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Yeast Metabolites of Glycated Amino Acids in Beer Michael Hellwig, Falco Beer, Sophia Witte, and Thomas Henle J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01329 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 12, 2018

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Journal of Agricultural and Food Chemistry

Yeast Metabolites of Glycated Amino Acids in Beer

Michael Hellwig, Falco Beer, Sophia Witte, Thomas Henle

Chair of Food Chemistry, Technische Universität Dresden, D-01062 Dresden, Germany

Corresponding author: T. Henle Tel.: +49-351-463-34647 Fax: +49-351-463-34138 Email: [email protected]

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Abstract

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Glycation reactions (Maillard reactions) during the malting and brewing processes are

3

important for the development of the characteristic color and flavor of beer. Recently, free

4

and protein-bound Maillard reaction products (MRPs) such as pyrraline, formyline, and

5

maltosine were found in beer. Furthermore, these amino acid derivatives are metabolized by

6

Saccharomyces cerevisiae via the Ehrlich pathway. In this study, a method was developed for

7

quantitation of individual Ehrlich intermediates derived from pyrraline, formyline, and

8

maltosine. Following synthesis of the corresponding reference material, the MRP-derived new

9

Ehrlich alcohols pyrralinol (up to 207 µg/L), formylinol (up to 50 µg/L) and maltosinol (up to

10

6.9 µg/L) were quantitated for the first time in commercial beer samples by RP-HPLC-

11

MS/MS in the MRM mode. This is equivalent to ca. 20-40% of the concentrations of the

12

parent glycated amino acids. The metabolites were almost absent from alcohol-free beers and

13

malt-based beverages. Two previously unknown valine-derived pyrrole derivatives were

14

characterized and qualitatively identified in beer. The metabolites investigated represent new

15

process-induced alkaloids that may influence brewing yeast performance due to structural

16

similarities to quorum sensing and metal-binding molecules.

17 18

Keywords

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Maillard reaction; glycation; beer; Saccharomyces cerevisiae; Ehrlich pathway; pyrraline;

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formyline; maltosine

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Introduction

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In the Maillard reaction (synonyms: non-enzymatic browning, glycation), amino and imino

24

groups at the N-termini and side-chains of free amino acids, peptides and proteins react with

25

the carbonyl groups of reducing sugars under formation of Amadori products (“sugar amino

26

acids”). These Amadori products can decompose in the second stage of the reaction, and 1,2-

27

dicarbonyl compounds such as 3-deoxyglucosone (3-DG) are generated. The final stage of the

28

reaction is characterized by the formation of stable “advanced glycation end products

29

(AGEs)” resulting from the reaction of 1,2-dicarbonyl compounds with amino acids and

30

proteins. AGEs comprise a heterogeneous group of chemically modified (“glycated”) amino

31

acids, mainly derivatives of lysine and arginine such as N-ε-carboxymethyllysine (CML),

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pyrraline 1a, formyline 1b, and maltosine 2 (Figure 1). High-molecular weight structures

33

responsible for the dark color of heated protein/sugar-mixtures, called melanoidins, are also

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formed in the late stage of the reaction.1,2

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The Maillard reaction is an important contributor to flavor and color of malt and beer. In the

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brewing process, there are several stages that allow for generation and degradation of

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Maillard reaction products (MRPs). First, dry heating of germinated cereal grains in the

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kilning process leads to the formation of protein-bound Amadori products, AGEs and

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melanoidins.3 Proteolytic processes during germination and mashing are responsible for

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significant amounts of free amino acids in malt and wort.4,5 The second process is wort

41

boiling, where malt components encounter an aqueous environment that allows chemical

42

reactions different to those in dry state. In this stage, the predominant part of 1,2-dicarbonyl

43

compounds, mainly 3-DG, is formed, and the reaction conditions also allow for isomerization

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of 3-DG to 3-deoxygalactosone (3-DGal) via the unsaturated intermediate 3,4-

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dideoxyglucosone-3-ene (3,4-DGE; 3-deoxyhexosone interconversion).6,7 Amino acids and

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dicarbonyl compounds can undergo Strecker degradation, i.e., the conversion of the amino 3 ACS Paragon Plus Environment


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acid to an aldehyde with one C-atom less than the parent amino acid. The most important

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Strecker aldehydes in malt are 2-methylbutanal (from isoleucine) and 3-methylbutanal (from

49

leucine). These aldehydes are present in low amounts in the free form in dry foods (ca. 1

50

mg/kg), but can be released from transient precursors through the addition of water yielding

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concentrations up to 63 mg/kg 3-methylbutanal in Munich malt.8 The same release of Strecker

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aldehydes from their precursors may take place during mashing.

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The third process with importance to MRPs is fermentation. Besides its ability to convert

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glucose to ethanol and carbon dioxide, brewer’s yeast Saccharomyces cerevisiae is able to

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transform free amino acids to higher alcohols via the Ehrlich pathway.9 The first step in this

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catabolic pathway is a transamination leading to an α-keto acid such as 3, which is shown for

57

the proposed pathway of metabolization of pyrraline 1a in Figure 1. The α-keto acid is then

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oxidatively decarboxylated to an “Ehrlich aldehyde” 4, which is identical to the above-

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mentioned Strecker aldehyde. The main pathway of metabolization of these aldehydes in beer

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is reduction to the respective Ehrlich alcohols 5; the oxidation to the Ehrlich acids 6 is of

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minor importance.10 Ehrlich alcohols originating from proteinogenic amino acids are found at

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high concentrations in beer. Quantitation of the compounds 2-methylbutanol (0.9-16.0

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mg/L),10 3-methylbutanol (5.6-61.1 mg/L),11,12 methylpropanol (14.5-23.1 mg/L),12 2-

64

phenylethanol (2.3-45 mg/L),11,12,13,14,15 tyrosol (5.3-21.9 mg/L),13,14 and tryptophol (0.1-12.1

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mg/L)13,14 was performed by GC-MS, GC-olfactometry or HPLC-UV. Ehrlich alcohols such

66

as 2-phenylethanol and 3-methylbutanol are among the key aroma compounds of all

67

fermented foods.16 With the Ehrlich pathway, yeast cells can (i) cover their nitrogen

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requirements, (ii) maintain their NAD+/NADH ratio by switching between Ehrlich aldehyde

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oxidation and reduction, and (iii) transform reactive aldehydes to less reactive products.17,18

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More than 50% of the free amino acids phenylalanine and tyrosine can be transformed into

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the respective Ehrlich aldehydes during fermentation of lager beer.14 Higher alcohols, also

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called “fusel alcohols”, were sometimes linked to the symptoms of “hangover” following 4 ACS Paragon Plus Environment

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excessive intake of alcoholic beverages, but this is most probably due to the ethanol

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metabolite acetaldehyde.19 Higher alcohols do not contribute significantly to the overall

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toxicity of alcoholic beverages; the major contributor is ethanol itself.20

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Recently, the metabolization of the lysine derivatives pyrraline 1a, formyline 1b, and

77

maltosine 2 was shown for two strains of S. cerevisiae.21 These products were mainly

78

converted to the corresponding Ehrlich alcohols such as 5 and 8 and α-hydroxy acids such as

79

7 and 9. Metabolization was obviously restricted by a transport phenomenon. While among

80

the free glycated amino acids, only formyline 1b was metabolized to a small extent,

81

dipeptide-bound pyrraline 1a and maltosine 2 were degraded nearly completely indicating a

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decisive impact of peptide transport (Figure 1).21 The occurrence of large amounts of free and

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protein-bound glycated amino acids in different types of beer strongly suggests that these

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products might enter the metabolism of S. cerevisiae.3 In the present work, we intended to get

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first insights into the occurrence of the metabolites in commercial beer samples, thereby

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assessing whether the metabolization of MRPs discovered in a model experiment21 is also of

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relevance in complex fermented foods. Therefore, the respective standard substances had to

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be synthesized and a method for quantitation of the compounds had to be established. Five

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pertinent metabolites and two previously unknown pyrrole compounds derived from valine

90

were synthesized as reference compounds. Pyrralinol 5a, formylinol 5b, and maltosinol 8

91

were quantitated in beer samples for the first time by HPLC-MS/MS in the MRM mode.

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Trace amounts of the α-hydroxy acids derived from pyrraline and formyline (7a and 7b) were

93

detected only in a limited number of samples.

94 95



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Materials and Methods

97 98

Chemicals. The following substances were commercially available: 2-(4-hydroxyphenyl)-

99

ethanol (tyrosol), 2-(indol-3-yl)-ethanol (tryptophol), 5-aminopentanol (Alfa Aesar,

100

Karlsruhe, Germany); trifluoroacetic acid (TFA), methanol MS grade, acetonitrile MS grade,

101

acetic acid (Fisher Scientific, Schwerte, Germany); oxalic acid, sodium acetate trihydrate

102

(Grüssing, Filsum, Germany); 2-phenylethanol, catalase from bovine liver (1 MU/mL), L-

103

amino acid oxidase from Crotalus adamanteus (Type I, dried venom, ≥ 0.3 U/mg), dimethyl

104

sulfoxide (DMSO),

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Karlsruhe, Germany); deuterium oxide, deuterated chloroform, nonafluoropentanoic acid

106

(NFPA),

107

gradient grade methanol and acetonitrile (VWR, Darmstadt, Germany); ninhydrin (Serva,

108

Heidelberg, Germany). Water used for the preparation of solutions and HPLC eluents was

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double-distilled before use (Destamat Bi 18E; QCS GmbH, Maintal, Germany). Pyrraline22 1a

110

and formyline23 1b were prepared according to the respective literature methods. The α-

111

hydroxy acids derived from pyrraline 1a, formyline 1b, and maltosine 3, namely (S)-6-(2’-

112

formyl-5’-hydroxymethylpyrrol-1’-yl)-2-hydroxyhexanoic acid 7a, (S)-6-(2’-formylpyrrol-1’-

113

yl)-2-hydroxyhexanoic

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hexanoic acid 9 and the Ehrlich alcohols 5-hydroxymethyl-1-(5’-hydroxypentyl)-1H-pyrrole-

115

2-carbaldehyde

116

(maltosinol, 8) had previously been synthesized and characterized.21

D-ribose,

D-xylose, L-valine

(Merck, Darmstadt, Germany); methanol-d4 (Roth,

5-aminopentanoic acid (Sigma-Aldrich, Steinheim, Germany); HPLC

acid

7b,

(S)-2-hydroxy-6-(3’-hydroxy-4’-oxo-1H-pyridin-1’-yl)-

(pyrralinol, 5a) and 3-hydroxy-1-(5’-hydroxypentyl)-1H-pyridin-4-one

117 118

Beer samples. Beer samples (20 barley beers, 12 wheat beers, 5 alcohol-free beers) and 7

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malt-based beverages (“malt beer”) were purchased from local retail stores. Such “malt beers”

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from the German market may also contain added sugars (e.g., glucose-fructose syrups, malt 6 ACS Paragon Plus Environment

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extract). Alcohol-free beers are brewed according to the German purity law and do not

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contain added sugars. Only samples that had not yet reached the indicated best-before date

123

were included in the study. All samples were degassed by sonication directly after opening.

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The samples were frozen at -18 °C until analysis.

125 126

Preparation of samples for analysis. Qualitative measurements of beer samples were

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performed after liquid-liquid extraction. Ethyl acetate (10 mL) was added to 25 mL of beer in

128

a 50-mL glass centrifuge tube (Kimax; Sigma-Aldrich). After shaking, the mixtures were

129

centrifuged (room temperature, 2000 rpm, 10 min). The organic layer was collected and the

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aqueous phase was extracted two further times with ethyl acetate in the same way. The

131

combined organic phases were evaporated to near dryness and taken up in 1 mL of a mixture

132

of methanol and water (50/50, v/v).

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For quantitation of Ehrlich alcohols in beer, samples were prepared as follows: Methanol (600

134

µL) was added to 600 µL of a degassed beer sample, and the mixture was stored for 1 h at 4

135

°C. After centrifugation (10,000 rpm, 10 min), the supernatants were removed and either

136

analysed directly by HPLC-UV or, after standard addition, by HPLC-MS/MS. All samples

137

were prepared at least in duplicate.

138 139

Analytical high-pressure liquid chromatography (HPLC) with UV-detection. Analysis of

140

2-phenylethanol, tyrosol, and tryptophol was performed using a low-pressure gradient system

141

consisting of a solvent organizer (K-1500; Knauer, Berlin, Germany), an autosampler (Basic

142

Marathon; Spark Holland, Emmen, Netherlands) a pump (Smartline 1000, Knauer), an online

143

degasser (Knauer), a column oven, and a diode array detector (DAD 2.1L, Knauer).

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Chromatograms were evaluated using the software ClarityChrom version 6.1.0.130 (Chrom

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Tech Inc., Apple Valley, MN). Samples were analyzed at room temperature on a stainless

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steel column (250 mm × 4.6 mm, 5 µm) filled with Eurospher-100 RP-18 material with an 7 ACS Paragon Plus Environment


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integrated guard column (5 mm × 4 mm) of the same material (Knauer). Solvent A was a

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mixture of water, acetonitrile, and TFA (90/10/0.1, v/v/v), and solvent B was a mixture of

149

acetonitrile, water and TFA (90/10/0.1, v/v/v). A gradient was applied (0 min, 10% B; 5 min,

150

10% B; 35 min, 25% B; 37 min, 80% B; 40 min, 80% B; 42 min, 10% B; 47 min, 10% B),

151

and the injection volume was 20 µL. The absorbance was read at 220 nm, 254 nm, and 280

152

nm, simultaneously, and UV spectra were recorded between 190 and 400 nm throughout the

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analytic run. Quantitation of the Ehrlich alcohols was based on external calibration with the

154

commercially available standards.

155 156

Analytical HPLC with DAD and mass-spectrometric detection. System 1 was employed

157

for quantitation of metabolites of pyrraline and formyline as well as tyrosol and tryptophol.

158

The high pressure system 1200 Series (Agilent Technologies, Böblingen, Germany),

159

consisting of a binary pump, an online degasser, a column oven, an autosampler, and a diode

160

array detector was used. Samples were analyzed at 35 °C on a Kinetex 5-µm Biphenyl 100 Å

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column (100 × 2.1 mm, 5 µm) from Phenomenex Ltd. (Aschaffenburg, Germany). As solvent

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A, a solution of 0.075% acetic acid in water and as solvent B, a mixture of 80% methanol and

163

20% solvent A were used at a flow rate of 0.25 mL/min in the gradient mode (0 min, 20% B;

164

16 min, 80% B; 19 min, 80% B; 20 min, 20% B; 28 min, 20% B). The injection volume was 5

165

µL. The mass spectrometer 6410 Triple Quad (Agilent) was connected to the HPLC device.

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Nitrogen was used as the nebulizing gas (nebulizer pressure, 35 psi; gas flow, 11 L/min; gas

167

temperature, 350 °C). The capillary voltage was set at +4000 V in the positive mode and at –

168

4000 V in the negative mode. During routine measurement of beer samples, quantitation of

169

the Ehrlich alcohols was performed in the multiple reaction monitoring (MRM) mode (Table

170

1). Data were acquired and evaluated with the software Mass Hunter B.02.00 (Agilent).

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Quantitation was performed by standard addition. The samples were analysed without

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addition and with two added concentrations of standards. In the first run, 100 µL of 8 ACS Paragon Plus Environment

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precipitated beer sample was mixed with 20 µL of water. In the second run, 100 µL of

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precipitated sample was mixed with 10 µL of water and 10 µL of a standard solution. In the

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last run, 100 µL of precipitated sample was mixed with 20 µL of standard solution. In the

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solution used for standard addition, Ehrlich alcohols had the following concentrations: tyrosol

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(84 mg/L), pyrralinol (1.01 mg/L), tryptophol (1.66 mg/L), formylinol (0.1 mg/L). This

178

system was also used for the qualitative identification of metabolites in the scan, product ion

179

scan and MRM modes (Table S1).

180

System 2 was employed for quantitation of maltosinol 8. The same HPLC and MS systems as

181

above were used. Analytes were separated at a column temperature of 35 °C on a stainless

182

steel column (Zorbax 100 SB-C18; 2.1 × 50 mm, 3.5 µm; Agilent) in the gradient mode (0

183

min, 10% B; 15 min, 66% B; 19 min, 66% B; 20 min, 10% B; 28 min, 10% B). The injection

184

volume was 5 µL. Between 6 and 10 min, the transition 212→126 (80 V, 20 eV) was

185

recorded for quantitation of the analyte, whereas the transitions 212→108 (80 V, 30 eV) and

186

212→ 69 (80 V, 20 eV) were recorded for confirming the presence of the analyte (fragmentor

187

voltage and collision energy are given in parentheses). The dwell time was 300 ms.

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Maltosinol 8 was quantitated via standard addition in the same way as described above for

189

other Ehrlich alcohols. The concentration of 8 in the standard addition solution was 20 µg/L.

190 191

Statistical treatment. The Kolmogorov-Smirnov test was applied for the evaluation of

192

normal distribution of values. Correlations between concentrations of individual Ehrlich

193

alcohols in different beer types were determined by Spearman’s rank correlation analysis.

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These statistical analyses were performed using the software PASW Statistics 18. The limits

195

of detection (LOD) and quantitation (LOQ), respectively, represent the concentrations of the

196

analytes that are necessary to show peaks with signal-to-noise ratios of 3 and 10, respectively.

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Characterization of synthesized compounds. Proton and

C NMR spectra were recorded

199

on an Avance III HDX 500 MHz Ascend device from Bruker (Rheinstetten, Germany) at

200

500.13 MHz (13C, 125.75 MHz) or on an Avance III HD Nanobay (Bruker) at 400.13 MHz

201

(13C, 100.61 MHz). Chemical shifts are given in parts per million (ppm) and referenced to the

202

internal HOD signal (δHOD = 4.70 ppm) or external tetramethylsilane (δTMS = 0.00 ppm).

203

Coupling constants (J) are reported in Hz. Assignments of 1H signals are based on

204

comparison with previously synthesized compounds21 as well as

205

spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), and distortionless

206

enhancement by polarization transfer (DEPT) experiments. Elemental analysis data were

207

obtained on a Vario Micro Cube CHNS elemental analyser (Elementar, Hanau, Germany).

208

Elemental analysis was used to calculate the product content of synthesized compounds. The

209

percentage of nitrogen in the preparation was divided by the theoretical percentage of nitrogen

210

and the content expressed in per cent by weight. Both HPLC-DAD-MS/MS systems were

211

employed for the determination of UV-maxima, molecular mass, and fragmentation behavior

212

of all synthesized compounds.

1

H–1H correlation

213 214

Semi-preparative high-pressure liquid chromatography. This was performed as published

215

previously21 on a Wellchrom system (Knauer) with two HPLC pumps K-1001, an online

216

degasser, a UV-detector K-2501, and a fraction collector K-16. An RP-18 column (Eurospher-

217

100, 300 mm × 8 mm, 5 µm, Knauer) with a guard column (30 mm × 8 mm) was used for

218

fractionations at room temperature. Four different eluents were used during the isolation

219

procedures: Eluent A was a solution of 0.075% acetic acid in water, Eluent B was a mixture

220

of 20% eluent A and 80% methanol, Eluent C was a mixture of water and acetonitrile (90/10,

221

v/v), and Eluent D was a mixture of acetonitrile and water (90/10, v/v). The flow rate was 1.4

222

mL/min. Mixtures for fractionations were dissolved appropriately prior to injection and

223

membrane filtered (0.45 µm). A volume of 2 mL was injected per run. Fractionation of the 10 ACS Paragon Plus Environment

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effluent was based on UV-detection at λ = 293 nm for N-substituted pyrrole-2-carbaldehyde

225

derivatives and at λ = 297 nm for N-substituted 5-hydroxymethylpyrrole-2-carbaldehyde

226

derivatives.

227 228

Action of L-amino acid oxidase on pyrraline 1a and formyline 1b. Based on a literature

229

method,24 either pyrraline22 1a (1.0 mg) or formyline23 1b (1.0 mg), respectively, were

230

dissolved in 1.2 mL 0.075 M TRIS buffer (pH 7.8). Then, 0.9 mg L-amino acid oxidase and

231

10 U catalase were added. The mixture was incubated for 4 h at 37 °C in a water bath.

232

Aliquots (100 µL) of the reaction mixture were taken during the incubation period and added

233

to methanol (100 µL). After centrifugation (10.000 U/min, 4 °C, 10 min), the samples were

234

directly subjected to HPLC-MS/MS (system 2) in the product ion scan mode.

235 236

Synthesis of 5-(2’-formyl-5’-hydroxymethylpyrrol-1’-yl)-pentanal (pyrralinal 4a). Based

237

on a literature method,25 20.0 mg (0.08 mmol) pyrraline 1a was dissolved in 6 mL 0.1 M

238

sodium acetate buffer, pH 5.0. The pH was adjusted to 5.0 with acetic acid. Ninhydrin (70.0

239

mg, 0.39 mmol) was added to the solution. The mixture was heated for 4 min in an oil bath

240

that had been preheated to 100 °C. After cooling, the purple suspension was diluted with 50

241

mL water and filtered. The filtrate was extracted at its pH 4.9 with ethyl acetate (4 × 30 mL),

242

and the combined organic phases were evaporated to dryness using a rotary evaporator. The

243

residue was dissolved in 10 mL of a mixture of water and methanol (50/50, v/v) and subjected

244

to semi-preparative HPLC using the solvents C and D. After injection, a linear gradient was

245

formed ascending from 20% B to 70% B in 20 min. The fraction eluting between 16 and 20.5

246

min was collected (16 runs). The combined fractions were evaporated to dryness, and the

247

white residue was first examined by NMR and then stored at -18 °C in solution.

248

Analytical data: 1H-NMR (500 MHz, CDCl3), δ [ppm]: 1.67 (m, 2H, H-3); 1.76 (m, 2H, H-4);

249

2.48 (td, 2H, J = 1.4 Hz, J = 7.2 Hz, H-2); 4.35 (m, 2H, H-5); 4.65 (s, 2H, H-6’); 6.19 (d, 1H, 11 ACS Paragon Plus Environment


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J = 4.0 Hz, H-4’); 6.84 (d, 1H, J = 4.0 Hz, H-3’); 9.47 (s, 1H, H-1’); 9.74 (t, 1H, J = 1.4 Hz,

251

H-1). HPLC-MS/MS (system 2): tR, 6.3 min; λmax, 297 nm; fragmentation (60 V, 10 eV) of

252

[M + H]+ (m/z 210): 164 (100), 120 (35), 136 (25), 134 (20), 146 (10), 94 (8), 108 (8), 152

253

(7). Yield: 13.0 mg (77%).

254 255

Synthesis of 5-(2’-formylpyrrol-1’-yl)-pentanal (formylinal 4b). The synthesis was

256

performed as for 4a, starting from formyline 1b (10.0 mg, 0.04 mmol) and ninhydrin (40.0

257

mg, 0.22 mmol) in 6 mL of 0.1 M sodium acetate buffer, pH 5.0. The mixture was heated for

258

5 min at 100 °C in a preheated oil bath. After cooling, the purple suspension was diluted with

259

water (50 mL), filtered and extracted at its pH 4.9 with ethyl acetate (3 × 30 mL). The

260

combined organic phases were evaporated to dryness using a rotary evaporator, and the

261

residue was dissolved in 10 mL of a mixture of water and methanol (80/20, v/v). Semi-

262

preparative HPLC was performed using the eluents A and B. After injection, a linear gradient

263

was formed ascending from 40% B to 100% B in 30 min. The fraction eluting between 28 and

264

33 min was collected (14 runs), evaporated to dryness, and stored at -18 °C.

265


266

2.47 (dt, 2H, J = 1.2 Hz, J = 7.2 Hz, H-2); 4.32 (m, 2H, H-5); 6.23 (dd, 1H, J = 2.6 Hz, J =

267

3.9 Hz, H-4’); 6.93 (dd, 1H, J = 1.5 Hz, J = 3.9 Hz, H-3’); 6.94 (d, 1H, J = 2.4 Hz, H-5’); 9.52

268

(d, 1H, J = 0.7 Hz, H-1’); 9.75 (t, 1H, J = 1.2 Hz, H-1). HPLC-MS/MS (system 2): tR, 8.7

269

min; λmax, 292 nm; fragmentation (80 V, 10 eV) of [M + H]+ (m/z 180): 134 (100), 152 (22),

270

85 (18), 68 (11), 67 (11), 57 (7). Yield: 4.3 mg (53%).

271 272

Synthesis of 5-(2’-formylpyrrol-1’-yl)-pentanol (formylinol 5b). Based on a literature

273

method,26 ribose (603.2 mg, 4.0 mmol) and 5-aminopentanol (436 µL, 4.0 mmol) were

274

suspended in 3 mL of DMSO, and 540 mg (6.0 mmol) of oxalic acid was added. The mixture

275

was heated under reflux for 30 min at 90 °C in an oil bath. After cooling, the mixture was 12 ACS Paragon Plus Environment

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dissolved in 40 mL of water and extracted with ethyl acetate (4 × 30 mL) at its pH 1.8. The

277

organic layers were combined, evaporated to dryness and taken up in 10 mL of a mixture of

278

water and methanol (50/50, v/v). Semi-preparative HPLC was performed using the eluents A

279

and B. After injection, a linear gradient was formed ascending from 40% B to 100% B in 30

280

min. The fraction eluting between 31 and 34 min was collected (32 runs) and evaporated to

281

dryness yielding formylinol 5b as a light brown liquid, which was stored at -18 °C.

282


283

1.79 (m, 2H, H-4); 3.63 (t, 2H, J = 6.5 Hz, H-1); 4.32 (m, 2H, H-5); 6.22 (dd, 1H, J = 2.5 Hz,

284

J = 4.0 Hz, H-4’); 6.93 (dd, 1H, J = 1.7 Hz, J = 4.0 Hz, H-3’); 6.94 (m, 1H, H-5’); 9.52 (d,

285

1H, J = 0.9 Hz, H-1’). HPLC-MS/MS (system 2): tR, 8.3 min; λmax, 292 nm; fragmentation

286

(80 V, 10 eV) of [M + H]+ (m/z 182): 136 (100), 154 (58), 80 (50), 108 (38), 69 (14), 68 (6),

287

96 (4). Elemental analysis: C10H15NO2 (MW = 181.23), calculated, C 66.27%, H 8.34%, N

288

7.73%; found, C 61.35%, H 7.34%, N 7.09%; content = 91.7%, based on nitrogen. Yield: 93.4

289

mg (12%).

290 291

Synthesis of 5-(2’-formyl-5’-hydroxymethylpyrrol-1’-yl)-pentanoic acid 6a. Glucose

292

(716.4 mg, 4.0 mmol) and 5-aminopentanoic acid (468.5 mg, 4.0 mmol) were dissolved in 3

293

mL DMSO, and oxalic acid (360 mg, 4.0 mmol) was added. The mixture was stirred for 30

294

min at 100 °C in an oil bath. After cooling, the thick brown suspension was diluted with 50

295

mL water. The filtrate was extracted at its pH 2.1 with ethyl acetate (3 × 30 mL), and the

296

combined organic phases were evaporated to dryness using a rotary evaporator. The residue

297

was dissolved in 15 mL of a mixture of water and methanol (50/50, v/v) and subjected to

298

semi-preparative HPLC with the eluents A and B. After injection, a linear gradient was

299

formed ascending from 40% B to 100% B in 30 min. The fraction eluting between 23 and

300

26.5 min was collected (20 runs), evaporated to dryness, and lyophilized to yield a light

301

brown powder, which was stored at -18 °C. 13 ACS Paragon Plus Environment


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302

Analytical data: 1H-NMR (400 MHz, D2O), δ [ppm]: 1.54 (m, 2H, H-3); 1.69 (m, 2H, H-4);

303

2.33 (t, 2H, J = 7.3 Hz, H-2); 4.26 (m, 2H, H-5); 4.64 (s, 2H, H-6’); 6.31 (d, 1H, J = 4.1 Hz,

304

H-4’); 7.08 (d, 1H, J = 4.1 Hz, H-3’); 9.29 (s, 1H, H-1’). HPLC-MS/MS (system 2): tR, 6.0

305

min; λmax, 297 nm; fragmentation (60 V, 10 eV) of [M + H]+ (m/z 226): 180 (100), 208 (37),

306

108 (27), 150 (19), 101 (16), 162 (10), 122 (10), 120 (6), 80 (6). Elemental analysis:

307

C11H15NO4 (MW = 225.24), calculated, C 58.66%, H 6.71%, N 6.22%; found, C 58.52%, H

308

6.41%, N 6.16%; content = 99.0%, based on nitrogen. Yield: 61.9 mg (7%).

309 310

Synthesis of 5-(2’-formylpyrrol-1’-yl)-pentanoic acid 6b. The synthesis was performed as

311

for 6a, starting from 5-aminopentanoic acid (471.2 mg, 4.0 mmol) and ribose (600.0 mg, 4.0

312

mmol) in 3 mL DMSO, and oxalic acid (360 mg, 4.0 mmol). The mixture was stirred at 90 °C

313

in an oil bath for 30 min. After cooling, the brown suspension was diluted with 30 mL water.

314

The filtrate was extracted at its pH 1.8 with ethyl acetate (4 × 30 mL), and the combined

315

organic phases were evaporated to dryness using a rotary evaporator. The residue was

316

dissolved in 15 mL of a mixture of water and methanol (50/50, v/v) and subjected to semi-

317

preparative HPLC. After injection, a linear gradient was formed ascending from 40% B to

318

100% B in 30 min. The fraction between 28 and 31 min was collected (14 runs), evaporated to

319

dryness, and lyophilized to yield a light brown powder, which was stored at -18 °C.

320


321

2.37 (t, 2H J = 7.3 Hz, H-2); 4.33 (m, 2H, H-5); 6.22 (dd, 1H, J = 2.6 Hz, J = 4.0 Hz; H-4’);

322

6.96 (dd, 1H, J = 4.0 Hz, H-3’); 6.97 (d, 1H, J = 2.6 Hz, H-5’); 9.52 (d, 1H, J = 0.9 Hz, H-1’).

323

HPLC-MS/MS (system 2): tR, 8.4 min; λmax, 292 nm; fragmentation (80 V, 10 eV) of [M +

324

H]+ (m/z 196): 150 (100), 106 (13), 122 (6), 168 (2). Elemental analysis: C10H13NO3 (MW =

325

195.22), calculated, C 61.53%, H 6.71%, N 7.18%; found, C 61.28%, H 6.43%, N 7.14%;

326

content = 99.4%, based on nitrogen. Yield: 80.8 mg (10%).

327 14 ACS Paragon Plus Environment

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328

Synthesis of 2-(2’-formyl-5’-hydroxymethylpyrrol-1’-yl)-3-methylbutanoic acid 10a. L-

329

valine (473.6 mg, 4.0 mmol), and D-glucose (722.7 mg, 4.0 mmol) were dissolved in 3 mL

330

DMSO, and 360 mg (4.0 mmol) oxalic acid was added. The resulting suspension was stirred

331

for 30 min at 100 °C in an oil bath. After cooling, the solution was diluted with water (50 mL)

332

and extracted at its pH 1.8 with ethyl acetate (4 × 30 mL). The organic layers were combined,

333

dried (Na2SO4), and evaporated to dryness. The residue was taken up in 10 mL of a mixture of

334

methanol and water (70/30, v/v) and subjected to semi-preparative HPLC. After injection, a

335

linear gradient was formed ascending from 60% B to 100% B in 30 min. The fraction eluting

336

between 16 and 20 min was collected (20 runs), evaporated to dryness and stored at -18 °C.

337

Analytical data: 1H-NMR (400 MHz, CDCl3), δ [ppm]: 1.06 (d, 3H, J = 6.9 Hz, CH3-A); 1.10

338

(d, 3H, J = 7.0 Hz, CH3-B); 2.34 (m, 1H, H-3); 5.35 (d, 1H, J = 15 Hz, H-6’A) ; 5.48 (d, 1H, J

339

= 15 Hz, H-6’B); 5.61 (d, 1H, J = 7.1 Hz, H-2); 6.21 (d, 1H, J = 4.0 Hz, H-4’); 6.99 (d, 1H, J

340

= 4.0 Hz, H-3’); 9.53 (s, 1H, H-1’). HPLC-MS/MS (system 1): tR, 11.8 min; λmax, 298 nm;

341

fragmentation (60 V, 10 eV) of [M + H]+ (m/z 226): 162 (100), 134 (60), 180 (16), 190 (8),

342

143 (5), 69 (5). Elemental analysis: C11H15NO4 (MW = 225.24), calculated, C 58.66%, H

343

6.71%, N 6.22%; found, C 61.86%, H 5.86%, N 6.56%; content = 105.5%, based on nitrogen

344

(a content of 100% was considered for quantitations). Yield: 31.5 mg (3.5%).

345 346

Synthesis of 2-(2’-formylpyrrol-1’-yl)-3-methylbutanoic acid 10b. This substance was

347

synthesized and extracted as described for 10a, but starting from 472.6 mg (4.0 mmol) of L-

348

valine and 603.6 mg (4.0 mmol) of D-xylose. Semi-preparative HPLC was performed using

349

the eluents A and B. After injection, a linear gradient was formed ascending from 60% B to

350

100% B in 30 min. The fraction eluting between 29 and 33 min was collected (29 runs) and

351

evaporated to dryness.

352

Analytical data: 1H-NMR (500 MHz, CDCl3), δ [ppm]: 0.82 (d, 3H, J = 6.8 Hz, CH3-A); 1.07

353

(d, 3H, J = 6.7 Hz, CH3-B); 2.48 (m, 1H, H-3); 5.84 (d, 1H, J = 8.9 Hz, H-2); 6.33 (dd, 1H, J 15 ACS Paragon Plus Environment


Page 16 of 42

354

= 2.7 Hz, J = 4.0 Hz, H-4’); 6.98 (dd, 1H, J = 1.6 Hz, J = 4.0 Hz, H-3’); 7.32 (m, 1H, H-5’);

355

9.49 (d, 1H, J = 1.0 Hz, H-1’). HPLC-MS/MS (system 1): tR, 16.2 min; λmax, 292 nm;

356

fragmentation (60 V, 10 eV) of [M + H]+ (m/z 196): 150 (100), 122 (91), 132 (76), 168 (31),

357

80 (7), 68 (4), 55 (4). Elemental analysis: C10H13NO3 (MW = 195.22), calculated, C 61.53%,

358

H 6.71%, N 7.18%; found, C 61.38%, H 6.13%, N 7.07%; content = 98.5%, based on

359

nitrogen. Yield: 109.5 mg (14%).

360

361

Results and discussion

362

Synthesis and qualitative analysis of pertinent metabolites. Brewer’s yeast Saccharomyces

363

cerevisiae can cover its nitrogen requirements in a nitrogen-deficient environment due to the

364

Ehrlich pathway.9,27 Only Ehrlich reaction products of proteinogenic amino acids have been

365

quantitated in fermented foods up to now. Ehrlich alcohols, primarily those derived from

366

phenylalanine and leucine, are important aroma compounds in beer.12,16 As yeast is able to

367

convert pyrraline 1a, formyline 1b, and maltosine 2 in the Ehrlich pathway,21 we

368

hypothesized that intermediates of this pathway should also be present in commercial beer

369

samples. Beer shows a special pattern of free amino acids. In a recent study, mean values

370

between 4 and 34 mg/L were determined for all proteinogenic amino acids except proline,

371

with alanine, arginine, and tyrosine predominating.28 Owing to the limited utilization of

372

proline by S. cerevisiae,29 a mean concentration of this amino acid of 146 mg/L was

373

determined.28 Compared to this, the concentrations of the free glycated amino acids pyrraline

374

1a (0.2–1.6 mg/L), formyline 1b (4–232 µg/L), and maltosine 2 (6–56 µg/L) are small,3 and

375

we assumed the concentrations of any metabolites to be even smaller. For qualitative

376

assessment of the presence of Ehrlich intermediates, a small set of beers was extracted with


Page 17 of 42


377

ethyl acetate. The extracts were concentrated 25-fold and directly analysed by RP-HPLC-

378

MS/MS in the product ion scan and multiple reaction monitoring modes.

379

Prior to the analyses, it was necessary to obtain the respective intermediates as standard

380

substances. Some compounds such as pyrralinol 5a, maltosinol 8 and the α-hydroxy acids 7a

381

and 9 had already been synthesized in a previous work.21 In the present work, further

382

metabolites of pyrraline 1a and formyline 1b were intended to be prepared. The preparation of

383

the α-keto acids started from the parent glycated amino acids by incubation in the presence of

384

L-amino

385

α-keto acids 3a and 3b during 4 h of incubation (Figure 2). Efforts to isolate the compounds

386

by semi-preparative HPLC failed, indicating low stability of the compounds. The MS/MS and

387

UV spectra of the α-keto acid 3a (Figures 2B and 2C) were identical to those obtained for a

388

significant peak in culture supernatants of two strains of S. cerevisiae that had been incubated

389

with dipeptide-bound pyrraline derivatives.21 This shows that yeast actually uses this α-keto

390

acid as an intermediate and is able to excrete it into the medium. However, product ion scan

391

measurements (Figures 2A and S1) revealed that the α-keto acids 3a and 3b were not present

392

in beer samples.

393

The Ehrlich aldehydes pyrralinal 4a and formylinal 4b were obtained in sufficient yield by a

394

preparative application of the ninhydrin reaction, using the Maillard reaction products as

395

starting materials. Synthesis of the Ehrlich acids 6a and 6b started from 5-aminovaleric acid,

396

which was allowed to react directly with glucose in DMSO in the presence of 1 equivalent of

397

oxalic acid.26 Formylinol 5b could be synthesized by this method only after adjustment of the

398

concentration of oxalic acid to 1.5 equivalents. With regard to quantitation of the metabolites

399

in beer samples, all standards were subjected to product ion scan experiments and significant

400

transitions were chosen for MRM measurements (Tables 1 and S1). The α-hydroxy acid 7a

401

was detected in two beer samples at its limit of detection (2.5 µg/L) and the α-hydroxy acid

402

7b was detected in four beer samples at its limit of detection (0.4 µg/L). Pyrralinal 4a and 17

acid oxidase.24 Pyrraline 1a and formyline 1b were both converted to the respective

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403

formylinal 4b were not detected in a subset of beer samples after optimization of the methods

404

in terms of MRM transitions (Figures 3 and S2).

405

A putative peak of the acid 6a in beer samples did not increase when a standard solution was

406

added (Figure 3). Changes in the gradient leading to better separation of the compounds

407

corroborated that 6a is not present in beer samples. However, the mass spectrum of the

408

unknown substance eluting shortly after 6a was very similar to that of 6a (Figure 4). The

409

same effect was observed for the formyline-derived acid 6b (Figure S2). We assumed that

410

these products might be structurally related to the respective Ehrlich acids. As the

411

aminopentyl residue of compounds 6a and 6b is a constitutional isomer of L-valine, the

412

respective pyrraline and formyline analogues derived from L-valine were synthesized and

413

characterized (Figure 5). The product 10b had already been synthesized as an intermediate in

414

the synthesis of heterocyclic compounds and was described as a possible aroma-relevant

415

compound in tobacco products.30,31 The derivative 10a had not yet been prepared. With the

416

synthesized valine derivatives, the peak of the unknown substance in Figure 3 could be

417

ascribed to the product 10a by its HPLC retention time and product ion spectrum. The product

418

10b was also qualitatively identified in beer samples (Figures 4 and 5).

419 420

Quantitation of yeast metabolites derived from glycated and proteinogenic amino acids

421

in beer. Based on the results of qualitative analysis, only the reliable quantitation of the

422

alcohols pyrralinol 5a, formylinol 5b, and maltosinol 8 was focused on. Sufficient separation

423

especially of 5a from coeluting matrix substances was achieved by use of a biphenyl-modified

424

RP-HPLC column (Figure 6). As the amounts of 5a and 5b were too low to be detected by

425

UV-detection, quantitation was based on MS/MS analysis in the MRM mode by application

426

of the standard addition method. Additionally, the Ehrlich alcohols tyrosol, phenylethanol and

427

tryptophol, whose occurrence in beer is already known, were considered to be included in the

428

MS/MS measurement. Unfortunately, phenylethanol was very badly ionizable in the positive 18 ACS Paragon Plus Environment

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429

and in the negative modes, which precluded its analysis by this method. The quantitation of

430

tyrosol was optimized in the negative mode, while tryptophol could be quantitated in the

431

positive mode along with pyrralinol and formylinol (Table 1). Since the concentrations of

432

tyrosol varied in a very broad range and linearity was no more given at high concentrations,

433

quantitation by standard addition was optimized only for small concentrations. A further

434

method was applied for quantitation of tyrosol, phenylethanol, and tryptophol by RP-HPLC

435

with UV-detection.14 The performance parameters for both methods are compiled in Tables 2

436

and 3. Recovery data for the sample pretreatment were obtained only for tyrosol,

437

phenylethanol, and tryptophol by applying the HPLC-UV method, but as these compounds

438

span a broad polarity range, the recoveries of pyrralinol and formylinol by use of the LC-MS

439

method are expected to be in the same range.

440

Pyrralinol 5a was determined in beer samples in concentrations up to 207 µg/L, and

441

formylinol 5b in concentrations up to 50 µg/L (Table 4). Both compounds could be

442

quantitated in all alcohol-containing beer samples along with the known Ehrlich alcohols

443

tyrosol, phenylethanol, and tryptophol. The latter three metabolites were quantitated in

444

concentration ranges known from the literature.11,12,13,14 The concentration of maltosinol 8

445

was determined for a broad range of beer samples in a preliminary study, but its concentration

446

did not exceed 2 µg/L in different beer types (Pilsner, dark, bock, alcohol-free beers).

447

However, the concentration was comparatively high in wheat beers, ranging from 0.8 to 6.9

448

µg/L (median, 2.0 µg/L). Most alcohol-free beers and malt-based beverages contain only

449

traces of the compounds 5a, 5b, and 8. These beer types can be fermented at low temperatures

450

in order to suppress alcohol formation. This underlines the necessity of sufficient action of

451

yeast for the formation of the metabolites. The appearance of high concentrations of Ehrlich

452

alcohols in some of the alcohol-free beers and malt-based beverages should be indicative for

453

particularities of individual steps in the fermentation process, e.g., the choice of special yeast

454

strains.32 The concentrations of pyrralinol, formylinol, tyrosol, and phenylethanol were ca. 19 ACS Paragon Plus Environment


455

1.2-1.6 times higher in wheat beers than in barley beers, whereas the tryptophol

456

concentrations were 13 times higher. Wheat beers are produced by top-fermenting yeasts at

457

higher fermentation temperatures, and higher fermentation temperatures promote the

458

synthesis of higher alcohols by yeast.33,34 Moreover, the protein content of wheat is higher.15

459

The concentrations of tyrosol, tryptophol and phenylethanol correlate well (Figure 7),

460

possibly because the amounts of the parent proteinogenic amino acids in the malt are similar

461

for each beer. As the types and concentrations of MRPs in general differ between pale and

462

dark malts,35 the amounts of pyrraline 1a, formyline 1b, and maltosine 2 in wort should

463

strongly depend on the malting and mashing conditions and on the malt mixture used for

464

brewing.3 The ratios between individual MRPs and those between MRPs and proteinogenic

465

amino acids will also depend on the malting conditions. Consequently, weaker correlations

466

between the new AGE-derived Ehrlich alcohols were found (Figures 7 and S3). Beers with a

467

higher original wort content tended to contain higher concentrations of Ehrlich alcohols which

468

should result from their higher concentrations of MRPs.3

469

In a previous study, the concentration of free and protein-bound MRPs was examined in

470

different beer types. The ratio between free and protein-bound pyrraline 1a, formyline 1b and

471

maltosine 2 indicated a possible degradation of these glycated amino acids during

472

fermentation.3 When the median metabolite concentrations determined in the present study

473

are compared with the concentrations of the parent amino acids,3 it becomes clear that

474

actually 20–40% of these free glycation compounds are present as the corresponding

475

metabolites. This might help explaining the unexpectedly low concentrations of the lysine

476

derivatives in their free form in beer. Further comprehensive studies need to be performed in

477

order to elucidate the formation and possible further reactions of these compounds covering

478

the entire brewing process as well as the storage. Detailed studies are also necessary for the

479

elucidation of the influence of the newly found metabolites on yeast physiology, since the

480

Ehrlich alcohols tryptophol and phenylethanol—and possibly tyrosol—have been described 20 ACS Paragon Plus Environment

Page 20 of 42

Page 21 of 42


481

as quorum sensing molecules in S. cerevisiae with potential implications in wine

482

production.36,37

483

In this context, it is interesting to mention that several pyrrole alkaloids exist in nature,

484

bearing the 2-formylpyrrole structure, such as hemerocallisamine I 11 (Figure 5) isolated from

485

a daylily species (Hemerocallis fulva)38 or magnolamide 12 isolated from a Magnolia species

486

(Magnolia coco).39 Immunostimulatory and hepatoprotective properties were ascribed to 4-

487

(2’-formyl-5’-hydroxymethylpyrrol-1’-yl)-butanoic acid 13,40,41 and antioxidant properties

488

have been reported for acortatarin A 14.42 Compounds 13 and 14 were also described as bitter

489

substances in whole wheat bread.43 The hepatoprotective effect of 13 was evident at

490

concentrations between 0.01 and 1 µM in cultured liver cells. Regarding the median

491

concentrations, 7 mg of tyrosol, 9 mg of phenylethanol, and 4 mg of tryptophol are ingested

492

with one portion of 500 mL of wheat beer along with ca. 37 µg (0.17 µmol) of pyrralinol 5a

493

and 6.5 µg of formylinol 5b. Assuming full bioavailability of 5a and its distribution in the

494

systemic circulation, a plasma concentration of 0.03 µM could be reached after the ingestion

495

of this amount of wheat beer. Maltosine 2 and maltosinol 8 belong to the class of 3-

496

hydroxypyridin-4-ones, which are chelators for iron and other metal ions such as aluminium,

497

zinc and copper.44 Both compounds were characterized in vitro with regard to use them as

498

iron chelators.45 N-Hydroxyalkyl 3-hydroxypyridin-4-ones similar to 8 can cross the blood-

499

brain barrier only to a small extent, which is considered advantageous for general chelation

500

therapy.46 However, there are no human or animal studies available for compound 8. Thus,

501

further knowledge on possible physiological effects of the new Ehrlich alcohols 5a, 5b and 8

502

needs to be gained. Moreover, due to the high similarity of the compounds 10a and 10b to

503

bioactive pyrrole alkaloids (Figure 5), studies also on these compounds are necessary.

504

In the present work, yeast metabolites of the Maillard reaction products pyrraline 1a and

505

formyline 1b have been synthesized and characterized. A method for quantitation of the new

506

Ehrlich alcohols pyrralinol 5a, formylinol 5b and maltosinol 8 was established and the 21 ACS Paragon Plus Environment


Page 22 of 42

507

metabolites were quantitated for the first time in beer samples. The presence of the valine-

508

derived pyrrole derivatives 10a and 10b was confirmed qualitatively. All these compounds

509

represent new pyrrole alkaloids in food. Further work is necessary in order to evaluate the

510

conditions of formation of these metabolites during the brewing process and their influence on

511

yeast metabolism as well as human physiology.

512 513 514

Abbreviations Used

515

3,4-DGE,

516

deoxygalactosone; AGE, advanced glycation end product; CML, N-ε-carboxymethyllysine;

517

DMSO, dimethyl sulfoxide; MRM, multiple reaction monitoring; MRP, Maillard reaction

518

product; NFPA, nonafluoropentanoic acid; PIS, product ion spectrum; TFA, trifluoroacetic

519

acid

3,4-dideoxyglucosone-3-ene;

3-DG,

3-deoxyglucosone;

3-DGal,

3-

520 521 522

Acknowledgments

523

We are grateful to the members of the Chair of Inorganic Molecular Chemistry (Prof. J.J.

524

Weigand), namely Dr. Sivathmeehan Yogendra and Dr. Kai Schwedtmann, for recording the

525

NMR spectra and Philipp Lange for performing the elemental analyses.

526 527

Supporting Information Description

528

Supporting information available: Operating conditions for qualitative analysis of products of

529

the Ehrlich pathway in beer and malt-based beverages (Table S1), qualitative analysis of the

530

α-keto acid derived from formyline (Figure S1), qualitative analysis of different further

531

formyline metabolites in beer (Figure S2), and correlations between individual Ehrlich 22 ACS Paragon Plus Environment

Page 23 of 42


532

alcohols in beer samples (Figure S3). This material is available free of charge via the Internet

533

at http://pubs.acs.org.

534 535

Notes

536

The authors declare no competing financial interest.

537

Parts of this manuscript were presented as a lecture at the 8th International Conference on

538

Chemical Reactions in Foods (CRF VIII) in Prague, Czech Republic, in February 2017.

539 540 541



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542 References [1] Ledl, F.; Schleicher, E. New aspects of the Maillard reaction in foods and in the human body. Angew. Chem. Int. Ed. Engl. 1990, 29, 597–626. [2] Hellwig, M.; Henle, T. Baking, ageing, diabetes: a short history of the Maillard reaction. Angew. Chem. Int. Ed. Engl. 2014, 53, 10316–10329. [3] Hellwig, M.; Witte, S.; Henle, T. Free and protein-bound Maillard reaction products in beer: method development and a survey of different beer types. J. Agric. Food Chem. 2016, 64, 7234–7243. [4] Enari, T.-M.; Sopanen, T. Mobilisation of endospermal reserves during the germination of barley. J. Inst. Brew. 1986, 92, 25–31. [5] Fumi, M.D.; Galli, R.; Lambri, M.; Donadini, G.; De Faveri, D.M. Impact of full-scale brewing process on lager beer nitrogen compounds. Eur. Food Res. Technol. 2009, 230, 209– 216. [6] Bravo, A.; Herrera, J.C.; Scherer, E.; Ju-Nam, Y.; Rübsam, H.; Madrid, J.; Zufall, C.; Rangel-Aldao, R. Formation of α-dicarbonyl compounds in beer during storage of Pilsner. J. Agric. Food Chem. 2008, 56, 4134–4144. [7] Hellwig, M.; Nobis, A.; Witte, S.; Henle, T. Occurrence of (Z)-3,4-dideoxyglucoson-3-ene in different types of beer and malt beer as a result of 3-deoxyhexosone interconversion. J. Agric. Food Chem. 2016, 64, 2746–2753. [8] Buhr, K.; Pammer, C.; Schieberle, P. Influence of water on the generation of Strecker aldehydes from dry processed foods. Eur. Food Res. Technol. 2010, 230, 375–381. [9] Hazelwood, L.A.; Daran, J.-M.; van Maris, A.J.A.; Pronk, J.T.; Dickinson, J.R. The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism. Appl. Environ. Microbiol. 2008, 74, 2259–2266. 24 ACS Paragon Plus Environment

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[10] Matheis, K.; Granvogl, M.; Schieberle, P. Quantitation and enantiomeric ratios of aroma compounds formed by an Ehrlich degradation of L-isoleucine in fermented foods. J. Agric. Food Chem. 2016, 64, 646–652. [11] Schieberle, P. Primary odorants of pale lager beer. Z. Lebensm.-Unters. Forsch. 1991, 193, 558–565. [12] Langos, D.; Granvogl, M.; Schieberle, P. Characterization of the key aroma compounds in two Bavarian wheat beers by means of the sensomics approach. J. Agric. Food Chem. 2013, 61, 11303–11311. [13] Szlavko, C.M. Tryptophol, tyrosol and phenylethanol—the aromatic higher alcohols in beer. J. Inst. Brew. 1973, 79, 283–288. [14] Li, M.; Yang, Z.; Hao, J.; Shan, L.; Dong, J. Determination of tyrosol, 2-phenethyl alcohol, and tryptophol in beer by high-performance liquid chromatography. J. Am. Soc. Brew. Chem. 2008, 66, 245–249. [15] Faltermaier, A.; Waters, D.; Becker, T.; Arendt, E.; Gastl., M. Common wheat (Triticum aestivum L.) and its use as a brewing cereal – a review. J. Inst. Brew. 2014, 120, 1–15. [16] Dunkel, A.; Steinhaus, M.; Kotthoff, M.; Nowak, B.; Krautwurst, D.; Schieberle, P.; Hofmann, T. Nature’s chemical signatures in human olfaction: A foodborne perspective for future biotechnology. Angew. Chem. Int. Ed. Engl. 2014, 53, 7124–7143. [17] Vuralhan, Z.; Morais, M.A.; Tai, S.-L.; Piper, M.D.W.; Pronk, J.T. Identification and characterization of phenylpyruvate decarboxylase genes in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 2003, 69, 4534–4541. [18] Styger, G.; Jacobson, D.; Bauer, F.F. Identifying genes that impact on aroma profiles produces by Saccharomyces cerevisiae and the production of higher alcohols. Appl. Microbiol. Biotechnol. 2011, 91, 713–730.



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[19] Yokoyama, M.; Yokoyama, A.; Yokoyama, T.; Funazu, K.; Hamana, G.; Kondo, S.; Yamashita, T.; Nakamura, H. Hangover susceptibility in relation to aldehyde dehydrogenase2 genotype, alcohol flushing, and mean corpuscular volume in Japanese workers. Alcoholism Clin. Exp. Res. 2005, 29, 1165–1171. [20] Lachenmeier, D.W.; Haupt, S.; Schulz, K. Defining maximum levels of higher alcohols in alcoholic beverages and surrogate alcohol products. Regul. Toxicol. Pharmacol. 2008, 50, 313–321. [21] Hellwig, M.; Börner, M.; Beer, F.; van Pée, K.-H.; Henle, T. Transformation of free and dipeptide-bound glycated amino acids by two strains of Saccharomyces cerevisiae. Chembiochem 2017, 18, 266–275. [22] Hellwig, M.; Geissler, S.; Peto, A.; Knütter, I.; Brandsch, M.; Henle, T. Transport of free and peptide-bound pyrraline at intestinal and renal epithelial cells. J. Agric. Food Chem. 2009, 57, 6474–6480. [23] Hellwig, M.; Henle, T. Formyline, a new glycation compound from the reaction of lysine and 3-deoxypentosone. Eur. Food Res. Technol. 2010, 230, 903–914. [24] Wellner, D.; Meister, A. Crystalline L-amino acid oxidase of crotalus adamanteus. J. Biol. Chem. 1960, 235, 2013–2018. [25] Van Slyke, D.D.; Dillon, R.T.; MacFayden, D.A.; Hamilton, P. Gasometric determination of carboxyl groups in free amino acids. J. Biol. Chem. 1941, 141, 627–669. [26] Das Adhikary, N.; Kwon, S.; Chung, W.-J.; Koo, S. One-pot conversion of carbohydrates into pyrrole-2-carbaldehydes as sustainable platform chemicals. J. Org. Chem. 2015, 80, 7693–7701. [27] Large, P.J. Degradation of organic nitrogen compounds by yeasts. Yeast 1986, 2, 1–34.


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[28] Kabelová, I.; Dvořáková, M; Čížková, H.; Dostálek, P.; Melzoch, K. Determination of free amino acids in beers: A comparison of Czech and foreign beers. J. Food Comp. Anal. 2008, 21, 736–741. [29] Jones, M.; Pierce, J.S. Absorption of amino acids from wort by yeasts. J. Inst. Brew. 1964, 70, 307–315. [30] Nenadjenko, V.G.; Reznichenko, A.L.; Balenkova, E.S. Diastereoselective Ugi reaction without chiral amines: the synthesis of chiral pyrroloketopiperazines. Tetrahedron 2007, 63, 3031–3041. [31] Dickerson, J.P.; Roberts, D.L.; Miller, C.W.; Lloyd, R.A.; Rix, C.E. Flue-cured tobacco flavor. II. Constituents arising from amino acid-sugar reactions. Tobacco Int. 1976, 178, 71– 77. [32] Brányik, T.; Silva, D.P.; Baszczyňski, M.; Lehnert, R.; Almeida e Silva, JB. A review of methods of low alcohol and alcohol-free beer production. J. Food Eng. 2012, 108, 493–506. [33] Landaud, S.; Latrille, E.; Corrieu, G. Top pressure and temperature control the fusel alcohol/ester ratio through yeast growth in beer fermentation. J. Inst. Brew. 2001, 107, 107– 117. [34] Beltran, G.; Novo, M.; Guillamón, J.M.; Mas, A.; Rozès, N. Effect of fermentation temperature and culture media on the yeast lipid composition and wine volatile compounds. Int. J. Food Microbiol. 2008, 121, 169–177. [35] Coghe, S.; Gheeraert, B.; Michiels, A.; Delvaux, F.R. Development of Maillard reaction related characteristics during malt roasting. J. Inst. Brew. 2006, 112, 148–156. [36] Avbelj, M.; Zupan, J.; Kranjc, L.; Raspor, P. Quorum-sensing kinetics in Saccharomyces cerevisiae: A symphony of ARO genes and aromatic alcohols. J. Agric. Food Chem. 2015, 63, 8644–8550.



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[37] Avbelj, M.; Zupan, J.; Raspor, P. Quorum-sensing in yeast and its potential in wine making. Appl. Microbiol. Biotechnol. 2016, 100, 7841–7852. [38] Wood, J.M.; Furkert, D.P., Brimble, M.A. Total synthesis and stereochemical revision of the 2-formylpyrrole alkaloid hemerocallisamine I. J. Nat. Prod. 2017, 80, 1926–1929. [39] Yu, H.-J.; Chen, C.-C.; Shieh, B.-J. Two new constituents from the leaves of Magnolia coco. J. Nat. Prod. 1998, 61, 1017–1019. [40] Kim, S.B.; Chang, B.Y.; Jo, Y.H.; Lee, S.H.; Han, S.-B.; Hwang, B.Y.; Kim, S.Y., Lee, M.K. Macrophage activating activity of pyrrole alkaloids from Morus alba fruits. J. Ethnopharmacol. 2013, 145, 393–396. [41] Chin, Y.-W.; Lim, S.W.; Kim, S.-H.; Shin, D.-Y.; Suh, Y.-G.; Kim, Y.-B.; Kim, Y.C., Kim, J. Hepatoprotective pyrrole derivatives of Lycium chinese fruits. Bioorg. Med. Chem. Lett. 2003, 13, 79–81. [42] Geng, H.M.; Chem, J. L.-Y.; Furkert, D.P.; Jiang, S.; Brimble, M.A. A convergent synthesis of the 2-formylpyrrole spiroketal natural product acortatarin A. Synlett 2012, 23, 855–858. [43] Jiang, D.; Peterson, D.G. Identification of bitter compounds in whole wheat bread. Food Chem. 2013, 141, 1345–1353. [44] Santos, M.A.; Marques, S.M.; Chaves, S. Hydroxypyridinones as “privileged” chelating structures for the design of medicinal drugs. Coord. Chem. Rev. 2012, 256, 240–259. [45] Brady, M.C.; Lilles, K.S.; Treffry, A.; Harrison, P.M.; Hider, R.C.; Taylor, P.D. Release of iron from ferritin molecules and their iron-cores by 3-hydroxypyridinone chelators in vivo. J. Inorg. Biochem. 1989, 35, 9–22. [46] Habgood, M.D.; Liu, Z.D.; Dehkordi, L.S.; Khodr, H.H.; Abbott, J.; Hider, R.C. Investigation into the correlation between the structure of hydroxypyridinones and bloodbrain barrier permeability. Biochem. Pharmacol. 1999, 57, 1305–1310. 28 ACS Paragon Plus Environment

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Figure captions

Figure 1. (A) Proposed pathways of formation of metabolites of pyrraline 1a, and formyline 1b in the Ehrlich pathway. (B) Metabolites of maltosine 2.

Figure 2. Analysis of the α-keto acid 3a derived from pyrraline. (A) RP-HPLC with UVdetection of (a) a pyrraline standard immediately after the addition to a buffered solution of Lamino acid oxidase and catalase and (b) of the same reaction mixture after 4 h of oxidation, (c) the latter reaction mixture with MS/MS detection in the product ion scan mode at the m/z of the protonated molecular ion of 3a. (d) Wheat beer sample with MS/MS detection in the product ion scan mode at the m/z of the protonated molecular ion of 3a. (B) UV spectrum of 3a. (C) Product ion spectrum of 3a (fragmentor voltage, 60 V; collision energy, 10 eV).

Figure 3. Biphenyl-RP-HPLC followed by MS/MS detection in the MRM mode of pyrraline metabolites in a wheat beer sample. (a) Detection at the most intense transition of the Ehrlich acid 6a and (b) the same sample after addition of a standard of 6a. (c) Detection at the most intense transition of the α-hydroxy acid 7a and (d) the same sample after addition of a standard of 7a. (e) Detection at the most intense transition of pyrralinal 4a and (f) chromatogram of a standard of 4a.

Figure 4. Product ion spectra recorded during Biphenyl-HPLC-MS/MS measurements of (A) a standard solution of 5-(2’-formyl-5’-hydroxymethylpyrrol-1’-yl)-pentanoic acid 6a (tR = 11.6 min), (B) a standard solution of 2-(2’-formyl-5’-hydroxymethylpyrrol-1’-yl)-3methylbutanoic acid 10a (tR = 11.8 min), (C) the peak eluting at tR = 11.8 min in a wheat beer sample (cf. Figure 3), (D) a standard solution of 5-(2’-formylpyrrol-1’-yl)-pentanoic acid 6b 29 ACS Paragon Plus Environment


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(tR = 16.3 min), (B) a standard solution of 2-(2’-formylpyrrol-1’-yl)-3-methylbutanoic acid 10a (tR = 16.4 min), (C) the peak eluting at tR = 16.4 min in a wheat beer sample (cf. Figure S2). The positions of the molecular ions are shown by asterisks.

Figure 5. Chemical structures of the valine-derived MRPs 10a and 10b as well as the naturally occurring pyrrole alkaloids hemerocallisamine I 11, magnolamide 12, 5-(2’-Formyl5’-hydroxymethylpyrrol-1’-yl)-butanoic acid 13, and acortatarin A 14.

Figure 6. Biphenyl-RP-HPLC-MS/MS (system 1) with MRM detection of (A) pyrralinol 5a and (B) formylinol 5b in (a) a sample of alcohol-free beer, and (b) a sample of wheat beer with standard additions (c, d) at ascending concentrations. (C) RP-HPLC-MS/MS (system 2) with MRM detection of maltosinol 8 in the same samples.

Figure 7. Correlation of the concentrations of tyrosol and tryptophol (A), tyrosol and phenylethanol (B), pyrralinol 5a and formylinol 5b (C), and pyrralinol 5a and maltosinol 8 (D) in 11 wheat beers. Correlation coefficients are deduced from Spearman’s rank correlation analysis.


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Tables Table 1. Transitions Recorded During MRM Measurement of Ehrlich Alcohols in Beer and Malt-Based Beverages.[a] Compound

Time

polarity

frame

Precursor

Product

Collision

Dwell

ion [m/z]

ion [m/z]

energy

time

[eV]

[ms]

250

[min]

Tyrosol

Pyrralinol

Tryptophol

3.5–6

6–12.5

12.5–

negative

positive

positive

137

119

0

137

106

0

212

166

10

212

148

10

162

144

10

162

117

20

182

136

10

182

80

20

Q/q[b]

Q q

100

Q q

200

Q

15.5

Formylinol

15.5–18

positive

q 200

Q q

[a] General conditions: Fragmentor voltage, 60 V. [b] Q, transition used for quantitation; q, transition used to confirm the presence of the analyte.



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Table 2. Performance Parameters of the HPLC-MS/MS Method for the Measurement of Ehrlich Alcohols in Beer and Malt-Based Beverages by Standard Addition.

Compound

Tyrosol

LOD[a]

LOQ[a]

cV[b]

[µg/L]

[µg/L]

[%]

60

340

5.4

Linear

Mean R2 Intercept

range[c]

[d]

accuracy[e] [%]

0.2-29.7

0.9990

105 ± 5

0.9997

103 ± 6

0.9991

100 ± 1

1–38 µg/L 0.9996

101 ± 3

mg/L Pyrralinol

1.1

4.6

7.3

2.3–240 µg/L

Tryptophol

—

—

7.7

0.01–6.3 mg/L

Formylinol

0.5

1.8

5.9

[a] Limits of detection (LOD) and limits of quantitation (LOQ) are based on matrix calibration. No data can be given for tryptophol because no analyte-free sample was available. [b] Coefficients of variation (cV) were determined by repeated measurements of beer samples (n = 2-5) of different beer types. [c] Range between the LOQ in methanol-precipitated beer samples and the highest concentration after standard addition. In the case of tryptophol, the smallest sample concentration is applied instead of the LOQ, and wheat beers were excluded. [d] Correlation coefficient of the regression function of standard addition. [e] Calculated as the quotient of the intercept of the regression line (peak area vs. concentration) obtained by standard addition and the peak area of the analyte measured in the sample without standard addition (expressed in percent, mean ± S.D.).


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Table 3. Performance Parameters of the HPLC-UV Method for the Measurement of Ehrlich Alcohols in Beer and Malt-Based Beverages by External Calibration.

Compound

LOD[a]

LOQ[a]

cV[b]

Linear

R2 [c]

Recovery[d]

range[c] [mg/L]

[mg/L]

[%]

[µM]

[%]

Tyrosol

0.04

0.12

0.8–3.0

0.1–42

0.9996

103 ± 7

Phenylethanol

0.60

1.80

1.4–3.3

1.1–39

0.9998

93 ± 1

Tryptophol

0.03

0.10

2.7–11.9

0.1–42

1.0000

98 ± 1

[a] Limits of detection (LOD) and limits of quantitation (LOQ) are calculated on the basis of the signal-to-noise ratio. [b] Coefficients of variation (cV) were determined by repeated measurements of selected beer samples (n = 3). [c] Linear range and R2 of the calibration curve used for external calibration. [d] Recovery was determined by adding different concentrations of alcohols (2 mg/L and 7 mg/L) to beer and malt-based beverages before precipitation.



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Table 4. Concentrations of Ehrlich Alcohols Derived from Glycated and Proteinogenic Amino Acids in Beer and Malt-Based Beverages.[a] Beer type

Barley

n

20

Beer Wheat beer

Pyrralinol

Formylinol Tyrosol

Phenylethanol Tryptophol

HPLC-

HPLC-

HPLC-

HPLC-UV

HPLC-UV

MS/MS

MS/MS

MS/MS

[µg/L]

[µg/L]

[mg/L]

[mg/L]

[mg/L]

14–204

6–21 (10)

3–21 (10)

6–22 (14)

0.3–1.7

(46) 12

(0.6)

33–207

10–50 (13)

7–34 (14)

11–32 (18)

2–22 (8)

n.d.–7 (tr)

tr–11 (1.3) n.d.–20 (tr)

n.d.–1.2

(73) Alcohol-

5

free beer Malt-based beverages[b]

n.d.–22 (tr)

7

(0.3)

n.d.–6 (tr)

n.d.

n.d.–1.4

n.d.

(tr)

n.d.–0.5 (n.d.)

[a] Data are given as ranges with the medians in parentheses. Individual values were determined at least twice. n, number of samples; tr, trace amounts between LOD and LOQ; n.d., not detectable. [b] All malt-based beverages were produced from barley malt.


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Figures Figure 1



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Figure 2


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Figure 3



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Figure 4


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Figure 5



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Figure 6


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Figure 7



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TOC graphic


Yeast Metabolites of Glycated Amino Acids in Beer - Journal of

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