plus Environment


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conditions on the release of phenolic acids into the mash and wort, with special focus

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on cinnamic acid, p-coumaric acid, and ferulic acid, as well as on styrene formation

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from cinnamic acid during fermentation.13,22-25 However, up to now, no studies are

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available reporting about a simultaneous quantitation of free cinnamic, p-coumaric,

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and ferulic acid and their corresponding vinyl aromatics styrene, 4-vinylphenol, and 2-

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methoxy-4-vinylphenol in the malts, the intermediates (unboiled wort, cast wort,

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green beer) of the brewing process, and the ready-to-drink beer.

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Thus, the aim of the current study was, first, to quantitate aroma-active 2-methoxy-

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4-vinylphenol and 4-vinylphenol as well as toxicologically relevant styrene in

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commercial wheat beers via GC-MS or GC/GC-MS using stable isotope dilution

61

assays (SIDAs). Secondly, to evaluate the contribution of each brewing process step

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(Figure 2) to the formation of the decarboxylation products, both free phenolic acids

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and their corresponding decarboxylated phenolics were quantitated in barley and

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wheat malts, in unboiled wort (after mashing), in cast wort (after boiling), in green

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beer (after first fermentation), and in the ready-to-drink beer (after secondary

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fermentation) on the basis of SIDAs using a recently developed HPLC-MS/MS

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method for the free phenolic acids26 and again GC-MS or GC/GC-MS for the vinyl

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aromatics. Further, the influence of the yeast type on the formation of the vinyl

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aromatics was investigated using two different strains of the top-fermenting brewing

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yeast Saccharomyces cerevisiae (normal and without Pof+-activity).

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MATERIALS AND METHODS

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Commercial Wheat Beers. Samples were bought in local supermarkets.

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Kiln-dried Malts and Wheat Beer Process Intermediates. Kiln-dried wheat and

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barley malts as well as the unboiled wort, cast wort, green beer, and ready-to-drink 4 ACS Paragon Plus Environment

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beer produced thereof were obtained from a Bavarian brewery. Thus, all samples

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were produced at industrial scale and under industrial conditions. Two series of

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samples (from malt to beer) were produced with different yeast strains of

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Saccharomyces cerevisiae: one with the strain W68 (normal Pof+-activity) and the

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other with a special strain (without Pof+-activity).

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Chemicals. The following compounds were commercially obtained: p-coumaric

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acid and styrene (Fluka, Neu-Ulm, Germany), cinnamic acid, ferulic acid, 4-

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vinylphenol, and zinc sulfate monohydrate (Aldrich; Sigma-Aldrich Chemie,

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Taufkirchen, Germany), acetonitrile, ethyl acetate, formic acid, methanol, and sodium

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sulfate

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Mühlheim/Main, Germany), potassium hexacyanoferrate (II) trihydrate (Carl Roth,

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Karlsruhe, Germany), and diethyl ether (VWR, Darmstadt). Ethyl acetate and diethyl

88

ether were of p.A. grade and were distilled on a Vigreux column before usage.

(Merck,

Darmstadt,

Germany),

2-methoxy-4-vinylphenol

(Lancaster,

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Stable Isotopically Labeled Compounds. The following compounds were

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commercially obtained: [13C3]-p-coumaric acid, [2H3]-ferulic acid, and [2H8]-styrene

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(Aldrich) and [2H6]-cinnamic acid (C/D/N Isotopes, Québec, Canada). [2H4]-4-

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Vinylphenol27 and [2H3]-2-methoxy-4-vinylphenol28 were prepared as previously

93

described.

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Sensory Analysis. The sensory panel consisting of 20 experienced assessors

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participating in weekly sensory training sessions intended to train their abilities to

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recognize and describe different aroma qualities was asked to hedonically evaluate

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the wheat beers according to the attribute “pronounced wheat beer aroma” on a

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scale from 1 (very weak) to 10 (very strong). Sensory analyses were performed in a

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sensory room at 21 ± 1 °C equipped with single booths. Samples (15 mL) were

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presented in covered glass vessels (40 mm i.d., total volume = 45 mL).



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Extraction of Free Phenolic Acids from Kiln-dried Malts and Quantitation by

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Stable Isotope Dilution Assays (SIDAs). A recently developed method was used

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for the extraction and quantitation of free phenolic acids.26 Briefly, malt samples were

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frozen with liquid nitrogen prior to grinding with an IKA M20 universal mill (Jahnke &

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Kunkel, Staufen im Breisgau, Germany). To the finely ground malt powder (10 g),

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methanol (200 mL) and the stable isotopically labeled internal standards [2H6]-

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cinnamic acid, [13C3]-p-coumaric acid, and [2H3]-ferulic acid (amounts determined in

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preliminary experiments) were added. Afterwards, the samples were stirred for 200

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min at room temperature, centrifuged (4000 rpm, 5 min at 15 °C; Multifuge X3 FR;

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Thermo Scientific, Schwerte, Germany), and filtered. After removal of the solvent by

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means of a rotary evaporator (35 °C, 20 mbar), the residue was dissolved in water

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(20 mL), and ultrasonified for 2 min. This solution was extracted with ethyl acetate (4

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x 20 mL), the combined organic extracts were dried over anhydrous sodium sulfate,

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filtered, and finally evaporated again to dryness. Prior to HPLC-MS/MS analysis, the

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residue was dissolved in water/acetonitrile (1 mL; 9/1, v/v) by ultrasonification and the

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sample was membrane filtered (Whatman Spartan® 13, 0.45 µm; GE Healthcare,

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Freiburg, Germany).

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Extraction of Free Phenolic Acids from Process Intermediates (Unboiled

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Wort, Cast Wort, Green Beer) and Ready-to-drink Beer and Quantitation by

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Stable Isotope Dilution Assays (SIDAs). For the extraction of free phenolic acids

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from liquid samples, a method developed by Czerny et al.29 was modified. Briefly,

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unboiled wort, cast wort, green beer, or ready-to-drink beer (20 - 50 mL) were first

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filtered and, afterwards, the isotopically labeled internal standards [2H6]-cinnamic

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acid, [13C3]-p-coumaric acid, and [2H3]-ferulic acid (amounts determined in preliminary

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experiments) were added and stirred for equilibration for 15 min at room temperature.

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To avoid gel formation inhibiting phase separation during solvent extraction, the 6 ACS Paragon Plus Environment

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original method was modified by adding a protein precipitation step with Carrez

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solution I (aqueous solution of K4[Fe(CN)6] * 3 H2O; c = 0.172 g/mL) and Carrez

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solution II (aqueous solution of ZnSO4 * H2O; c = 0.534 g/mL; addition of 2 - 5 mL of

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each solution). After 20 min, the samples were filtered and the filtrate (10 - 20 mL,

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depending on the sample) was filled up to a volume of approximately 25 mL with

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water and extracted with ethyl acetate (4 x 25 mL). Finally, the combined extracts

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were dried over anhydrous sodium sulfate, filtered, and the supernatant was removed

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by means of a rotary evaporator (35 °C, 20 mbar). Prior to HPLC-MS/MS analysis,

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the residue was dissolved in water/acetonitrile (1 mL; 9/1, v/v) by ultrasonification and

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the sample was membrane filtered (Whatman Spartan® 13, 0.45 µm).

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Extraction of Volatile Vinyl Aromatics from Kiln-dried Malts and Quantitation

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by Stable Isotope Dilution Assays (SIDAs). To the finely ground malt powder (50

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g), diethyl ether (3 x 200 mL) and the isotopically labeled internal standards [2H3]-2-

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methoxy-4-vinylphenol, [2H8]-styrene, and [2H4]-4-vinylphenol (amounts determined in

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preliminary experiments) were added and the sample was stirred for extraction (3 x 2

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h) at room temperature. The combined extracts were dried over anhydrous sodium

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sulfate, filtered, and the solution was concentrated to ~100 mL by distilling off the

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solvent at 38 °C using a Vigreux column (60 cm x 1 cm i.d.). To remove the non-

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volatile material, the extract was subjected to a high vacuum distillation using the

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solvent assisted flavor evaporation (SAFE) technique.30 Finally, the distillate was

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concentrated to ~250 µL by micro-distillation,31 and the vinyl aromatics were

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quantitated via GC-MS (for 2-methoxy-4-vinylphenol and 4-vinylphenol) or GC/GC-

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MS (for styrene), respectively. GC/GC-MS was used due to the fact that styrene was

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present in low amounts and that in some samples co-elution with other major

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components was observed. In this way, low limits of detection and of quantitation

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were achieved. 7 ACS Paragon Plus Environment


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Extraction of Volatile Vinyl Aromatics from Process Intermediates (Unboiled

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Wort, Cast Wort, Green Beer) and Ready-to-drink Beer and Quantitation by

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Stable Isotope Dilution Assays (SIDAs). For extraction of vinyl aromatics, the liquid

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samples unboiled wort, cast wort, green beer, and ready-to-drink beer (2 - 10 mL for

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the determination of 2-methoxy-4-vinylphenol and 4-vinylphenol, filled up to

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approximately 25 mL with water; 250 mL for the determination of styrene) were

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filtered and the isotopically labeled internal standards [2H3]-2-methoxy-4-vinylphenol,

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[2H8]-styrene,

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experiments) were added. After extraction with diethyl ether (4 x 25 mL and 4 x 250

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mL, respectively), the combined organic phases were dried over anhydrous sodium

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sulfate, filtered, and for styrene quantitation concentrated to ~100 mL by distilling off

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the solvent at 38 °C using a Vigreux column (60 cm x 1 cm i.d.). After removing the

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non-volatile material by SAFE distillation,30 the extracts were concentrated to ~250

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µL by micro-distillation31 and used for quantitation via GC-MS (2-methoxy-4-

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vinylphenol and 4-vinylphenol) or GC/GC-MS (styrene), respectively.

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and

[2H4]-4-vinylphenol

(amounts

determined

in

preliminary

High Performance Liquid Chromatography-Tandem Mass Spectrometry

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(HPLC-MS/MS)

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HPLC-MS/MS was performed by means of a triple-quadrupole tandem mass

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spectrometer (TSQ Quantum Discovery; Thermo Electron, Dreieich, Germany)

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coupled to a Surveyor high-performance liquid chromatography system (Thermo

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Finnigan, Egelsbach, Germany) equipped with a thermostated (20 °C) autosampler

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(Thermo Finnigan). Separation was performed using a Synergi 4u Fusion RP column

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(150 mm x 2 mm i.d.) connected to a C18 pre-column (4 mm x 2 mm i.d.; both

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Phenomenex, Aschaffenburg, Germany) and a mobile phase consisting of an

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aqueous formic acid solution (0.1%; A) and a formic acid solution in acetonitrile

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(0.1%; B). The flow rate was 0.2 mL/min with an elution gradient of 90% A and 10% 8 ACS Paragon Plus Environment

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B at the beginning, raised to 65% B and 35% A during 30 min, and finally to 100% B

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within 2 min and kept for 3 min. Precursor ions were generated using positive

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electrospray ionization (ESI+) with a spray voltage of 3500 V, sheath gas pressure of

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35 arb, auxiliary gas pressure of 5 arb, and a capillary temperature of 320 °C. The

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mass spectrometer was operated in the single reaction monitoring (SRM) mode. The

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most intensive product ion for each compound was used as quantifier and the

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second intensive one was used as qualifier (Table 1).

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Response factors (Rf; Table 1) were calculated by analyzing mixtures of known

187

amounts of the unlabeled analyte and the corresponding labeled standard in five

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different ratios (5:1, 3:1, 1:1, 1:3, 1:5) showing a good linearity for all analytes (R2 =

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0.99) in the applied range.

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Gas Chromatography-Mass Spectrometry (GC-MS) and Two Dimensional

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Gas Chromatography-Mass Spectrometry (GC/GC-MS). For the quantitation of 2-

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methoxy-4-vinylphenol and 4-vinylphenol, GC-MS was performed using a Varian 431

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GC (Darmstadt) equipped with a DB-FFAP column (30 m x 0.32 mm i.d., 0.25 µm

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film thickness; J&W Scientific; Agilent, Waldbronn, Germany) and coupled to a Varian

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MS 220. Mass spectra were generated in the chemical ionization mode at 70 eV

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using methanol as the reactant gas. Samples were injected by means of a Combi

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PAL autosampler (CTC Analytics, Zwingen, Switzerland).

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For quantitation of styrene, GC/GC-MS was applied using a ThermoQuest GC

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Trace 2000 (Frankfurt, Germany) equipped with a DB-FFAP column in the first

200

dimension coupled to a Varian GC CP 3800 equipped with an OV-1701 column (both

201

30 m x 0.25 mm i.d., 0.25 µm film thickness; J&W Scientific) in the second

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dimension. Heart cuts were transferred to the second column by means of a moving

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capillary stream switching system and the volatiles were cryo-focused with a cold trap

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cooled to - 100 °C with liquid nitrogen. The second GC was coupled to a Varian ion 9 ACS Paragon Plus Environment


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trap MS Saturn 2000 and mass spectra were generated in the chemical ionization

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mode at 70 eV using methanol as reactant gas. Samples were injected by means of

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a Combi PAL autosampler (CTC Analytics).

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Response factors (Rf; Table 1) were determined by analyzing mixtures of known

209

amounts of the labeled and unlabeled compound in five different ratios (5:1, 3:1, 1:1,

210

1:3, 1:5; v/v) either by GC-MS or GC/GC-MS, respectively.

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Determination of Dry Mass of Kiln-dried Malts. Finely ground malt powder (10

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g) was added to sea sand (25 g) in an evaporating dish and mixed thoroughly. The

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sample was dried in a drying oven at 102 °C for 5 h. After cooling down to room

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temperature in an exsiccator, the dry mass was determined by weight difference of

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the sample before and after drying.

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RESULTS AND DISCUSSION

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First of all, the concentrations of the key odorants8 2-methoxy-4-vinylphenol and 4-

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vinylphenol as well as the toxicologically relevant styrene were analyzed in eight

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commercial wheat beers. Interestingly, beers showing higher amounts of the two

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desired aroma-active phenols also revealed elevated concentrations of undesired

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styrene (samples 1-6; Table 2). In contrast, sample 7 showing a very low

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concentration of styrene, revealed in parallel very low concentrations of the odorants.

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These data pointed to a similar formation mechanism of styrene from its precursor

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free cinnamic acid analogous to the well-known pathway of 4-vinylphenol from p-

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coumaric acid and of 2-methoxy-4-vinylphenol from ferulic acid, already discussed in

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the literature.13,25 Additionally, to the high concentrations of the desired aroma-active

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phenols, sample 1 elicited the most pronounced overall wheat beer aroma, while

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sample 7 with the low amounts of 2-methoxy-4-vinylphenol and 4-vinylphenol showed 10 ACS Paragon Plus Environment

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the least pronounced aroma, evaluated by a sensory panel. It is also noteworthy that

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sample 8 (Table 2) revealed comparatively low concentrations of the desired aroma-

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active phenols, while the styrene concentration (24.4 µg/L) was almost as high as in

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sample 1. Sample 8 was an alcohol reduced wheat beer, thus, it might be assumed

234

that it was manufactured by limited fermentation. An explanation might be that

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cinnamic acid can be quickly metabolized by yeasts, while the formation of 2-

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methoxy-4-vinylphenol and 4-vinylphenol from their respective precursors need

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longer times.13,25

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These results prompted us to have a deeper insight into the brewing process

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starting with the malts as raw materials, followed by the process intermediates

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(unboiled wort, cast wort, green beer) and the ready-to-drink beer (Figure 2), to i)

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evaluate the input of free phenolic acids as well as of vinyl aromatics from the malts

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into the brewing process, ii) to identify the key steps responsible for the

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decarboxylation of the free phenolic acids acting as precursors during the

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manufacturing process, and iii) to determine the contribution of each step to the final

245

amounts of vinyl aromatics in the beer. Theoretically, the decarboxylation might occur

246

during the thermal influence during mashing and wort boiling or due to the enzymatic

247

influence during fermentation. Consequently, two different wheat beers were

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produced in co-operation with an industrial brewery using either the yeast

249

Saccharomyces cerevisiae strain W68 (normal Pof+-activity; WB A) or a special yeast

250

without Pof+-activity (WB B).

251

Concentrations of Free Phenolic Acids and Vinyl Aromatics in Kiln-dried

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Malts. To evaluate the input of free phenolic acids and their decarboxylation products

253

from the malts into the brewing process, the phenolic acids as well as the volatile

254

vinyl aromatics were analyzed in kiln-dried wheat and barley malts. For the

255

quantitation of free phenolic acids, a new HPLC-MS/MS method on the basis of 11 ACS Paragon Plus Environment


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stable isotope dilution assays were developed.32 Application of this method to kiln-

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dried wheat malt and kiln-dried barley malt of WB A revealed ferulic acid as the most

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abundant free phenolic acid (4.43 mg/kg dry mass and 2.94 mg/kg dry mass,

259

respectively; Table 3). Its decarboxylation product 2-methoxy-4-vinylphenol was also

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the most abundant vinyl aromatic in the kiln-dried wheat malt (42.2 µg/kg dry mass)

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and in the kiln-dried barley malt (46.4 µg/kg dry mass) (Table 3). p-Coumaric acid

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revealed concentrations of 0.73 mg/kg dry mass (kiln-dried wheat malt) and 0.91

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mg/kg dry mass (kiln-dried barley malt), respectively, while the amounts of its

264

decarboxylation product 4-vinylphenol were low with 5.52 µg/kg dry mass in the

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wheat malt and 3.54 µg/kg dry mass in the kiln-dried barley malt. The concentration

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of cinnamic acid was higher compared to p-coumaric acid, with 1.17 mg/kg dry mass

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in the kiln-dried wheat malt and 1.34 mg/kg dry mas in the kiln-dried barley malt.

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Styrene, the toxicologically relevant decarboxylation product of cinnamic acid, also

269

showed higher concentrations compared to 4-vinylphenol (about 11 µg/kg dry mass

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for both malts) (Table 3).

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Ferulic acid was also the most abundant free phenolic acid in the kiln-dried wheat

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malt (2.35 mg/kg dry mass) and in the kiln-dried barley malt (2.73 mg/kg dry mass) in

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the batches used for the production of WB B, but the concentrations were lower

274

compared to the malts used for WB A (cf. Tables 3 and 4).

275

Input of Free Phenolic Acids and Vinyl Aromatics from the Malts into the

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Brewing Process. Next, the process intermediates produced from the previously

277

described malts were analyzed in regard to their free phenolic acids as well as the

278

corresponding decarboxylation products. In the unboiled wort of WB A, 2400 µg of

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ferulic acid/L, 1290 µg of p-coumaric acid/L, and 276 µg of cinnamic acid/L were

280

found (Table 5), while in WB B the amounts were 2290 µg of ferulic acid/L, 1230 µg

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of p-coumaric acid/L, and 259 µg of cinnamic acid/L (Table 6). In consideration of the 12 ACS Paragon Plus Environment

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ratios of wheat to barley malt and of water to malt used for mashing as well as the

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volume of water used for sparging, these analyzed concentrations were clearly higher

284

as they could be explained only by a transfer of the free phenolic acids from the

285

malts, even if the transfer rate would be 100%. Thus, during the process of mashing,

286

an extensive release of bound phenolic acids took place as already reported in many

287

studies.5,22,23,33,34 It is noteworthy that ferulic acid and p-coumaric acid were either

288

released to a much higher extent during mashing in comparison to cinnamic acid or

289

that bound cinnamic acid is less abundant in the malts and fragments solubilized in

290

the mash compared to ferulic and p-coumaric acid. Although the concentrations of

291

free cinnamic and ferulic acid were higher in the wheat and barley malts of WB A

292

compared to WB B (cf. Tables 3 and 4), their concentrations in the unboiled wort of

293

WB A and WB B were almost similar (cf. Tables 5 and 6). Hence, there is not

294

necessarily a correlation between the amounts of free phenolic acids in the malts and

295

in the unboiled wort, most likely due to the extensive release of bound phenolic acids

296

during mashing.

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Regarding the vinyl aromatics, 62.8 µg of 2-methoxy-4-vinylphenol/L and 20.8 µg

298

of 4-vinylphenol/L were detected in the unboiled wort of WB A (Table 5), while WB B

299

showed concentrations of 55.4 µg of 2-methoxy-4-vinylphenol/L and 23.5 µg of 4-

300

vinylphenol/L, respectively (Table 6). The concentration of styrene was < LoD in both

301

samples. However, according to the recipe, the theoretically (based on a simple

302

transfer from the malt into the unboiled wort) calculated concentrations of 2-methoxy-

303

4-vinylphenol and 4-vinylphenol in the unboiled wort were lower compared to the

304

analyzed ones. Obviously, a moderate formation of these compounds occurred

305

already during mashing in both samples. In contrast, the calculated amount of

306

styrene was higher than the analyzed one, which indicated that it was probably

307

evaporated during the mashing process. 13 ACS Paragon Plus Environment


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Formation of Vinyl Aromatics from Free Phenolic Acids during Wheat Beer

309

Production. To determine the contribution of the further steps of wheat beer

310

production to the formation of the aroma-active vinyl aromatics 2-methoxy-4-

311

vinylphenol and 4-vinylphenol as well as of the toxicologically relevant styrene, the

312

concentrations of these compounds and of their precursors were analyzed in the cast

313

wort, green beer, and ready-to-drink beer.

314

In the cast wort of WB A, the amounts of all vinyl aromatics moderately increased

315

compared to the unboiled wort, while at the same time the concentrations of the

316

respective free phenolic acids decreased (Table 5). This formation of vinyl aromatics

317

was possibly due to a thermal decarboxylation of the free phenolic acids. Hence, the

318

concentrations of 2-methoxy-4-vinylphenol increased by a factor of 2.5 to 158 µg/L

319

and of 4-vinylphenol by a factor of 2 to 44.5 µg/L, while the amount of the undesired

320

styrene was < LoQ. Of course, also the evaporation of water during wort boiling has

321

to be taken into account, but it only explains an increase of vinyl aromatics by

322

approximately a factor of 1.1 (according to the process).

323

In the green beer of WB A, the concentrations of the vinyl aromatics considerably

324

increased to 1770 µg of 2-methoxy-4-vinylphenol/L (factor 11) and to 888 µg of 4-

325

vinylphenol/L (factor 20). Due to their positive impact on the overall aroma, these

326

high concentrations are desired.8 However, also the amount of the toxicologically

327

relevant styrene clearly increased to 33.7 µg/L (Table 5).

328

At the same time, the concentrations of the respective precursors decreased to

329

only 10.4 µg of ferulic acid/L and to 10.5 µg of p-coumaric acid/L, while cinnamic acid

330

was not detectable any more. This conversion of free phenolic acids to vinyl

331

aromatics can only be explained by the decarboxylase activity7,10,35 during

332

fermentation using the top fermenting yeast Saccharomyces cerevisiae strain W68.

333

Comparing the influence of thermal (unboiled wort to cast wort) and enzymatic (cast 14 ACS Paragon Plus Environment

Page 14 of 35

Page 15 of 35


334

wort to green beer) decarboxylation on the formation of vinyl aromatics, it was clearly

335

demonstrated that the enzyme was mainly responsible for this reaction, while

336

elevated temperatures only showed a minor contribution.

337

In the ready-to-drink beer, which was fermented a second time, but now with a

338

bottom fermenting yeast, the concentrations of vinyl aromatics did not increase any

339

more. This might be explained with the low amounts of free phenolic acids remaining

340

for a further decarboxylation and with the fact that the bottom fermenting yeast was

341

not able to convert free phenolic acids due to the lack of the decarboxylase.12

342

In the cast wort of WB B, also a moderate increase of the aroma-active vinyl

343

aromatics was observed to 195 µg of 2-methoxy-4-vinylphenol/L (factor 3.5) and to

344

67.1 µg of 4-vinylphenol/L (factor 2.9), respectively. Styrene was still not detectable

345

in the cast wort (Table 6). Consequently, also the amount of free cinnamic acid did

346

not decrease; quite the contrary, even a slight increase was observed in comparison

347

to the unboiled wort. While the amount of free p-coumaric acid (1260 µg/L) remained

348

almost unchanged, the concentration of free ferulic acid clearly declined to 1810 µg/L

349

(Table 6).

350

Contrary to WB A, the amounts of vinyl aromatics in the green beer of WB B did

351

not increase, revealing concentrations of 199 µg of 2-methoxy-4-vinylphenol/L and

352

62.4 µg of 4-vinylphenol/L, while styrene was still not detectable (Table 6). The much

353

lower concentrations of vinyl aromatics in the green beer of WB B were based on the

354

special strain (without decarboxylase activity) of Saccharomyces cerevisiae used for

355

fermentation. Again, two perspectives have to be considered: the positive one, the

356

reduction of the toxicologically relevant styrene, but also the negative one, the

357

simultaneously lowered amounts of the desired aroma-active compounds 2-methoxy-

358

4-vinylphenol and 4-vinylphenol. Consequently, this beer lacked the special and

359

unique wheat beer aroma attributes induced by these two compounds ending in a 15 ACS Paragon Plus Environment


Page 16 of 35

360

less pronounced overall wheat beer aroma. In parallel with the low concentrations of

361

the decarboxylation products, high amounts of the corresponding free phenolic acids

362

(1820 µg of ferulic acid/L, 998 µg of p-coumaric acid/L, and 154 µg of cinnamic

363

acid/L) remained in the green beer of WB B, which were considerably higher in

364

comparison to the green beer of WB A (Tables 5 and 6).

365

In the ready-to-drink beer of WB B, the concentrations of free phenolic acids as

366

well as of vinyl aromatics did not change remarkably, simultaneous to WB A. Styrene

367

could be detected in this sample, but its amount was below the limit of quantitation

368

(0.80 µg/L). The amounts of the other vinyl aromatics, 2-methoxy-4-vinylphenol (158

369

µg/L) and 4-vinylphenol (46.7 µg/L), were very low, indicating that no further

370

decarboxylation of free phenolic acids took place during the secondary fermentation.

371

Thus, the amounts of the corresponding free phenolic acids stayed comparatively

372

high (Table 6).

373

Summing up, the formation of vinyl aromatics during wheat beer brewing mainly

374

occurred during fermentation and to a much lesser extent during mashing and wort

375

boiling. The amounts of the desired aroma-active compounds 2-methoxy-4-

376

vinylphenol and 4-vinylphenol as well as of the undesired toxicologically relevant

377

styrene were mainly dependent on the yeast applied for fermentation. The use of a

378

Saccharomyces

379

decarboxylase activity, yielded in a clearly lower styrene concentration in the ready-

380

to-drink beer, but, in parallel, lowered concentrations of 2-methoxy-4-vinylphenol and

381

4-vinylphenol ending up with odor activity values (= ratio of concentration divided by

382

the odor threshold) of 2 and < 1, respectively. Thus, a reduced clove-like aroma

383

impression and a less pronounced overall wheat beer aroma were proven by the

384

sensory panel.

cerevisiae strain without Pof+-activity, meaning


a

lack

of

Page 17 of 35


385

The input of free phenolic acids from malts into the brewing process was shown to

386

be less important, as an extensive release of bound phenolic acids occurred during

387

mashing. The analyzed concentrations of free phenolic acids and vinyl aromatics in

388

the malts were in accordance with literature data. For example, Samaras et al.36

389

detected 3.58 mg of ferulic acid/kg dry mass and 0.85 mg of p-coumaric acid/kg dry

390

mass in pale malt as well as 3.76 mg of ferulic acid/kg dry mass and 1.03 mg of p-

391

coumaric

392

decarboxylation product of ferulic acid, was only detected in roasted malts, e.g., black

393

malt (336 µg/kg dry mass), chocolate malt (267 µg/kg dry mass), and roasted malt

394

(439 µg/kg).36 These concentrations were clearly higher compared to our study.

395

However, the roasted malts were kiln-dried at high temperatures (220 - 229 °C),

396

which facilitates the decarboxylation of free phenolic acids.9 In pale and lager malts,

397

which were similar to those analyzed in our study, Samaras et al., contrary to our

398

work, did not detect any 2-methoxy-4-vinylphenol, which might be explained by the

399

capillary electrophoresis-diode array used in their study. Also, other groups reported

400

about p-coumaric and ferulic acid concentrations in malts, which were comparable to

401

our work.20,37,38

acid/kg

dry

mass

in

lager

malt.

2-Methoxy-4-vinylphenol,

the

402

Studies about cinnamic acid in cereals are scarcely available, however, Hithamani

403

and Srinivasan39 detected very high concentrations in native wheat (27.4 mg/kg) and

404

in roasted wheat (34.1 mg/kg), while Comino et al.40 were not able to detect any

405

cinnamic acid in the insoluble cell wall fraction from wheat and hull less barley

406

endosperm flours. The reasons for these different results could be the use of different

407

wheat and barley varieties for analysis, the use of different analytical methods

408

(HPLC-diode array detector and GC-MS after derivatization, respectively), or the lack

409

of an isotopically labeled internal standard used for quantitation.



410

Regarding the input and release of phenolic acids during mashing, several studies

411

already showed that this process is dependent on technological parameters. For

412

example, McMurrough et al.34 demonstrated that the optimum mashing-in

413

temperature for the enzymatic release of ferulic acid was 45 °C, while a temperature

414

of 65 °C led to a significant decrease. Similar results were obtained by Schwarz et

415

al.,24,25 reporting that a temperature of 45 °C and a pH value of 5.4 - 6.6 were optimal

416

conditions for the release of cinnamic, p-coumaric, and ferulic acid during mashing,

417

with the highest concentrations of free phenolic acids in the mash after 120 and 180

418

min. Vanbeneden et al.23 showed that the optimal pH value for an enzymatic release

419

of ferulic acid during mashing was 5.8 at an optimal temperature of 40 °C. Thereby,

420

not only feruloyl esterase or rather cinnamoyl esterase activity was reported to be

421

important, but also the activity of other arabinoxylan degrading enzymes, like

422

endoxylanase, α-L-arabinofuranosidase, and β-D-xylosidase. Furthermore, the

423

stirring regime, grist coarseness, and mash thickness influenced the release of bound

424

phenolic acids as well.23

425

Coghe et al.33 reported that the amount of ferulic acid in the mash was also

426

dependent on the malted wheat cultivar. Further, they found that from barley malt,

427

ferulic acid was mainly released during mashing, while from wheat malt the release

428

mainly occurred during the fermentation process. Likewise, Vanbeneden et al.22

429

found that the amount of free p-coumaric and ferulic acid transferred into the

430

congress wort was strongly dependent on the barley variety used for brewing.

431

Regarding ferulic acid, they calculated that only 10% of the bound acid was

432

transferred from the malts into the mash and wort, out of which 6 - 9% were still

433

present in bound form as soluble esters, and, hence, not available for

434

decarboxylation. These findings were supported by Nardini and Ghiselli,21 proving

435

that the concentrations of free p-coumaric acid and ferulic acid clearly increased in a 18 ACS Paragon Plus Environment

Page 18 of 35

Page 19 of 35


436

German beer after alkaline hydrolysis of phenolic acid esters to 1.96 mg/L and 16.75

437

mg/L, respectively. In contrast, before hydrolysis, the amounts of the free phenolic

438

acids were only 0.34 mg/L and 1.36 mg/L.

439

However, in the present study, the release of p-coumaric acid and ferulic acid

440

during mashing was shown to be much higher compared to the one of cinnamic acid.

441

In a recent study,26 we proved that some free phenolic acids were unstable during

442

aqueous extraction from malts. Hence, as mashing is also an aqueous extraction of

443

malts, it can be concluded that during this procedure a degradation of some free

444

phenolic acids may also occur, while, at the same time, bound phenolic acids are

445

released. This would suggest that phenolic acids undergo a very dynamic process

446

during mashing, consisting of a degradation of some free phenolic acids on the one

447

side and of a release of bound phenolic acids to different degrees on the other. A

448

significant input of phenolic acids from the hop has not to be expected. For example,

449

Wackerbauer et al.3 determined only low amounts of ferulic acid (13.5 µg/g) and of p-

450

coumaric acid (2.5 µg/g) in hop pellets or in hop umbels of the variety Hallertauer

451

Nordbrauer (14.1 µg of ferulic acid/g and 2.8 µg of p-coumaric acid/g for both before

452

and after alkaline hydrolysis).

453

Up to now, published studies about the decarboxylation of free phenolic acids

454

mainly emphasized specific steps of beer brewing showing that during wort boiling, a

455

moderate, thermally induced decarboxylation occurred,33,34 and that the main

456

formation of vinyl aromatics took place by an enzymatic decarboxylation during

457

fermentation with Saccharomyces cerevisiae strains with Pof+-activity.4,5,33,34,41 Both

458

aspects were impressively proven in our study, which was focused on samples

459

produced under industrial conditions and in industrial scale. Also, to the best of our

460

knowledge, we were the first quantitating the free phenolic acids cinnamic, p-

461

coumaric, and ferulic acid as well as their corresponding decarboxylation products 19 ACS Paragon Plus Environment


462

styrene, 4-vinylphenol, and 2-methoxy-4-vinylphenol throughout the whole brewing

463

process, starting with the raw materials wheat and barley malts, via the process

464

intermediates to the corresponding ready-to-drink-beer produced thereof. All applied

465

methods were based on stable isotopically labeled standards, ensuring very reliable

466

results.

467

In summary, the evaluation of the whole brewing process clearly demonstrated

468

that the decarboxylation is clearly enzymatically driven and not thermally. A reduction

469

of the undesired styrene in wheat beer was possible using a Saccharomyces

470

cerevisiae strain without Pof+-activity. However, this resulted in a less pronounced

471

wheat beer aroma due to significantly lowered amounts of the important aroma

472

compounds 2-methoxy-4-vinylphenol and 4-vinylphenol. As the decarboxylase of

473

Pof+ strains seems to be very unselective and is able to degrade cinnamic acid, p-

474

coumaric acid, and ferulic acid similarly,10 a reduction of styrene in wheat beer by

475

maintaining the typical and by consumers desired aroma remains a challenge. One

476

strategy might be the use of a special fermentation management, e.g., varying

477

parameters like the pitching rate, the fermentation temperature, and the fermentation

478

tank (open or closed).13,14 Another possibility might be the use of malts produced with

479

special malting parameters, as recently suggested.32

480 481 482 483

ACKNOWLEDGMENTS The authors thank Miss Ines Otte and Mr. Sami Kaviani-Nejad for performing the HPLC-MS/MS experiments.

484

This IGF Project of the FEI was supported via AiF within the program for

485

promoting the Industrial Collective Research (IGF) of the German Ministry of

486

Economics and Energy (BMWi), based on a resolution of the German Parliament. 20 ACS Paragon Plus Environment

Page 20 of 35

Page 21 of 35


487

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488

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Hemminki, K. Biomarkers of styrene exposure in lamination workers: levels of O6-

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guanine

phosphoribosyltransferase

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T-lymphocytes.

Artuso, M.; Angotzi, G.; Bonassi, S.; Bonatti, S.; De Ferrari, M.; Gargano, D.;

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Barros, A. A. Antioxidant properties of free, soluble ester and insoluble-bound

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beer. Food Chem. 2004, 84, 137-143.

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of free and bound hydroxycinnamic acids from diverse malted barley (Hordeum

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vulgare L.) cultivars during wort production. J. Agric. Food Chem. 2007, 55, 11002-

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11010.

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of phenolic flavour precursors during wort production: influence of process

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parameters and grist composition on ferulic acid release during brewing. Food Chem.

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acids during mashing dependent on temperature, pH, time, and raw materials. J. Am.

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dilution assays for the quantitation of free phenolic acids in wheat and barley and

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malts produced thereof. Eur. Food Res. Technol. 2015, 241, 637-645.

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Vanbeneden, N.; Gils, F.; Delvaux, F.; Delvaux, F. R. Variability in the release

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Jezussek, M. Aroma formation during boiling of brown rice (Oryza sativa L.) as

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well as of leaves of Pandanus amaryllifolius Roxb. (in German). Ph.D. thesis,

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Technical University of Munich, Munich, Germany, 2002

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beef juice by instrumental analyses and sensory studies. J. Agric. Food Chem. 1994,

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42, 2862-2866.

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potent flavor precursor in sourdough (in German). Getreidetechnologie 2006, 60,

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new and versatile technique for the careful and direct isolation of aroma compounds

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from complex food matrices. Eur. Food Res. Technol. 1999, 209, 237-241.

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in Flavor Research; Land, G. G., Nursten, H. E., Eds.; Applied Science Publisher:

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Barking, UK, 1979, pp 79-98.

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modifications on the concentrations of free phenolic acids in wheat and barley malts.

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Brew. Sci. 2015, 68, 93-101.

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acid release and 4-vinylguaiacol formation during brewing and fermentation:

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indications for feruloyl esterase activity in Saccharomyces cerevisiae. J. Agric. Food

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Chem. 2004, 52, 602-608.

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G.; McNulty, N.; Smyth, M. R. Control of ferulic acid and 4-vinyl guaiacol in brewing.

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J. Inst. Brew. 1996, 102, 327-332.

Guth, H.; Grosch, W. Identification of the character impact odorants of stewed

Czerny, M.; Brandt, M. J.; Hammes, W. P.; Schieberle, P. Ferulic acid - a

Engel, W.; Bahr, W.; Schieberle, P. Solvent assisted flavour evaporation - a

Bemelmans, J. Review of isolation and concentration techniques. In Progress

Langos, D.; Granvogl, M.; Gastl, M.; Schieberle, P. Influence of malt

Coghe, S.; Benoot, K.; Delvaux, F.; Vanderhaegen, B.; Delvaux, F. R. Ferulic

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35.

Clausen, M.; Lamb, C. J.; Megnet, R.; Doerner, P. W. PAD1 encodes

590

phenylacrylic acid decarboxylase which confers resistance to cinnamic acid in

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Saccharomyces cerevisiae. Gene 1994, 142, 107-112.

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Antioxidant properties of kilned and roasted malts. J. Agric. Food Chem. 2005, 53,

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8068-8074.

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37.

596

kilning on the antioxidant and pro-oxidant activities of pale malts. J. Agric. Food

597

Chem. 2002, 50, 4925-4933.

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38.

599

modification of the kilning regimen on levels of free ferulic acid and antioxidant

600

activity in malt. J. Agric. Food Chem. 2011, 59, 9335-9343.

601

39.

602

(Triticum aestivum), sorghum (Sorghum bicolor), green gram (Vigna radiata), and

603

chickpea (Cicer arietinum) as influenced by domestic food processing. J. Agric. Food

604

Chem. 2014, 62, 11170-11179.

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40.

606

Characterisation of soluble and insoluble cell wall fractions from rye, wheat and hull-

607

less barley endosperm flours. Food Hydrocolloids 2014, 41, 219-226.

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41.

609

4-ethyl derivatives from hydroxycinnamic acids: occurrence of volatile phenolic

610

flavour compounds in beer and distribution of Pad1-activity among brewing yeasts.

611

Food Chem. 2008, 107, 221-230.

Samaras, T. S.; Camburn, P. A.; Chandra, S. X.; Gordon, M. H.; Ames, J. M.

Woffenden, H. M.; Ames, J. M.; Chandra, S.; Anese, M.; Nicoli, M. C. Effect of

Inns, E. L.; Buggey, L. A.; Booer, C.; Nursten, H. E.; Ames, J. M. Effect of

Hithamani, G.; Srinivasan, K. Bioaccessibility of polyphenols from wheat

Comino, P.; Collins, H.; Lahnstein, J.; Beahan, C.; Gidley, M. J.

Vanbeneden, N.; Gils, F.; Delvaux, F.; Delvaux, F. R. Formation of 4-vinyl and

612



FIGURE CAPTIONS 613

Figure 1. Thermally or enzymatically induced decarboxylation of free phenolic acids

614

to their corresponding vinyl aromatics.

615 616

Figure 2. Scheme of wheat beer production.

617


Page 26 of 35

Page 27 of 35


Table 1. Selected Ions (m/z) of Analytes and Stable Isotopically Labeled Standards as well as Response Factors (Rf) used in Stable Isotope Dilution Assays analyte compound

standard Rf

precursor

product

isotope

precursor

product

ion (m/z)

ions (m/z)a

labeling

ion (m/z)

ions (m/z)a

cinnamic acidb

149

103, 131

2

155

108, 136

0.99

p-coumaric acidb

165

119, 147

13

C3

168

121, 150

0.85

ferulic acidb

195

145, 177

2

H3

198

145, 180

0.87

analyte

H6

isotope

standard Rf

(m/z)

labeling

(m/z)

styrenec

105

2

H8

113

0.74

4-vinylphenold

121

2

H4

125

0.85

2-methoxy-4-vinylphenold

151

2

H3

154

0.94

a c

Product ions in bold were used for quantitation.

b

Analyzed via HPLC-MS/MS.

Analyzed via GC/GC-MS. d Analyzed via GC-MS.



Page 28 of 35

Table 2. Concentrations of Aroma-Active Vinyl Aromatics and Toxicologically Relevant Styrene in Commercial Wheat Beers concna (µg/L) of sample


4-vinylphenol

styrene

1

2020

882

27.7

2

1650

719

32.6

3

1360

1020

25.1

4

1270

639

29.3

5

1050

695

18.0

6

626

620

15.0

7

159

8

795

a

59.8

355

Mean values of duplicates, differing not more than ± 8%.


1.67

24.4

Page 29 of 35


Table 3. Concentrations of Free Phenolic Acids and Vinyl Aromatics in Kiln-dried Wheat and Barley Malt Used for the Production of WB A concna (µg/kg dry mass) in analyte kiln-dried wheat malt

kiln-dried barley malt

1170 (± 35.2)

1340 (± 104)

730 (± 78.1)

907 (± 162)

ferulic acid

4430 (± 223)

2940 (± 143)

styrene

10.6 (± 0.7)

11.0 (± 1.0)

cinnamic acid p-coumaric acid

4-vinylphenol 2-methoxy-4-vinylphenol a

5.52 (± 0.40) 42.2 (± 7.9)

3.54 (± 0.78) 46.4 (± 0.8)

Mean values of triplicates. Relative standard deviations in parentheses.



Table 4. Concentrations of Free Phenolic Acids and Vinyl Aromatics in Kiln-dried Wheat and Barley Malt Fermented Used for the Production of WB B concna (µg/kg dry mass) in analyte kiln-dried wheat malt

kiln-dried barley malt

cinnamic acid

535 (± 44.6)

1150 (± 58.4)

p-coumaric acid

750 (± 55.6)

1120 (± 67.1)

2350 (± 302)

2730 (± 105)

ferulic acid

styrene

3.68 (± 0.23)

4.30 (± 0.42)

4-vinylphenol

3.85 (± 0.27)

3.08 (± 0.40)

2-methoxy-4-vinylphenol a

12.9 (± 0.6)

22.1 (± 1.0)



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Table 5. Concentrations of Free Phenolic Acids and Vinyl Aromatics in Process Intermediates of Wheat Beer A (Yeast Strain W68) concna (µg/L) in analyte unboiled wort cinnamic acid

cast wort

green beer

ready-to-drink beer

< LoDb

< LoQc

276 (± 7)

258 (± 1)

p-coumaric acid

1290 (± 80)

1130 (± 90)

10.5 (± 1.2)

20.9 (± 6.1)

ferulic acid

2400 (± 400)

1880 (± 30)

10.4 (± 1.0)

16.8 (± 4.1)

styrene

< LoDd

< LoQe

4-vinylphenol

20.8 (± 0.8)

44.5 (± 1.2)


62.8 (± 1.0)

33.7 (± 1.8)

158 (± 2)

a


e

LoQ = 0.80 µg/L.

b

888 (± 58)

736 (± 49)

1770 (± 22)

1790 (± 170)

LoD = 4.0 µg/L.

31

ACS Paragon Plus Environment

28.3 (± 1.1)

c

LoQ = 6.6 µg/L.

d

LoD = 0.50 µg/L.


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Table 6. Concentrations of Free Phenolic Acids and Vinyl Aromatics in Process Intermediates of Wheat Beer B (Special Yeast Strain) concna (µg/L) in analyte unboiled wort

cast wort

259 (± 6)

282 (± 9)

p-coumaric acid

1230 (± 60)

1260 (± 100)

998 (± 59)

1020 (± 60)

ferulic acid

2290 (± 300)

1810 (± 160)

1820 (± 150)

1770 (± 160)

< LoDb

< LoDb

< LoDb

< LoQc

67.1 (± 4.5)

62.4 (± 4.2)

46.7 (± 1.8)

cinnamic acid

styrene 4-vinylphenol

23.5 (± 1.3)


55.4 (± 0.9)

a

195 (± 15)

green beer 154 (± 9)

199 (± 1)

ready-to-drink beer 148 (± 11)

158 (± 2)

Mean values of triplicates. Relative standard deviations in parentheses. b LoD = 0.50 µg/L. c LoQ = 0.80 µg/L.

32

ACS Paragon Plus Environment

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COOH HO

HO OCH3

OCH3


ferulic acid COOH HO

HO

p-coumaric acid p-cumaric acid

4-vinylphenol

COOH

cinnamic acid

styrene

Figure 1 618



619 620

Figure 2


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TOC graphic


Studies on the Simultaneous Formation of Aroma-Active and

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