Activity-Guided Identification of <i>in Vitro</i> Antioxidants in Beer

In order to locate the key antioxidants contributing to oxidative stability of beer, activity-guided fractionation in combination with the oxygen radical absorbance capacity (ORAC) assay, hydrogen peroxide scavenging (HPS) assay, and linoleic acid (LA) assay was applied to a pilsner-type beer. LC-MS and 1D/2D NMR experiments led to the identification of a total of 31 antioxidants, among which 3-methoxy-4-hydroxyphenyl-β-d-glucopyranoside (tachioside), 4-(2-formylpyrrol-1-yl)­butyric acid, 4-[2-formyl-5-(hydroxymethyl)­pyrrol-1-yl]­butyric acid, <i>n-</i>multifidol-3-<i>O</i>-β-d-glucoside, quercetin-3-<i>O</i>-(6″-malonyl)-glucoside, 4-feruloylquinic acid, syringaresinol, saponarin, and hordatines A–C have been isolated from beer for the first time. On a molar comparison, the hordatines A–C, saponarin, and quercetin-3-<i>O-β</i>-d-(6″-malonyl)­glucoside were evaluated with the highest antioxidant activities of all identified beer constituents, reaching values of 10–17.5 (ORAC), 2.0–4.1 (HPS), and 1.1–6.1 μmol TE/μmol (LA) for hordatines A–C

Compositional and Sensory Characterization of Red Wine Polymers

After isolation from red wine by means of ultrafiltration and gel adsorption chromatography, the composition of the highly astringent tasting high-molecular weight polymers was analyzed by means of HPLC–MS/MS, HPLC–UV/vis, and ion chromatography after thiolytic, alkaline, and acidic depolymerization and, on the basis of the quantitative data obtained as well as model incubation experiments, key structural features of the red wine polymers were proposed. The structural backbone of the polymers seems to be comprised of a procyanidin chain with (−)-epicatechin, (+)-catechin, (−)-epicatechin-3-<i>O</i>-gallate units as extension and terminal units as well as (−)-epigallocatechin as extension units. In addition, acetaldehyde was shown to link different procyanidins at the A-ring via an 1,1-ethylene bridge and anthocyanins and pyranoanthocyanins were found to be linked to the procyanidin backbone via a C–C-linkage at position C(6) or C(8), respectively. Alkaline hydrolysis demonstrated the polymeric procyanidins to be esterified with various organic acids and phenolic acids, respectively. In addition, the major part of the polysaccharides present in the red wine polymeric fraction were found not to be covalently linked to procyanidins. Interestingly, sensory evaluation of individual fractions of the red wine polymers did not show any significant difference in the astringent threshold concentrations, nor in the astringency intensity in supra-threshold concentrations and demonstrated the mean degree of polymerization as well as the galloylation degree not to have an significant influence on the astringency perception

Label-free quantitative ¹H NMR spectroscopy to study low-affinity ligand–protein interactions in solution: A contribution to the mechanism of polyphenol-mediated astringency

<div><p>Nuclear magnetic resonance (NMR) spectroscopy is well-established in assessing the binding affinity between low molecular weight ligands and proteins. However, conventional NMR-based binding assays are often limited to small proteins of high purity and may require elaborate isotopic labeling of one of the potential binding partners. As protein–polyphenol complexation is assumed to be a key event in polyphenol-mediated oral astringency, here we introduce a label-free, ligand-focused ¹H NMR titration assay to estimate binding affinities and characterize soluble complex formation between proteins and low molecular weight polyphenols. The method makes use of the effects of NMR line broadening due to protein–ligand interactions and quantitation of the non-bound ligand at varying protein concentrations by quantitative ¹H NMR spectroscopy (qHNMR) using electronic reference to access in vivo concentration (ERETIC 2). This technique is applied to assess the interaction kinetics of selected astringent tasting polyphenols and purified mucin, a major lubricating glycoprotein of human saliva, as well as human whole saliva. The protein affinity values (<i>BC</i>_<i>50</i>) obtained are subsequently correlated with the intrinsic mouth-puckering, astringent oral sensation imparted by these compounds. The quantitative NMR method is further exploited to study the effect of carboxymethyl cellulose, a candidate “anti-astringent” protein binding antagonist, on the polyphenol–protein interaction. Consequently, the NMR approach presented here proves to be a versatile tool to study the interactions between proteins and low-affinity ligands in solution and may find promising applications in the discovery of bioactives.</p></div

Sensomics-Assisted Elucidation of the Tastant Code of Cooked Crustaceans and Taste Reconstruction Experiments

Sensory-guided fractionation by means of ultrafiltration and cation-exchange chromatography, followed by MS/MS quantitation, and taste re-engineering experiments revealed the key taste molecules coining the characteristic taste profile of the cooked meat of king prawns. Furthermore, quantitative analysis demonstrated that the taste differences between crustaceans are due to quantitative differences in the combinatorial code of tastants, rather than to qualitative differences in the tastant composition. Besides the amino acids glycine, l-proline, and l-alanine, the characteristic seafood-like sweet profile was found to be due to the sweet modulatory action of quaternary ammonium compounds, among which betaine, homarine, stachydrin, and trimethylamine-<i>N</i>-oxide were found as the key contributors on the basis of dose–activity considerations. Knowledge of this combinatorial tastant code provides the foundation for the development of more sophisticated crustacean flavors that are lacking any heavy metal ions and allergenic proteins present when using crustacean extracts for food flavoring

Unbound (-)-epigallocatechin gallate (EGCG) in pure buffer (reference), 10% (v/v) human whole saliva, 1% (w/v) carboxymethyl cellulose (CMC), and a combination of both.

<p>Unbound EGCG was quantified by single qHNMR measurements and is expressed as mole fractions. Error bars denote the standard deviation as obtained from integrating all the distinct, non-overlaid proton signals of EGCG. Data points behind means are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184487#pone.0184487.s003" target="_blank">S3 Table</a>.</p

Label-free quantitative ¹H NMR spectroscopy to study low-affinity ligand–protein interactions in solution: A contribution to the mechanism of polyphenol-mediated astringency - Fig 1

<p><b>Molecular structures of catechins and structurally related polyphenolic compounds.</b> (-)-epicatechin (a), (-)-epigallocatechin (b), (-)-epicatechin-3-gallate (c), (-)-epigallocatechin-3-gallate (d), methyl gallate (e), and quercetin-3-O-rutinoside (f).</p

Quantitation of Sweet Steviol Glycosides by Means of a HILIC-MS/MS-SIDA Approach

Meeting the rising consumer demand for natural food ingredients, steviol glycosides, the sweet principle of Stevia rebaudiana Bertoni (Bertoni), have recently been approved as food additives in the European Union. As regulatory constraints require sensitive methods to analyze the sweet-tasting steviol glycosides in foods and beverages, a HILIC-MS/MS method was developed enabling the accurate and reliable quantitation of the major steviol glycosides stevioside, rebaudiosides A–F, steviolbioside, rubusoside, and dulcoside A by using the corresponding deuterated 16,17-dihydrosteviol glycosides as suitable internal standards. This quantitation not only enables the analysis of the individual steviol glycosides in foods and beverages but also can support the optimization of breeding and postharvest downstream processing of <i>Stevia</i> plants to produce preferentially sweet and least bitter tasting <i>Stevia</i> extracts

Taste-Active Maillard Reaction Products in Roasted Garlic (<i>Allium sativum</i>)

In order to gain first insight into candidate Maillard reaction products formed upon thermal processing of garlic, mixtures of glucose and <i>S</i>-allyl-l-cysteine, the major sulfur-containing amino acid in garlic, were low-moisture heated, and nine major reaction products were isolated. LC-TOF-MS, 1D/2D NMR, and CD spectroscopy led to their identification as acortatarin A (<b>1</b>), pollenopyrroside A (<b>2</b>), <i>epi</i>-acortatarin A (<b>3</b>), xylapyrroside A (<b>4</b>), 5-hydroxymethyl-1-[(5-hydroxymethyl-2-furanyl)­methyl]-1<i>H</i>-pyrrole-2-carbalde-hyde (<b>5</b>), 3-(allylthio)-2-(2-formyl-5-hydroxymethyl-1<i>H</i>-pyrrol-1-yl)­propanoic acid (<b>6</b>), (4<i>S</i>)-4-(allylthiomethyl)-3,4-dihydro-3-oxo-1<i>H</i>-pyrrolo­[2,1-<i>c</i>]­[1,4]­oxazine-6-carbaldehyde (<b>7</b>), (2<i>R</i>)-3-(allylthio)-2-[(4<i>R</i>)-4-(allylthiomethyl)-6-formyl-3-oxo-3,4-dihydropyrrolo-[1,2-<i>a</i>]­pyrazin-2­(1<i>H</i>)-yl]­propanoic acid (<b>8</b>), and (2<i>R</i>)-3-(allylthio)-2-((4<i>S</i>)-4-(allylthiomethyl)-6-formyl-3-oxo-3,4-dihydropyrrolo-[1,2-<i>a</i>]­pyrazin-2­(1<i>H</i>)-yl)­propanoic acid (<b>9</b>). Among the Maillard reaction products identified, compounds <b>5</b>–<b>9</b> have not previously been published. The thermal generation of the literature known spiroalkaloids <b>1</b>–<b>4</b> is reported for the first time. Sensory analysis revealed a bitter taste with thresholds between 0.5 and 785 μmol/kg for <b>1</b>–<b>5</b> and <b>7</b>–<b>9</b>. Compound <b>6</b> did not show any intrinsic taste (water) but exhibited a strong mouthfullness (kokumi) enhancing activity above 186 μmol/kg. LC-MS/MS analysis showed <b>1</b>–<b>9</b> to be generated upon pan-frying of garlic with the highest concentration of 793.7 μmol/kg found for <b>6</b>, thus exceeding its kokumi threshold by a factor of 4 and giving evidence for its potential taste modulation activity in processed garlic preparations

Sensomics Analysis of Key Bitter Compounds in the Hard Resin of Hops (<i>Humulus lupulus</i> L.) and Their Contribution to the Bitter Profile of Pilsner-Type Beer

Recent brewing trials indicated the occurrence of valuable bitter compounds in the hard resin fraction of hop. Aiming at the discovery of these compounds, hop’s ε-resin was separated by means of a sensory guided fractionation approach and the key taste molecules were identified by means of UV/vis, LC-TOF-MS, and 1D/2D-NMR studies as well as synthetic experiments. Besides a series of literature known xanthohumol derivatives, multifidol glucosides, flavon-3-on glycosides, and <i>p</i>-coumaric acid esters, a total of 11 bitter tastants are reported for the first time, namely, 1″,2″-dihydroxanthohumol F, 4′-hydroxytunicatachalcone, isoxantholupon, 1-methoxy-4-prenylphloroglucinol, dihydrocyclohumulohydrochinone, xanthohumols M, N, and P, and isoxanthohumols M, N, and P, respectively. Human sensory analysis revealed low bitter recognition threshold concentrations ranging from 5 (co-multifidol glucopyranoside) to 198 μmol/L (<i>trans</i>-<i>p</i>-coumaric acid ethyl ester) depending on their chemical structure. For the first time, LC-MS/MS quantitation of these taste compounds in Pilsner-type beer, followed by taste re-engineering experiments, revealed the additive contribution of iso-α-acids and the identified hard resin components to be truly necessary and sufficient for constructing the authentic bitter percept of beer. Finally, brewing trails using the ε-resin as the only hop source impressively demonstrated the possibility to produce beverages strongly enriched with prenylated hop flavonoids

Pairwise comparison matrix to assess the relative astringency of polyphenols.

<p>Pairwise comparison matrix to assess the relative astringency of polyphenols.</p