Amino Acids In Beer Research Articles

Fast Determination of Main Bioamines and Precursor Amino Acids in Beer by Miniaturized Electrophoresis Using Gold Nanoparticle Composite Electrode

A gold nanoparticle/reduced graphene oxide composite electrode (AuNP/rGO) was fabricated for the determination of the main bioamines and their precursor amino acids in beer based on a miniaturized capillary electrophoresis with amperometric detection system (mini-CE-AD). The surface morphology and structure of the synthetic AuNP/rGO material were observed by scanning electron microscope, energy dispersive X-ray spectroscope, X-ray diffraction, and Fourier transform infrared spectroscopy. The analytical performance of the composite electrode was verified by cyclic voltammetry and mini-CE-AD approaches. Four target analytes were separated within 7 min in 50 mM Na2B4O7-NaOH buffer (pH 9.35) at a separation voltage of 3 kV. This developed method has been applied to the beer samples, and acceptable recovery could be obtained ranging from 84.5 to 108%. The assay results indicated that this AuNP/rGO composite electrode has the advantages of simple preparation, low working potential (+ 0.65 V vs. SCE), good sensitivity (0.13~0.87 μM), and satisfactory reproducibility. This proposed method provides an alternative for rapid analysis of food safety.

Yeast Metabolites of Glycated Amino Acids in Beer.

Glycation reactions (Maillard reactions) during the malting and brewing processes are important for the development of the characteristic color and flavor of beer. Recently, free and protein-bound Maillard reaction products (MRPs) such as pyrraline, formyline, and maltosine were found in beer. Furthermore, these amino acid derivatives are metabolized by Saccharomyces cerevisiae via the Ehrlich pathway. In this study, a method was developed for quantitation of individual Ehrlich intermediates derived from pyrraline, formyline, and maltosine. Following synthesis of the corresponding reference material, the MRP-derived new Ehrlich alcohols pyrralinol (up to 207 μg/L), formylinol (up to 50 μg/L), and maltosinol (up to 6.9 μg/L) were quantitated for the first time in commercial beer samples by reverse phase high performance liquid chromatography tandem mass spectrometry in the multiple reaction monitoring mode. This is equivalent to ca. 20-40% of the concentrations of the parent glycated amino acids. The metabolites were almost absent from alcohol-free beers and malt-based beverages. Two previously unknown valine-derived pyrrole derivatives were characterized and qualitatively identified in beer. The metabolites investigated represent new process-induced alkaloids that may influence brewing yeast performance due to structural similarities to quorum sensing and metal-binding molecules.

Rapid Determination of Amino Acids in Beer, Red Wine, and Donkey-Hide Gelatin by Gradient Elution of HPLC: From Micellar Liquid Chromatography to High Submicellar Liquid Chromatography.

Amino acids (AAs) in beer, red wine, and donkey-hide gelatin were rapidly determined by gradient LC elution from micellar LC to high submicellar LC. Mobile phase A was a 0.075 M sodium dodecyl sulfate solution containing 20 mM ammonium acetate with a pH value adjusted to 3.5 with acetic acid solution, and mobile phase B was acetonitrile. Optimized chromatographic conditions were as follows: mobile phase B increased from 25 to 60% (v/v) in 30 min and the use of a Venusil XBP C18 column (5 µm, 250 × 4.6 mm) as the stationary phase, with a column temperature of 35°C, flow rate of 1.2 mL/min, and detection wavelength of 266 nm. The results indicated good linearity (r2 ≥ 0.9924). The intraday precision of the retention time was RSD ≤ 1.1%, whereas interday was RSD ≤ 3.2%; intraday precision of the peak area was RSD ≤ 3.3%, whereas interday was RSD ≤ 4.9%. The range of recovery was 94.6-102.4%. The RSDs of the retention time for the AAs for the different samples were 0.04-0.31%.

Free and Protein-Bound Maillard Reaction Products in Beer: Method Development and a Survey of Different Beer Types

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.

Selection of Protease for Increased Solubilization of Protein Derived Thiols during Mashing with Limited Release of Free Amino Acids in Beer

Extraction of protein-derived thiols by protease treatment during mashing for improvement of flavor stability in beer has previously been shown to cause concomitant increase in free amino acid concentrations and thereby increased levels of unwanted Maillard reaction products during aging. The previous methodology, with addition of protease dosed at 50 mg of enzyme/kg of malt at the beginning of the protein rest during mashing, resulted in increased amino acid concentrations of approximately 40% and increased free thiol concentrations of approximately 50% in wort compared with a control without protease addition. The objective of this study was to reduce the amount of amino acids in wort obtained by protease treatment but still obtain a significant increase in protein-derived thiols. In this paper we demonstrate that by omitting the protein rest during mashing in combination with addition of a protease with a higher temperature optimum dosed at only 3 mg of enzyme/kg of malt, it is possible to increase thiol concentrations in wort by 30% and with only a maximum 10% increase in amino acid concentration compared with a control. Pilot brewing showed that beer brewed with addition of protease resulted in a 30% increase in total thiol concentration, along with a decrease in or no effect on the concentration of some amino acids but also an increase in the concentration of other amino acids. Sulfite concentration was increased by 37%, so the effect of increasing free thiol concentration on flavor stability during storage could not be evaluated. Overall, similar brewing and sensory characteristics were obtained compared with a control beer brewed without addition of protease. Foam stability was decreased by protease treatment, and formation of haze was reduced by protease treatment.

Effect of Protease Treatment during Mashing on Protein-Derived Thiol Content and Flavor Stability of Beer during Storage

The ability of a protease added during mashing in a pilot-scale brew to release protein-derived thiols as antioxidants and thereby improve flavor stability during storage was investigated. The protease treatment resulted in increased protein-derived thiol concentrations in fresh beer produced at a pilot scale compared with a control beer without protease treatment, and electron spin resonance lag phase analysis also demonstrated improved oxidative stability of the protease-treated beer. However, the protease treatment also resulted in high sulfite levels and, therefore, it was not possible to conclude that the improvement in oxidative stability was solely a direct effect of increased levels of protein-derived thiol concentrations. The development of papery character and volatile linear aldehydes was inhibited when the protease-treated beer was stored at 35°C but the protease treatment also resulted in increased concentrations of free amino acids in beer, resulting in a more fruity aged or vinous character of the beer during storage due to increased formation of Maillard reaction products. Thiols were found to be irreversibly oxidized during storage, which indicates that thiols cannot be regenerated during storage; and, if thiols are to act as antioxidants in beer, increased concentrations must be present in the fresh beer.

High-performance liquid chromatographic method for determination of amino acids by precolumn derivatization with 4-chloro-3,5-dinitrobenzotrifluoride

This work presents an high-performance liquid chromatography method for the determination of amino acids after precolumn derivatization with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF) which can readily react with both primary and secondary amines. The precolumn derivatization conditions, including the CNBF concentration, reaction pH, temperature and reaction time were investigated for method optimization. In pH 9.0 borate buffer, the reaction of amino acids with CNBF was carried out at 60 °C for 30 min, the optimized concentration of CNBF was 70 mmol L −1 and the molar ratio of amino acids to CNBF was 1:5.25. The chromatographic separation of 19 amino acids derivatives was performed on a Kromasil ODS C 18 column (250 mm × 4.6 mm, 5 μm) with good reproducibility, and ultraviolet detection was applied at 260 nm. The mobile phase was a mixture of phase A (acetonitrile) and phase B (acetate buffer, acetonitrile, triethylamine; 82.8:17:0.2, pH 4.9), and the flow rate was 0.4 mL min −1. The separation of all the labeled amino acids was achieved within 45 min at room temperature by gradient elution mode. The method linearity, calculated for each amino acid, had a correlation coefficient higher than 0.9979, in concentrations ranging from 9.60 to 3330.00 μmol L −1. The detection limits of amino acids were 2.40–6.50 μmol L −1, at a signal-to-noise ratio of 3. The proposed method was applied for the determination of amino acids in beer with recoveries of 97.0–103.9% and relative standard deviations of 2.62–4.22%, respectively. This method showed good accuracy and repeatability that can be used for the quantification of amino acids in real samples.

Analysis of Protein and Total Usable Nitrogen in Beer and Wine Using a Microwell Ninhydrin Assay

In this study we present a ninhydrin based microwell assay that can be utilized in place of the traditional Kjeldahl method for the determination of the protein content of beer or wine. In addition, the assay is ideal for the determination of free amino acids in beer (FAN), a term understood and used by brewers, and yeast assimilable nitrogen (YAN) used by enologists. The assay only measures alpha amino acids and ammonia so other nitrogen sources are not detected, resulting in a 30% reduction in total protein of a variety of beers compared to the Kjeldahl method, which measures nitrogen from all sources. The results also showed that only 25% of the total “protein” in beer is actually derived from peptides larger than 3,500 Kd. Analysis of beer or wine with the microwell assay for total usable nitrogen was compared to the standard FAN and YAN methods and conditions were determined for maximal efficiency and precision. Superior results were obtained with low reaction volumes and a stable sodium acetate buffered ninhydrin reagent at pH 5.5. As an alternative, for use with cuvettes, a reduced volume FAN assay using the same pH 5.5 sodium acetate buffered ninhydrin reagent gave comparable results. The assay is economical, rapid, accurate and applicable to large numbers of samples.

Simultaneous analysis of riboflavin and aromatic amino acids in beer using fluorescence and multivariate calibration methods

The study demonstrates an application of the front-face fluorescence spectroscopy combined with multivariate regression methods to the analysis of fluorescent beer components. Partial least-squares regressions (PLS1, PLS2, and N-way PLS) were utilized to develop calibration models between synchronous fluorescence spectra and excitation–emission matrices of beers, on one hand, and analytical concentrations of riboflavin and aromatic amino acids, on the other hand. The best results were obtained in the analysis of excitation–emission matrices using the N-way PLS2 method. The respective correlation coefficients, and the values of the root mean-square error of cross-validation (RMSECV), expressed as percentages of the respective mean analytic concentrations, were: 0.963 and 14% for riboflavin, 0.974 and 4% for tryptophan, 0.980 and 4% for tyrosine, and 0.982 and 19% for phenylalanine.

Quantification of organic and amino acids in beer by 1H NMR spectroscopy.

The quantification of organic and amino acids in beer using 1H NMR spectroscopy is demonstrated. Quantification was made both by integration of signals in the spectra together with use of calibration references and by use of partial least-squares (PLS) regression. Results from the NMR quantifications were compared with those obtained from determinations by amino acid analysis on HPLC and organic acid analysis by capillary electrophoresis. The described NMR-based methods could satisfactorily be used for quantification of several of the investigated metabolites in beer down to approximately 10 mg/L and for most with a good to high accuracy compared to results obtained by HPLC and capillary electrophoresis (R2 0.90-0.99). This was achieved with a simple sample preparation and one-dimensional 1H NMR spectra obtained in a few minutes. The use of PLS clearly improves the accuracy of the quantifications, based on comparison to results obtained by HPLC and capillary electrophoresis, and furthermore permits the determination of components with partially overlapped signals in the spectrum. NMR spectroscopy in combination with PLS will be a useful tool for the quantification of metabolites, not only in beer but also in other beverages and biofluids.

Isocratic ion chromatographic determination of underivatized amino acids by electrochemical detection

An isocratic chromatographic separation with amperometric detection of underivatized amino acids at a copper oxyhydroxide modified glassy carbon electrode is described. A simple and sensitive quantitation procedure of amino acids without the need of tedious and time-consuming derivatization step was achieved by coupling anion-exchange chromatography with electrochemical detection. The effects of hydroxide, nitrate and acetonitrile concentration in the mobile phase on the capacity factors and peak resolution was evaluated. Under the optimized isocratic chromatographic conditions (i.e. 60 mM NaOH) and under constant applied potentials (i.e. 0.55 V versus Ag/AgCl) the detection limit ranged between 4 and 24 pmol injected and the linear dynamic range spanned generally over three or four order of magnitude for all investigated amino acid compounds. Direct determination of several common free amino acids in beer and milk samples were performed.

Hypolipidemic Effect of Beer Proteins in Experiment on Rats

Amino acid analysis, electrophoretic separation and Fourier transform-infrared spectra (FT-IR) were used to determine and characterize proteins and amino acids in two types of beer: with a high content of proteins (Beer 1) and with a low content of proteins (Beer 2). The concentration of total proteins, albumin and of most studied amino acids in Beer 1 were significantly higher than in Beer 2 ( P<0.05–0.0005). Thirty-six rats were divided in three groups, each 12. The rats of the control group were fed basal diet (BD) only and the BD of the two experimental groups (Beer 1 and Beer 2) was supplemented with lyophilized, polyphenol-free Beer 1 and Beer 2, respectively. Before and after completion of the 4 w feeding period the total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol, triglycerides (TG), lipid peroxides (LP) and total radical-trapping antioxidative potential (TRAP) in all three groups were examined. In Beer1 group a significant decrease in the level of TC, LDL-C and TG was registered ( P<0.05, 0.05 and 0.005, respectively). No differences in the level of LP and TRAP in all three groups were found. This investigation proves that beer with high protein and essential amino acid concentrations does affect the plasma lipids level in rats.

Analysis of amino acids by capillary zone electrophoresis with electrochemical detection.

The precapillary derivatization of 20 amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) and CN(-) was investigated. All these derivatized amino acids could be oxidized on the carbon fiber microdisk bundle electrode except proline. Capillary zone electrophoresis with electrochemical detection was employed for the analysis of 19 amino acids. The optimum conditions of separation and detection were borate, pH 9.48, for the electrolyte, 18 kV for the separation voltage and 1.15 V versus a saturated calomel electrode for the detection potential. Limits of detection of concentration or mass for individual amino acids were between 1.7 x 10(-7) and 1.8 x 10(-6) mol/L or 84 and 893 amol (according to the signal-to-noise ratio of 3) for the injection voltage of 6 kV and injection time of 10 s. The relative standard deviations were between 0.80 and 2.3% for the migration times and 1.4 and 6.4% for the electrophoretic peak currents. From a mixture of 19 amino acids, 10 amino acids (Arg, Lys, Orn, Try, Ser, Ala, Gly, Cys, Glu, Asp) could be well separated. The other 9 amino acids appeared on three electrophoretic peaks. From the samples, in which the nine amino acids do not exist simultaneously, some of them could also be detected. The method was applied to the determination of amino acids in beer by the standard addition method. The recovery for the amino acids in beer was 91-109%.

Proteins of beer affect lipid levels in rats

Consumption of dry matter of alcoholic beverages leads to improved lipid metabolism and increased antioxidant activity in experiments on rats. Proteins and amino acids are part of the dry matter. Are proteins and amino acids playing a role in these changes? Amino acid analysis, electrophoretic separation and Fourier transform-infrared spectra (FT-IR) were used to determine and characterize proteins and amino acids in beer and white wine. The contents of total proteins, albumin and of most studied amino acids in beer were significantly higher than in white wine (P < 0.05–0.0005). Thirty-six rats were divided in 3 groups, each 12. The rats of the Control group were fed basal diet (BD) only and the BD of the two experimental groups (B and WW) was supplemented with lyophilized, polyphenol-free beer and white wine, respectively. Before and after completion of the 4 weeks feeding period, total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol, triglycerides (TG) and lipid peroxides (LP) were examined. Only in the group of rats (B) fed diet, supplemented with beer a significant decrease in the level of TC, LDL-C and TG was observed (P < 0.05, 0.05 and 0.005, respectively). No differences in the level of LP in all 3 groups were found. Therefore, only diet supplemented with lyophilized, polyphenol-free beer, which has significantly higher concentration of proteins and essential amino acids than white wine does affect the level of plasma lipids in rats.