Free and Protein-Bound Maillard Reaction Products in Beer: Method

Article pubs.acs.org/JAFC

Free and Protein-Bound Maillard Reaction Products in Beer: Method Development and a Survey of Different Beer Types Michael Hellwig, Sophia Witte, and Thomas Henle* Institute of Food Chemistry, Technische Universität Dresden, D-01062 Dresden, Germany S Supporting Information *

ABSTRACT: The Maillard reaction is important for beer color and flavor, but little is known about the occurrence of individual glycated amino acids in beer. Therefore, seven Maillard reaction products (MRPs), namely, fructosyllysine, maltulosyllysine, pyrraline, formyline, maltosine, MG-H1, and argpyrimidine, were synthesized and quantitated in different types of beer (Pilsner, dark, bock, wheat, and nonalcoholic beers) by HPLC-ESI-MS/MS in the multiple reaction monitoring mode through application of the standard addition method. Free MRPs were analyzed directly. A high molecular weight fraction was isolated by dialysis and hydrolyzed enzymatically prior to analysis. Maltulosyllysine was quantitated for the first time in food. The most important free MRPs in beer are fructosyllysine (6.8−27.0 mg/L) and maltulosyllysine (3.7−21.8 mg/L). Beer contains comparatively high amounts of late-stage free MRPs such as pyrraline (0.2−1.6 mg/L) and MG-H1 (0.3−2.5 mg/L). Minor amounts of formyline (4−230 μg/L), maltosine (6−56 μg/L), and argpyrimidine (0.1−4.1 μg/L) were quantitated. Maltulosyllysine was the most significant protein-bound MRP, but both maltulosyllysine and fructosyllysine represent only 15−60% of the total protein-bound lysine-derived Amadori products. Differences in the patterns of protein-bound and free individual MRPs and the ratios between them were identified, which indicate differences in their chemical, biochemical, and microbiological stabilities during the brewing process. KEYWORDS: Maillard reaction, glycation, beer, HPLC-MS/MS, Amadori product, maltulosyllysine

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INTRODUCTION The production of beer is unique among beverages because it comprises several steps with thermal treatment of materials that contain substantial amounts of reducing sugars and amino acids. First, malt is produced by allowing cereals (predominantly barley) to germinate, followed by kilning of sprouted grain. Malt is then extracted with water under application of a carefully optimized temperature program in the mashing process, and wort is generated by filtration. Boiling of wort after the addition of hops followed by fermentation with brewer’s yeast provides the final product beer. Chemical reactions between ingredients such as reducing sugars and amino compounds can proceed throughout the process. Maillard reaction products (MRPs) play an especially important role for flavor and color of beer. The Maillard reaction, also termed “non-enzymatic browning” or “glycation”, is a network of chemical reactions starting with the addition of a reducing sugar to an amine to form an Amadori rearrangement product (ARP).1,2 In malt, wort, and beer, amino acids can be present in proteins, in peptides of various lengths, and in the free form.3,4 Proline, alanine, arginine, and tyrosine are the dominating free amino acids.3 Reducing sugars can therefore react at the α-amino groups of free amino acids and peptides,5,6 at the imino group of free proline, and at the ε-amino group of lysine. By the latter reaction, fructosyllysine and maltulosyllysine (Figure 1) are formed from glucose and maltose as the most important saccharides in mash.7 The ARPs generated in the first stage of the Maillard reaction are degraded to 1,2-dicarbonyl compounds in the second stage. Such 1,2-dicarbonyl compounds contribute to the formation and stability of beer flavor.8 Beyond © 2016 American Chemical Society

Figure 1. Chemical structures of the Maillard reaction products analyzed in this study.

aroma-active Strecker aldehydes and furanones, a structurally heterogeneous group of protein-bound MRPs, also called Received: Revised: Accepted: Published: 7234

June 13, 2016 August 30, 2016 September 3, 2016 September 3, 2016 DOI: 10.1021/acs.jafc.6b02649 J. Agric. Food Chem. 2016, 64, 7234−7243

Article

Journal of Agricultural and Food Chemistry “advanced glycation end products (AGEs)”, is formed in the late stage of the reaction (Figure 1). Important compounds among these AGEs are pyrroles such as pyrraline and formyline, which are formed by Paal−Knorr reaction from 1,2-dicarbonyl compounds. Pyrraline, formyline, and maltosine are typical products in reaction systems with low water activity.9,10 The arginine derivatives MG-H1 and argpyrimidine are formed by the reaction of methylglyoxal with the guanidino group of arginine in solution.11,12 Upon further reaction, which also include polyphenols, pigmented high molecular weight (HMW) structures (melanoidins) are formed. They are well soluble in water and determine the color of the final beer.13−15 Moreover, structural elements of melanoidins that remain to be elucidated are responsible for pro- and antioxidant properties of roasted malts.16 The analysis of MRPs in food consistently has shown that the concentrations of individual MRPs vary strongly in different product groups (e.g., milk products, pasta, and bakery products).10,17−21 Differences in MRP concentrations are caused not only by differences in the chemical composition of the ingredients but also by variations in the production process.18 Up to now, studies on the Maillard reaction in beer have been focusing mainly on its impact on beer flavor and aging,8,22−24 melanoidins, and color development.15 Differences in the pattern of ARPs at the α-amino group of free amino acids in beer were described and traced back to differences in the kilning process.5 Little information has yet been gained regarding the formation in beer of protein-bound lysine and arginine derivatives such as fructosyllysine, maltulosyllysine, maltosine, pyrraline, formyline, or MG-H1, although it is known that the Maillard reaction positively influences the foaming properties of certain barley proteins.25 Free argpyrimidine was quantitated in lager beers in concentrations up to 6.9 μg/L, and the respective concentrations were correlated with the wort concentration and the beer color.26 For proteinbound adducts, only a few preliminary data have been published for maltosine, pyrraline, and formyline showing that their concentration in the beer protein can be as high as in bread protein.10,19 Moreover, no information is available in the literature on the extent of lysine and arginine modification in different types of beer (e.g., Pilsner, dark, bock, wheat, and nonalcoholic beers). We hypothesized that differences in the production processes of different beer types should be reflected in the pattern of free and protein-bound MRPs in the final product. A deeper knowledge on the chemistry of the Maillard reaction of proteins in beer can possibly allow retrospective conclusions about details of the malting and brewing processes. This might lead to the identification of expedient indicators of process parameters. It was therefore the aim of the present study to investigate the extent of amino acid modification in beer by quantitating the MRPs fructosyllysine, maltulosyllysine, pyrraline, formyline, maltosine, MG-H1, and argpyrimidine (Figure 1) in free and protein-bound form. First insight should be gained into the “fate” of the products during the brewing process, and the contribution of beer to the dietary intake of MRPs should be estimated.

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(Steinheim, Germany), and formic acid was purchased from Grüssing (Filsum, Germany). N-α-tert-Butyloxycarbonyllysine (Boc-lysine) from Fluka was employed, and acetic acid was from Roth (Karlsruhe, Germany). Maltose, hydrochloric acid, and Pronase E (4000 PU/mg protein) from Merck (Darmstadt, Germany) were used, and pyridine was from Acros (Geel, Belgium). The water for the preparation of buffers and solutions was obtained by a Purelab Plus purification system (USF Elga, Ransbach-Baumbach, Germany). Water for HPLCMS/MS was double-distilled (first over potassium permanganate). Fructosyllysine,27 pyrraline,28 formyline,9 maltosine,29 MG-H1,30 and argpyrimidine30 (Figure 1) were synthesized according to the literature stated. Beer Samples. All beer samples were purchased in local retail stores. Aliquots of beer samples were degassed in an ultrasonic bath prior to all analyses. Free amino acids and free individual MRPs were analyzed in the degassed samples after membrane filtration (0.45 μm) without further workup. For the isolation of a high molecular weight fraction, 50 g of degassed beer samples was dialyzed against water at 4 °C for 4 days (cellulose tubings, MWCO, 14 kDa, Sigma).10,19 The water was changed twice per day. The retentates were lyophilized. Enzymatic hydrolysis was performed using 20 mg of the lyophilized HMW fractions, representing 2−3 mg of protein, according to the literature.9,10,19 The samples were first dissolved in 1.05 mL of 0.02 M hydrochloric acid containing 356 U of pepsin. The solution was incubated at 37 °C for 24 h in a drying chamber. Then, 300 μL of 2 M TRIS buffer, pH 8.2, containing 400 PU of Pronase E was added. After further incubation for 24 h, 0.4 U of leucine-aminopeptidase and 1 U of prolidase were added. After 24 h, the samples were freeze-dried, and the lyophilisates were taken up in 1000 μL of 10 mM NFPA. After centrifugation (10600g, 10 min) and membrane filtration (0.45 μm), the samples were subjected to HPLC analysis. For acid hydrolysis, 20 mg of the HMW fractions was dissolved in 2 mL of 6 M HCl and incubated at 110 °C for 23 h in a drying chamber. An aliquot (1 mL) of the hydrolysate was then evaporated to dryness in vacuo and suspended in 1 mL of the loading buffer for amino acid analysis (0.12 N lithium citrate, pH 2.20). Characterization of Beer Samples and HMW Fractions. Nitrogen was determined by using the Kjeldahl method. The protein content was calculated from the N contents using the factor 6.25 for cereal products. The pH values of the beer samples were measured at 20 °C using the pH meter InoLab Level 2 (Mettler Toledo, Gießen, Germany). For the estimation of beer color according to the respective EBC method,31 aliqouts of beer samples were centrifuged (10600g, 10 min), and the clear supernatant was transferred to a cuvette (d = 10 mm). The absorption at 430 nm (E430) was measured photometrically (Spekol 1500, Analytik Jena, Germany) against water as the blank. If necessary, beer samples were diluted to keep the absorption below a value of 0.8. The EBC value was calculated as follows: EBC = E430 × 25 × dilution factor

Amino Acid Analysis. Amino acids were separated on a PEEK column filled with the cation exchange resin LCA K07/Li (150 mm × 4.6 mm, 7 μm) using the amino acid analyzer S 433 (Sykam, Fürstenfeldbruck, Germany). Loading and running buffers for this lithium system were obtained from Sykam and employed for different gradient programs utilized previously.30,32 After postcolumn derivatization with ninhydrin, amino acids were detected with a two-channel photometer simultaneously working at 440 and 570 nm, respectively. Furosine was quantitated by use of a further detector operating at 280 nm.27 External calibration was performed with a commercial amino acid mixture (Sigma-Aldrich, Steinheim, Germany). Amino acid analysis was also used for estimation of the efficiency of enzymatic hydrolysis. Enzymatic hydrolysates (100 μL) were diluted with 400 μL of the loading buffer, and 50 μL of samples was injected. The percentage release due to enzymatic hydrolysis was calculated by taking the release during acid hydrolysis as 100%. High-Pressure Liquid Chromatography (HPLC) with Tandem Mass Spectrometric Detection. The analysis of free and proteinbound MRPs was performed on the high-pressure gradient system 1200 series (Agilent Technologies, Böblingen, Germany), consisting of

MATERIALS AND METHODS

Chemicals. HPLC gradient grade acetonitrile was obtained from VWR Prolabo (Leuven, Belgium). Nonafluoropentanoic acid (NFPA), N,N-dimethylformamide (DMF), deuterium oxide, prolidase (208 U/ mg protein), pepsin (3555 U/mg protein), and leucine aminopeptidase (19 U/mg protein) were purchased from Sigma-Aldrich 7235

DOI: 10.1021/acs.jafc.6b02649 J. Agric. Food Chem. 2016, 64, 7234−7243

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Journal of Agricultural and Food Chemistry

heated under reflux for 4 h in a mixture of 30 mL of N,Ndimethylformamide (DMF) and 70 mL of methanol.9,27 Methanol and DMF were then removed in vacuo by repeated coevaporation with water. The residue was taken up in water (75 mL), and the pH of the solution was adjusted to 2.0 with 6 M HCl. The solution was applied to a column (2.5 cm × 20 cm) filled with the strong cation exchange resin Lewatit S100 (Bayer, Leverkusen, Germany), which had previously been equilibrated with 250 mL of 6 M HCl and 250 mL of water. The column was washed with 250 mL of water to remove sugar and uncharged byproducts. After 16 h, 300 mL of pyridinium acetate buffer, pH 5.5, was used to elute the crude product. After evaporation to dryness, the residue was taken up in 30 mL of 0.1 N pyridinium formate buffer, pH 3.0. The pH of the solution was adjusted to 3.0 with formic acid. The solution was applied to a column (1.5 cm × 50 cm) filled with the strong cation exchange resin DOWEX 50 WX-8, which had previously been equilibrated with 250 mL of 6 M HCl, 250 mL of water, 250 mL of 2 M pyridine, 250 mL of water, and 250 mL of 0.1 N pyridinium formate buffer, pH 3.0. After the column had been rinsed with 200 mL of 0.1 N pyridinium formate buffer, pH 3.0, maltulosyllysine was eluted with 400 mL of 0.4 N pyridinium formate buffer, pH 4.35, while the eluate was collected in 10 mL fractions using a fraction collector (RediFrac, Pharmacia Biotech, Uppsala, Sweden). The presence of products in the fractions was monitored by spotting 1 μL aliquots on TLC plates and spraying with a mixture of ninhydrin, acetic acid, and ethanol (0.1:3:100, w/v/ v). Spots were developed at 50 °C in a drying chamber. In parallel, aliquots of the fractions spotted on another TLC plate were sprayed with a 1% solution of triphenyl tetrazolium chloride in 1 M NaOH. Spots indicating reducing ARPs developed at room temperature. Maltulosyllysine was found to elute between 50 and 90 mL of the elution buffer. The respective fractions were pooled and repeatedly evaporated under reduced pressure after the addition of water. Finally, the residue was taken up in water and lyophilized to yield maltulosyllysine as an off-white powder, which was stored at −18 °C. Analytical data: ESI-MS, positive mode, [M + H]+ m/z 471.2; 1H NMR (500 MHz, D2O, β-pyranose form) δ 1.39 (m, 2H, Lys-H4), 1.68 (m, 2H, Lys-H5), 1.81 (m, 2H, Lys-H3), 3.05 (m, 2H, Lys-H6), 3.23 (s, 2H, H1′), 3.35 (m, 1H, H5″), 3.53 (m, 1H, H4″), 3.65 (m, 2H, H6A′, H6A″), 3.66 (m, 2H, Lys-H2), 3.68 (m, 1H, H3″), 3.75 (m, 2H, H2″, H6B″), 3.84 (d, 1H, 9.8 Hz, H3′), 3.88 (dd, 1H, 3.2 Hz, 9.8 Hz, H4′), 3.94 (d, 1H, 12.8 Hz, H6B′), 4.10 (s, br, H5′), 5.14 (d, 1H, 3.9 Hz, H1″); 13C NMR (125 MHz, D2O, β-pyranose form) δ 21.5 (CH2, Lys-C4), 24.6 (CH2, Lys-C5), 29.8 (CH2, Lys-C3), 48.0 (CH2, Lys-C6), 52.7 (CH2, C1′), 54.4 (CH, Lys-C2), 60.5 (CH2, C6″), 64.0 (CH2, C6′), 68.8 (CH, C5′), 68.9 (CH, C3′), 69.5 (CH, C5″), 71.6 (CH, C4″), 72.4 (CH, C2″), 72.7 (CH, C3″), 77.5 (CH, C4′), 95.5 (Ci, C2′), 100.5 (CH2, C1″), 174.5 (Ci, Lys-C1). Elemental analysis:

a binary pump, an online degasser, a column oven, an autosampler, and a diode array detector. The HPLC was coupled to the mass spectrometer 6410 Triple Quad (Agilent) working in the positive mode. The method details are compiled in Table 1, and the operating

Table 1. Operating Conditions for the Measurement of Free and Protein-Bound Maillard Reaction Products in Beer by HPLC-MS/MS HPLC

column: column temperature: mobile phases:

Zorbax 100 SB-C18 (Agilent), 2.1 × 50 mm, 3.5 μm 35 °C

gradient: flow rate: Injection volume:

A, 10 mM NFPA in double-distilled water B, 10 mM NFPA in acetonitrile from 5 to 50% B in 25 min 0.25 mL/min 10 μL

ESI

nebulizing gas: capillary voltage gas temperature: gas flow: nebulizer pressure:

N2 (nitrogen generator 5183-2003, Agilent) 4000 V 350 °C 11 L/min 35 psi

MS/MS

MRM:

see Table 2

conditions for the detector working in the multiple reaction monitoring (MRM) mode are listed in Table 2. These conditions were optimized prior to the measurements by scan and product ion scan measurements of the respective standards (Figure 2). Data acquisition and evaluation were performed with the software Mass Hunter B.02.00 (Agilent). Quantitation of MRPs was performed by standard addition. In the first run, 100 μL of sample was mixed with 20 μL of water. In the second run, 100 μL of sample was mixed with 10 μL of water and 10 μL of a standard solution. In the third run, 100 μL of samples was mixed with 20 μL of standard solution. In the standard solution, maltulosyllysine (55.9 μg/mL), fructosyllysine (87.6 μg/mL), pyrraline (20.1 μg/mL), MG-H1 (5.0 μg/mL), formyline (1.9 μg/mL), maltosine (0.39 μg/mL), and argpyrimidine (66.0 ng/mL) were dissolved in water. Synthesis and Isolation of N-ε-(1-Deoxy-D-maltulosyl)-Llysine (Maltulosyllysine). On the basis of a previously published protocol for the synthesis of lactulosyllysine, 753.7 mg (3.0 mmol) of Boc-lysine and 6.53 g (18.1 mmol) of maltose monohydrate were

Table 2. Transitions Recorded during MRM Measurement of MRPs in Beer time frame 3−8.5 min

compound

precursor ion (m/z)

product ion (m/z)

fragmentor voltage (V)

collision energy (eV)

dwell time (ms)

Q/qa

maltulosyllysine

471 471 309 309

128 225 84 225

140 140 120 120

20 20 30 10

125 125 125 125

q Q Q q

255 255 229 229 255 255 225 225 255 255

148 175 70 114 84 126 134 161 70 140

80 80 120 120 120 120 80 80 80 80

20 10 20 10 20 10 20 10 30 10

70 70 70 70 70 70 70 70 70 70

q Q q Q Q q Q q Q q

fructosyllysine

8.5−19.5 min

pyrraline MG-H1 maltosine formyline argpyrimidine

a

Q, transition used for quantitation; q, transition used for the confirmation of the presence of the analyte. 7236


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Figure 2. Product ion spectra obtained during HPLC-MS/MS measurement of a standard solution of (A) maltulosyllysine, (B) pyrraline, and (C) formyline and of a sample of free MRPs in a dark beer sample, (D) maltulosyllysine, (E) pyrraline, and (F) formyline, with denotation of the quantifier (bold numbers) and qualifier ions (italic numbers). Operating conditions are those for the quantifier transitions (see Table 2).

proline, alanine, arginine, and tyrosine3 are the predominating free amino acids in beer, the most significant reaction sites on proteins are the ε-amino group of lysine and the guanidino group of arginine. Therefore, the present study focused on individual reaction products of these two amino acids. Maillard reactions of the beer protein have only scarcely been addressed in the literature, and studies on the occurrence of individual derivatives of lysine and arginine in malt and beer are not available. Differences in the patterns of free and protein-bound MRPs were expected particularly for different types of beer. The beers chosen in this study differed in the type of malt used, the wort concentration, and the cereal source. Various free MRP amino acids were then sought by recording product ion spectra by HPLC-MS/MS using an eluent system with nonafluoropentanoic acid as published previously.35,36 The product ion spectra of standards were compared with those obtained in beer samples (Figure 2). For quantitation in the MRM mode, the most abundant and/or most selective transitions were chosen (Table 2). Because 3-DG and 3-DGal are the most important 1,2-dicarbonyl compounds in beer,8,37−39 pyrraline was analyzed along with formyline and maltosine, the occurrence of which in beer protein has already been established.10,19 Finally, two adducts resulting from the reaction of methylglyoxal with the guanidino group of arginine, namely, MG-H1 and argpyrimidine, were included. Argpyrimidine had been quantitated earlier in beer as the free amino acid.26 The ARPs fructosyllysine and maltulosyllysine were determined, because glucose and maltose are the predominating sugars in mash and wort.7 Maltulosyllysine had not yet been quantitated in foods and synthesized only in a peptide-bound form.27 The free form of maltulosyllysine was synthesized on the basis of established methods9,27 and characterized by twodimensional NMR measurements. Three signal groups were identified, and the most prominent form was assigned to the βpyranose isomer (62−65%) by comparison with published data for chemical shifts and coupling constants of fructosyl

C18H34N2O12 (MW = 470.47), calcd C, 45.95%; H, 7.28%; N, 5.95%. Found: C, 43.15%; H, 7.70%; N, 4.93%. Content = 82.9%, based on nitrogen. Yield = 736 mg (molar yield = 43.2%). Characterization of Maltulosyllysine. A PerSeptive Biosystems Mariner time-of-flight mass spectrometry instrument equipped with an electrospray ionization source (ESI-TOF-MS, Applied Biosystems, Stafford, TX, USA) working in the positive mode was used for mass spectroscopy. The sample was injected into the ESI source by a syringe pump at a concentration of 0.01 mg/mL in a mixture of 1% formic acid in 50% aqueous acetonitrile. Spray tip potential, nozzle potential, quadrupole RF voltage, and detector voltage were adjusted to 4812.3, 80, 1000, and 2400 V, respectively. 1H and 13C NMR spectra were recorded on an Avance III HDX 500 MHz Ascend instrument from Bruker (Rheinstetten, Germany) at 500.13 and 125.75 MHz, respectively. Maltulosyllysine (22.2 mg) was dissolved in 750 μL of deuterium oxide and stored at room temperature overnight to allow mutarotation. All chemical shifts are given in parts per million (ppm), those of protons relative to the internal HOD signal (4.70 ppm) and those of carbon atoms relative to external standard tetramethylsilane. Assignments of 1H and 13C signals are based on 1H−1H correlation spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), heteronuclear multiple-bond correlation (HMBC), and distortionless enhancement by polarization transfer (DEPT) experiments. Elemental analysis data were obtained by use of a Euro EA 3000 elemental analyzer (Eurovector, Milano, Italy). Statistical Treatment. For correlation analysis, Spearman’s rank correlation coefficients (rS) and significance of correlations were calculated using the software PASW Statistics 18. Limits of detection (LOD) and quantitation (LOQ) were calculated as the concentrations of MRPs necessary to show peaks at signal-to-noise ratios of 3 and 10, respectively. All samples were analyzed at least in duplicate.

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RESULTS AND DISCUSSION Determination of Individual MRPs in Beer. Investigations of beer have been a major driving force in the research on nonenzymatic browning even before the works of Maillard.1,2,33 It was shortly after Maillard’s fundamental publication that Strecker degradation of leucine in the presence of sugars was found to be a significant determinant of beer flavor due to the formation of 3-methylbutanal.34 Although 7237


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Journal of Agricultural and Food Chemistry Table 3. Performance Parameters of the HPLC-MS/MS Method Used for the Analysis of MRPs in Beer Samplesa free MRPs

protein-bound MRPs

MRP

LOD (μg/L)

LOQ (μg/L)

cV (%)

LOD (mg/kg protein)

LOQ (mg/kg protein)

cV (%)

fructosyllysine maltulosyllysine pyrraline formyline maltosine MG-H1 argpyrimidineb

2.5 8.2 0.4 0.1 0.6 1.6 0.1

9.0 28.5 2.8 0.4 2.3 6.0 0.4

0.7−2.2 2.1−2.9 0.4−7.0 0.9−1.1 0.4−3.2 0.6−1.4 4.0−4.5

6.3 22.8 0.6 0.1 0.3 3.8

2.1 75.9 2.1 0.2 1.1 12.5

3.9−8.3 7.9−17.0 5.7−8.3 2.8−4.9 4.1−12.1 6.5−10.2

a

Limits of detection (LOD) and limits of quantitation (LOQ) are given in mg/kg protein for protein-bound MRPs based on a protein amount of 4 mg per enzymatic hydrolysis. Coefficients of variation (cV) were determined by triplicate analyses of free and protein-bound MRPs in three different beer samples. bArgpyrimidine was not detectable in enzymatic hydrolysates.

Table 4. Characterization of Beer Samplesa beer type

n

pH

EBC

Nbeerb (g/kg)

wHMWc (g/kg)

NHMWb (g/kg)

Ntransfd (%)

Pilsner dark bock wheat alcohol-free

5 5 5 5 5

4.3−4.7 4.3−4.7 4.6−4.9 4.2−4.4 4.2−5.0

6.4−7.0 34.3−98.4 20.4−95.5 11.3−16.6 6.5−11.8

0.5−0.7 0.5−0.7 1.1−1.2 0.7−0.9 0.2−0.6

5.1−9.6 7.9−10.4 6.8−12.8 9.5−11.7 4.1−6.7

15−20 13−18 16−21 21−37 10−19

15−25 20−24 11−20 35−45 20−56

a

Data are given as ranges. bThe nitrogen content of the beer and of the HMW fractions was determined by using the Kjeldahl method. cwHMW denotes the yield of the retentate after lyophilization. dNtransf denotes the percentage of nitrogen transferred from the beer to the HMW fraction.

Table 5. Concentrations of Free MRPs in Different Types of Beera

a

beer type

n

fructosyllysine (mg/L)

maltulosyllysine (mg/L)

pyrraline (mg/L)

formyline (μg/L)

maltosine (μg/L)

MG-H1 (mg/L)

argpyrimidine (μg/L)


5 5 5 5 5

11.6−13.6 8.8−14.9 22.2−27.0 9.1−12.5 6.8−12.0

8.9−27.3 9.6−11.2 10.4−21.8 9.7−13.8 3.7−12.4

0.16−0.34 0.16−0.85 0.42−1.63 0.20−0.64 0.16−0.22

76−99 64−127 164−232 4−95 42−78

13−22 6−41 15−56 17−36 8−13

0.49−1.49 0.65−1.54 1.38−2.47 1.18−2.13 0.31−1.21

0.7−1.3 0.7−1.9 1.4−4.1 0.7−2.0 0.1−1.8

Data are given as ranges (n = 2−5).

amines.30,40,41 Comparison of these data with those obtained in the literature for 13C and 1H chemical shifts of the atoms of the fructosyl residue and the C1″ of the α-glucosyl residue in the isomers of maltulose indicates that the minor two forms are the α- and β-furanoses (each ca. 18%).42 This is the same distribution of pyranose and furanose forms as reported for the disaccharide-derived ARP lactulosyllysine.43 As stableisotope labeled standards were not available for all analytes, we decided to determine the MRP concentrations by the standard addition method. LODs and LOQs for all analytes were sufficiently low (Table 3) to enable quantitation of the analytes in the majority of samples. Free MRPs in Beer. Twenty-five beer samples representing five beer types (Pilsner, dark, bock, wheat, and alcohol-free beers) were included in the study. These beers showed typical pH values and colors.3 The differences in the nitrogen content reflect the original wort content, which is typically higher in bock beers (Table 4). Free MRPs were analyzed in these samples and expressed on a mass concentration basis (Table 5). Fructosyllysine and maltulosyllysine were the most abundant free MRPs with concentrations up to 27 mg/L, which is similar to low-concentrated proteinogenic amino acids (e.g., aspartic acid, serine, threonine).5 The contents of fructosyllysine and maltulosyllysine are higher than those reported for other glycated free amino acids, namely, ARPs resulting from

modification of the amino group of free amino acids such as N-α-fructosylvaline or N-α-fructosylleucine.5,6 Strong differences in the concentration of free lysine in beers are reported in the literature, ranging from “not detectable” to 36 mg/L.3,5 The mean concentration of free lysine in the beer samples of the present study was 26 mg/L. The mean concentration of fructosyllysine, therefore, corresponds to ca. 40% of the lysine concentration, and that of maltulosyllysine to 25% of the lysine concentration. Free amino acids are removed from wort and beer during fermentation and storage.5,44 The high abundance of fructosyllysine and maltulosyllysine could be caused by differences in their chemical stability during storage but also by differences in the metabolization by yeast. It is surprising that MG-H1 predominates among the free AGEs (Table 5) because the parent dicarbonyl compound, methylglyoxal (MGO), is of minor importance in beer, but this might be explained by the high reactivity of intermediate MGO.37 The second most important AGE is pyrraline, predominating over formyline and maltosine. The concentrations of pyrraline and MG-H1 are in the range of those reported for the prolinederived MRPs N-formylproline and N-carboxymethylproline.39 Argpyrimidine was quantitated in concentrations between 0.1 and 4.1 μg/L, which is similar to literature data.26 Variations in free MRP concentrations between beer types were comparable to differences found for beer samples of the same type. Significantly higher amounts of free amino acids 7238


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Journal of Agricultural and Food Chemistry Table 6. Concentrations of Protein-Bound MRPs in Different Types of Beera beer type

n

fructosyllysine (g/kg protein)

maltulosyllysine (g/kg protein)

pyrraline (mg/kg protein)

formyline (mg/kg protein)

maltosine (mg/kg protein)

MG-H1 (mg/kg protein)

lysine blockageb (%)


5 5 5 5 5

0.3−2.9 1.8−3.2 1.7−2.6 1.0−1.4 1.0−2.0

1.8−5.1 3.6−5.4 3.5−6.2 2.2−3.6 2.6−4.4

132−156 122−400 177−345 74−203 55−211

30.2−46.5 37.8−49.7 36.2−64.3 17.3−28.0 20.0−45.0

5.1−7.7 4.7−14.2 8.5−16.6 3.3−8.4 2.1−9.6

47.9−103.0 46.2−106.4 67.3−90.0 35.9−136.6 35.5−90.4

14.7−16.5 13.5−16.7 18.2−20.3 12.1−15.1 12.1−15.5

a

Data are given as ranges (n = 2−5). bLysine blockage is calculated from furosine concentrations taking as a basis the conversion factors for fructosyllysine (cf. Krause et al.27).

sine and fructosyllysine are converted to furosine in defined amounts (each ca. 30%).27 A comparison of the furosine contents measured by amino acid analysis with those expected from conversion of maltulosyllysine and fructosyllysine revealed that 40−85% of furosine must be formed from ARPs that were not measured in this study. Formation of ARPs resulting from the reaction of lysine with maltooligosaccharides such as maltotriose, maltotetraose, and maltopentaose, which abound in mash, wort, and beer,7 can be expected. Adducts of proteins and starch with the ε-amino group of lysine attached to a reducing end of amylose or amylopectin would also give rise to furosine during acid hydrolysis, but would not lead to fructosyllysine or maltulosyllysine during enzymatic hydrolysis. Whether such “polysaccharide ARPs” may explain the observed “Amadori product gap” remains to be elucidated. Moreover, the ratio of fructosyllysine to maltulosyllysine is different between the protein-bound and the free amino acids in a way that fructosyllysine is enriched in the free form. Besides differences in proteolytic release of the individual ARPs, which should be marginal according to our previous study,32 differences in the chemical stability and susceptibility to degradation by enzymes can be expected. In a study on maltooligosaccharide ARPs of glycine, the degradability of the trisaccharide ARP maltotriulosylglycine by pancreatic amylase and yeast α-glucosidases was investigated.47 The disaccharide-derived ARP maltulosylglycine was only very slowly degraded to fructosylglycine by yeast αglucosidase.47 Thus, it should be possible that a part of fructosyllysine in beer is hydrolyzed from maltulosyllysine by yeast α-glucosidase during fermentation. However, fructosyllysine could as well be formed by the action of amylases on higher oligosaccharide ARPs. Studies on the metabolization of such oligosaccharide ARPs by yeast are currently carried out in our laboratory. Pyrraline was the most important protein-bound AGE in beer with concentrations up to 400 mg/kg protein in dark beers, followed by MG-H1. The concentrations of formyline are half as high as those of MG-H1, whereas the maltosine concentrations are in the lower ppm range (Table 6). Thus, the order is diverse from that of free AGEs, where MG-H1 predominated over pyrraline. The concentrations of pyrraline, formyline, and maltosine in the HMW fractions confirm the results of earlier studies obtained with different methods.10,19 Due to its comparatively low concentration, maltosine is probably not a key structure for the modulation of pro- and antioxidant properties in melanoidins,16 despite its iron-binding 3-hydroxy-4(1H)-pyridinone structure.10 The ratio of pyrraline to formyline is about 10 to 1 in most food items (milk and bakery products) and can rise even higher in the presence of lactose.19 In the beer HMW isolates, this ratio is only at about 4 to 1, indicating that the HMW fraction of beer contains relatively large amounts of formyline. This can be explained by

were found only for bock beers, which have a higher nitrogen content due to the higher original wort content. However, amino acids seem to be affected differently, because in comparison with the other beer types, primarily the concentrations of free fructosyllysine and formyline are increased. Thus, it is not possible to use the concentrations of free MRPs as an indicator of beer types. The concentrations of free MRPs should rather reflect differences in technological process parameters, and their formation and potential as indicators should be further elucidated in experiments covering the brewing process completely from barley to beer. Protein-Bound MRPs in Beer. A protein-enriched fraction was isolated from beer by dialysis as in previous studies.10,19 Comparison of the nitrogen contents of the original beer with the lyophilized retentates revealed that 10−50% of beer nitrogen is bound in this HMW fraction with molecular weights above 14 kDa (Table 4). It has been reported that only ca. 25% of the beer amino nitrogen is bound in proteins with molecular weights >14 kDa.4 Conclusions concerning the total concentrations of MRPs in beer are limited by this approach, because an intermediate molecular weight fraction from dipeptides to 14 kDa proteins is not considered. However, when a fraction of 30% of non-amino nitrogen in beer is assumed (e.g., from nucleic acids),4 the free amino acids and those in the HMW fraction represent ca. 50% of the total amino nitrogen. The protein content of the retentates was a maximum of 20%, indicating that further beer constituents (e.g., intact starch, polysaccharide conjugates, and melanoidins) play a more important role in the HMW fraction compared to proteins. Dialysis has actually been used as a method for the isolation of beer melanoidins.14,15 Owing to a possible impairment of the proteolytic release of amino acids from such melanoidins, the efficiency of enzymatic hydrolysis was determined. Compared to acid hydrolysis, 70% of proline, 80− 95% of alanine, valine, isoleucine, and phenylalanine, and 95% of arginine are released by enzymatic hydrolysis from the HMW fractions. This points to a very efficient enzymatic hydrolysis and is in very good agreement with data published for enzymatic hydrolysates of other food items such as bread and peanut products.10,45 As known from the literature, the amino acid composition of the HMW fraction reflected mainly the amino acid composition of barley prolamins and glutelins.46 MRPs in enzymatic hydrolysates of HMW samples were measured with the same HPLC-MS/MS method as the free MRPs. Whereas the concentrations of free maltulosyllysine and free fructosyllysine were roughly equal in all beer types (Table 5), the concentrations of protein-bound maltulosyllysine were twice as high as those of protein-bound fructosyllysine, making maltulosyllysine the quantitatively most important proteinbound MRP determined in this study (Table 6). During hydrolysis with 6 M hydrochloric acid, the ARPs maltulosylly7239


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Figure 3. Results of correlation analysis for all beer samples (n = 25): (A) correlation of the concentrations of free pyrraline and pyrraline in the HMW fractions; (B) correlation of the concentrations of free fructosyllysine and fructosyllysine in the HMW fractions; (C) correlation between the concentrations of pyrraline and maltosine in the HMW fractions. Nonparametric correlation coefficients (Spearman’s rho) were calculated with the software PASW statistics 18.0; the significance of the correlation is indicated by asterisks (*, P < 0.01; otherwise not significant).

needs to be further explored in systematic studies covering the whole brewing process. As shown recently in our laboratory, one serving of beer can contribute significantly to the overall dietary intake of 1,2dicarbonyl compounds such as 3-DG and 3-DGal, with beer and malt beer being the main dietary source of 3-DGal.37 Compared to this, the ingestion of glycated amino acids through the consumption of beer is low. Assuming a similar distribution of MRPs in the not yet analyzed molecular weight fraction between dipeptides and 14 kDa as in the HMW fraction (Table 6), not more than 1.5 mg of pyrraline or MGH1 would be ingested with the consumption of 500 mL of beer, together with 0.2 mg of formyline and 0.1 mg of maltosine and up to 20 mg each of fructosyllysine and maltulosyllysine. This represents only about 10% of the daily intake of individual AGEs such as pyrraline, formyline, and maltosine and 5−10% of the daily intake of ARPs.10,19,51 However, 50−80% of these amounts are ingested as free MRPs, some of which are handled differently from protein-bound MRPs during human metabolism. Protein-bound MRPs can be released from proteins during intestinal digestion, and glycated dipeptides can pass the gut epithelium by the peptide transporter PEPT1, whereas free MRPs are not absorbed and are subject to degradation by the intestinal microbiota.28,32,36 MRPs such as pyrraline, which are normally absorbed and excreted via the urine,52 will thus largely pass into the large intestine following the consumption of beer. It remains to be elucidated whether this is of any physiological significance. A further general aspect related to the physiological handling of MRPs by yeast can be deduced from the comparison of the contribution of free and protein-bound amino acids to the summed concentration in Pilsner beer (Figure 4). Apparently, the major part of the amino acids is present in the free form; however, as stated above, not the whole beer protein was included in the analysis and the protein-bound amino acids should be underestimated. The concentrations of proline, alanine, and tyrosine, respectively, in the HMW fractions are higher by factors of 5, 2.5, and 1.7, respectively, than the concentration of the free amino acids. This reflects the known differences in the uptake of wort amino acids by brewer’s yeast: Proline uptake is strongly limited under brewing conditions, whereas the uptake of tyrosine and alanine is comparatively slow but complete.53,54 The fraction of free lysine can even be lower than that of protein-bound lysine (Figure 4), which can also be explained by the reported high rate of absorption from wort of this amino acid.44,53 It can be postulated that the

the fact that the HMW aggregates in beer can contain pentoses such as arabinose and xylose as the predominating sugars that can act as formyline precursors in close vicinity to the protein.46 Comparison of the Concentrations of Free and Protein-Bound MRPs. Correlation analysis was performed between the concentrations of free and protein-bound MRPs. A higher concentration of an MRP in the HMW fraction was usually accompanied by a higher concentration of the compound in the free form (Figure 3), but the correlations were not more than moderate (rS < 0.6, Figure S1). As shown by the example of fructosyllysine (Figure 3B), the interpretation of correlation coefficients can be biased by characteristics of individual beer types. However, significant correlations between the concentrations of individual protein-bound MRPs were revealed, primarily between the two ARPs and within the group of the lysine-derived AGEs pyrraline, maltosine, and formyline (Figure 3C and Figure S2). These correlations could be due to comparable pathways of formation, and in the case of the AGEs, the formation from the same precursor molecules. Especially the correlation between protein-bound pyrraline and maltosine shows that pyrraline in malt should be formed from disaccharides along with maltosine, and not from monosaccharides as in other foodstuffs.19 Interestingly, concerning a possible link between MRPs and color, a significant correlation was found only for free pyrraline (rS = 0.6, P < 0.01, Figure S3). Although the Maillard reaction is generally made responsible for nonenzymatic color formation, we conclude that color is generated in reaction pathways distant from those that lead to the formation of the MRPs analyzed in the present study. It remains an open question as to which circumstances ultimately lead to the occurrence of the relatively high concentrations of free MRPs in beer. In most food items, free MRPs do not play a significant role from a quantitative point of view, because proteins predominate over free amino acids.35,48 Furthermore, free amino acids react with dicarbonyl compounds such as methylglyoxal or 3-deoxyglucosone mainly via Strecker degradation, leading to the formation of aromaactive Strecker aldehydes, which has already been proven in malt.49 The proteolytic breakdown and release of free amino acids occur predominantly in the malting and mashing processes; proteolysis during fermentation is of lower importance.50 Thus, it should be possible to get insight into the malting and mashing processes by analyzing the pattern of protein-bound and free MRPs. The formation and degradation of MRPs along with the impact of proteolysis on the distribution of free and protein-bound glycated amino acids 7240



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AUTHOR INFORMATION

Corresponding Author

*(T.H.) Phone: +49-351-463-34647. Fax: +49-351-463-34138. E-mail: [email protected]. Funding

M.H. thanks the Deutscher Akademischer Austauschdienst (DAAD) for financial support through a travel grant. Notes

Parts of the manuscript were presented as a poster at the XVIII EuroFoodChem in Madrid, Spain, in October 2015. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the members of the Chair of Inorganic Molecular Chemistry (Prof. J. J. Weigand), namely, Kai Schwedtmann, for recording the NMR spectra. We thank Karla Schlosser, Institute of Food Chemistry, for performing the amino acid analyses and Anke Peritz, Institute of Organic Chemistry, for the elemental analysis.

Figure 4. Comparison of the concentrations of free (black squares) and protein-bound (gray squares) proteinogenic and glycated amino acids in Pilsner beer samples. Data are given as ranges (n = 5).

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ABBREVIATIONS USED 3-DG, 3-deoxyglucosone; 3-DGal, 3-deoxygalactosone; AGE, advanced glycation end product; ARP, Amadori rearrangement product; DMF, N,N-dimethylformamide; EBC, European Brewery Convention; HMW, high molecular weight; HPLC, high-pressure liquid chromatography; LOD, limit of detection; LOQ, limit of quantitation; MG-H1, methylglyoxal-derived hydroimidazolone 1; MGO, methylglyoxal; MRM, multiple reaction monitoring; MRP, Maillard reaction product; MS, mass spectrometry; MWCO, molecular weight cutoff; NFPA, nonafluoropentanoic acid; RP, reversed-phase

inability of yeast to remove certain amino acids from wort leads to the accumulation of these amino acids in wort and beer, because proteolysis is observed predominantly during malting and mashing. This could indicate that individual MRPs are also removed from wort by yeast during fermentation, because the contribution of the free form of certain MRPs is only slightly higher than that of the protein-bound form (Figure 4), for example, pyrraline (2.6-fold), maltosine (3.0-fold), and formyline (2.6-fold). For other MRPs, this ratio can be considerably higher. The free forms predominate over the protein-bound ones by factors of 13.6 for fructosyllysine, 18.6 for MG-H1, and 4.0 for maltulosyllysine. Studies on the metabolic handling of individual MRPs by brewer’s yeast are required. Taken together, it was shown that individual MRPs play an important role in the amino acid composition of beer. The ARP maltulosyllysine, which was synthesized and determined for the first time in food, was the quantitatively most important compound among the protein-bound MRPs. However, both maltulosyllysine and fructosyllysine can explain only 15−60% of the furosine content of the HMW fractions. Pyrraline and MG-H1 are the predominating free and protein-bound AGEs in beer. The MRP concentrations vary considerably within the beer types, which precludes reliable differentiations of beer types by these products. However, the potential of free and protein-bound MRPs as indicators of technological processes should be further explored. Differences in the patterns of free and protein-bound MRPs indicate that brewer’s yeast might be capable of removing individual MRPs from wort during fermentation.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b02649. Correlations between free and protein-bound concentrations of MRPs (Figure S1), correlations between individual protein-bound MRPs (Figure S2), and correlations between color and free and protein-bound concentrations of MRPs (Figure S3) (PDF) 7241


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