Isolation, structure determination, synthesis, and sensory activity of N-phenylpropenoyl-L-amino acids from cocoa (Theobroma cacao)

Application of chromatographic separation and taste dilution analyses recently revealed besides procyanidins a series of N-phenylpropenoyl amino acids as the key contributors to the astringent taste of nonfermented cocoa beans as well as roasted cocoa nibs. Because these amides have as yet not been reported as key taste compounds, this paper presents the isolation, structure determination, and sensory activity of these amino acid amides. Besides the previously reported (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine (clovamide), (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-tyrosine (deoxyclovamide), and (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tyrosine, seven additional amides, namely, (+)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[4'-hydroxy-(E)-cinnamoyl]-L-aspartic acid, (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4'-hydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine, (+)-N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-aspartic acid, and (+)-N-[(E)-cinnamoyl]-L-aspartic acid, were identified for the first time in cocoa products by means of LC-MS/MS, 1D/2D-NMR, UV-vis, CD spectroscopy, and polarimetry, as well as independent enantiopure synthesis. Using the recently developed half-tongue test, human recognition thresholds for the astringent and mouth-drying oral sensation were determined to be between 26 and 220 micromol/L (water) depending on the amino acid moiety. In addition, exposure to light rapidly converted these [E]-configured N-phenylpropenoyl amino acids into the corresponding [Z]-isomers, thus indicating that analysis of these compounds in food and plant materials needs to be performed very carefully in the absence of light to prevent artifact formation.     

Sensory-guided decomposition of roasted cocoa nibs (Theobroma cacao) and structure determination of taste-active polyphenols

Sequential application of solvent extraction, gel permeation chromatography, and RP-HPLC in combination with taste dilution analyses, followed by LC-MS and 1D/2D-NMR experiments and thiolytic degradation, revealed that, besides theobromine and caffeine, the flavan-3-ols epicatechin, catechin, procyanidin B-2, procyanidin B-5, procyanidin C-1, [epicatechin-(4beta-->8)](3)-epicatechin, and [epicatechin-(4beta-->8)](4)-epicatechin were among the key compounds contributing to the bitter taste as well as the astringent mouthfeel imparted upon consumption of roasted cocoa. In addition, a series of quercetin, naringenin, luteolin, and apigenin glycopyranosides as well as a family of not previously identified amino acid amides, namely, (+)-N-[4'-hydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-aspartic acid, (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4'-hydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine, (+)-N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-aspartic acid, and (+)-N-(E)-cinnamoyl-L-aspartic acid, have been identified as key astringent compounds of roasted cocoa. Furthermore, (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-3-hydroxy-l-tyrosine (clovamide), (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-tyrosine (deoxyclovamide), and (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tyrosine, reported previously as antioxidants, have been found as contributors of cocoa's astringent taste. By means of the half-tongue test, the taste thresholds of flavan-3-ols and glycosides have been determined.     

Structures, sensory activity, and dose/response functions of 2,5-diketopiperazines in roasted cocoa nibs (Theobroma cacao)

The taste compounds inducing the blood-like, metallic bitter taste sensation reported recently for a dichloromethane extract prepared from roasted cocoa nibs were identified as a series of 25 diketopiperazines by means of HPLC degustation, LC-MS/MS, and independent synthesis. Among these 25 compounds, 13 cis-configured diketopiperazines, namely, cyclo(L-IIe-L-Phe), cyclo(L-Val-L-Leu), cyclo(L-Pro-L-Pro), cyclo(L-IIe-L-Pro), cyclo(L-Val-L-Tyr), cyclo(L-Ala-L-Tyr), cyclo(L-Phe-L-Ser), cyclo(L-Ala-L-IIe), cyclo(L-Leu-L-Phe), cyclo(L-Pro-L-Val), cyclo(L-Pro-L-Thr), cyclo(L-Pro-L-Tyr), and cyclo(L-Val-L-Val) were identified for the first time in cocoa. In addition, the taste recognition thresholds for the metallic as well as the bitter taste of the diketopiperazines were determined, and after quantitative analysis by using two diastereomeric diketopiperazines as the internal standards, the sensory impact of the diketopiperazines was evaluated on the basis of their dose-over-threshold (DoT) factors calculated as the ratio of the concentration and the threshold concentration of a compound. These data revealed DoT factors above 1.0 exclusively for cis-cyclo(L-Pro-L-Val), cis-cyclo(L-Val-L-Leu), cis-cyclo(L-Ala-L-Ile), cis-cyclo(L-Ala-L-Leu), and cis-cyclo(L-Ile-L-Pro), whereas all of the other diketopiperazines were present below their individual bitter taste threshold concentrations and should therefore not contribute to the cocoa taste. Because the DoT factors do not consider the nonlinear relationship between the concentration and gustatory response of an individual compound, we, for the first time, report on the recording of dose/response functions describing the human bitter taste perception of diketopiperazines more precisely.     

Molecular definition of the taste of roasted cocoa nibs (Theobroma cacao) by means of quantitative studies and sensory experiments

Sensory-guided decomposition of roasted cocoa nibs revealed that, besides theobromine and caffeine, a series of bitter-tasting 2,5-diketopiperazines and flavan-3-ols were the key inducers of the bitter taste as well as the astringent mouthfeel imparted upon consumption of roasted cocoa. In addition, a number of polyphenol glycopyranosides as well as a series of N-phenylpropenoyl-l-amino acids have been identified as key astringent compounds of roasted cocoa. In the present investigation, a total of 84 putative taste compounds were quantified in roasted cocoa beans and then rated for the taste contribution on the basis of dose-over-threshold (DoT) factors to bridge the gap between pure structural chemistry and human taste perception. To verify these quantitative results, an aqueous taste reconstitute was prepared by blending aqueous solutions of the individual taste compounds in their "natural" concentrations. Sensory analyses revealed that the taste profile of this artificial cocktail was very close to the taste profile of an aqueous suspension of roasted cocoa nibs. To further narrow down the number of key taste compounds, finally, taste omission experiments and human dose/response functions were performed, demonstrating that the bitter-tasting alkaloids theobromine and caffeine, seven bitter-tasting diketopiperazines, seven bitter- and astringent-tasting flavan-3-ols, six puckering astringent N-phenylpropenoyl-l-amino acids, four velvety astringent flavonol glycosides, gamma-aminobutyric acid, beta-aminoisobutyric acid, and six organic acids are the key organoleptics of the roasted cocoa nibs.     

Arylsulphatase activity of different latosol soils of Ghana cropped to cocoa (Theobroma cacao) and coffee (Coffea canephora var. robusta)

Summary A study was undertaken to investigate arylsulphatase activity in 15 soils cropped to cocoa (Theobroma cacao) and coffee (Coffea canephora var. robusta) in Ghana. The arylsulphatase activity was correlated positively and significantly with organic C, total N, and cation exchange capacity, and correlated negatively with acetate soluble sulphate. The enzyme was deactivated at an incubation temperature of over 60°C. Preheating and oven-drying of soils decreased arylsulphatase activity. Addition of 1.0 ml toluene during the assay resulted in a sharp decline in arylsuphatase activity. The addition of trace elements at a concentration of 1 ppm caused a reduction in soil arylsulphatase activity compared with that of the untreated samples.     

Identification of procyanidins in cocoa (Theobroma cacao) and chocolate using high-performance liquid chromatography/mass spectrometry

Monomeric and oligomeric procyanidins present in cocoa and chocolate were separated and identified using a modified normal-phase high-performance liquid chromatography (HPLC) method coupled with on-line mass spectrometry (MS) analysis using an atmospheric pressure ionization electrospray chamber. The chromatographic separation was achieved using a silica stationary phase in combination with a gradient ascending in polarity. This qualitative report confirms the presence of a complex series of procyanidins in raw cocoa and certain chocolates using HPLC/MS techniques. Although both cocoa and chocolate contained monomeric and oligomeric procyanidin units 2-10, only use of negative mode provided MS data for the higher oligomers (i.e., >pentamer). Application of this method for qualitative analysis of proanthocyanidins in other food products and confirmation of this method as a reliable quantitative tool for determining levels of procyanidins in cocoa, chocolate, and other food products are currently being investigated.     

Interactions between flavan-3-ols and poly(L-proline) studied by isothermal titration calorimetry: effect of the tannin structure

Interactions of proline-rich proteins (PRPs) with flavan-3-ols was studied using poly(L-proline) as a model protein by means of isothermal titration calorimetry (ITC). Several parameters were varied: (i) the galloylation and B-ring trihydroxylation of the flavan-3-ols (catechin, epicatechin, epicatechin gallate, and epigallocatechin gallate) and (ii) the degree of polymerization (monomers were compared to a mixture of oligomers with average degree of polymerization of 3.85). Large differences were observed between the flavan-3-ol monomers: no enthalpy change was measured when catechin and epicatechin were titrated by poly(L-proline), whereas thermodynamic parameters were determined in the case of galloylated monomers and mixture of oligomers. Stoichiometry ranged from 1 oligomer bound for each 12 proline units to 1 galloylated monomer for each 8 or 10 proline units. Association constants were in the range of 10(4)-10(5) M(-1), indicating a relatively high affinity of galloylated flavanols toward poly(L-proline), and the coexistence of both enthalpy- and entropy-driven phenomena was suggested. Finally, the binding of grape seed tannins to proteins was shown to be a cooperative process.     

Hydrogen peroxide and flavan-3-ols in storage roots of cassava (Manihot esculenta crantz) during postharvest deterioration

Cassava storage roots are an important staple food throughout the lowland humid tropics. However, cassava suffers from a poorly understood storage disorder, known as postharvest physiological deterioration (PPD), which constrains its exploitation. In an attempt to broaden the understanding of PPD, nine different cassava cultivars were analyzed for specific compounds accumulating during the process. The production of hydrogen peroxide (H(2)O(2)) is involved in the early stages of PPD in cassava roots. H(2)O(2) was quantified and localized histochemically at the tissue and cell level in deteriorating roots. This reactive oxygen species accumulated during the first 24 h after harvest, especially in the inner parenchymatic tissue. Three flavan-3-ols, (+)-catechin, (+)-catechin gallate, and (+)-gallocatechin, accumulated during the storage of cassava roots. However, these potential antioxidants cannot be related to early storage disorders or wound responses because they start to accumulate only after 4-6 days.     

High-performance liquid chromatography separation and purification of cacao (Theobroma cacao L.) procyanidins according to degree of polymerization using a diol stationary phase

A new chromatographic approach for separating cacao procyanidins according to their degree of polymerization has been developed. It utilizes diol stationary phase columns operating in normal phase mode with a binary gradient of acidified acetonitrile and methanol-water. Performance of the diol stationary phase was evaluated on an analytical scale utilizing classical chromatographic conditions for the normal phase separation of procyanidins according to their degree of polymerization. The new separation approach was developed on an analytical scale but further extended to the preparative scale. These newly developed analytical and preparative high-performance liquid chromatography procedures were successfully applied to the separation, as well as isolation, of cacao procyanidins from unfermented cacao seeds. The degree of polymerization associated with each molecular weight fraction was determined by mass spectrometry.     

Effect of zinc application on the dry matter yield uptake and distribution of zinc and other micronutrients in cocoa (Theobroma cacao. L.)

Abstract

The effects of application of zinc fertilizer on dry matter yield, uptake and distribution of zinc and other nutrients by Amazon and Amelonado cocoa cultivars grown in a soil of low zinc content in the greenhouse were investigated.

There was a differential response to zinc fertilization by the two cocoa cultivars. Maximum dry matter yields of Amazon and Amelonado were attained with 10 ppm Zn and 50 ppm Zn, respectively. Under similar experimental conditions Amelonado seedlings expressed zinc deficiency symptoms whereas Amazon did not.

Zinc concentrations in the leaves, stem and roots of both cuitivars did not give a good index of the zinc status of the crops. This was because of the existence of the “Piper‐Steenbjerg”; effect in that nil zinc rates often gave higher leaf concentration of the zinc than next higher rate. In general, the relative content of zinc followed the pattern; leaves > roots > stem with the Amazon cultivar containing more zinc than the Amelonado.

The distribution of absorbed Cu in the leaves, stem and roots did not differ in both varieties. Whereas Fe uptake was mostly concentrated in the roots, Mn absorbed was largely concentrated in the leaves of both varieties and only Mn uptake in the leaves of Amazon consistently increased with Zn application.

The differences in the uptake and distribution of nutrients between the two cocoa cultivars was attributed to differences in their ability to extract nutrients from the soil and in their requirements for metabolic processes.     

Antioxidant activity and radioprotective effects against chromosomal damage induced in vivo by X-rays of flavan-3-ols (Procyanidins) from grape seeds (Vitis vinifera): comparative study versus other phenolic and organic compounds

The quantitative distribution of several flavan-3-ols was determined using HPLC in a grape (Vitis vinifera) seed extract (GSE) of four cultivars grown in the region of Murcia. Polymer >/= C(4) units made up the largest group of procyanidins in the GSE (90.92%, expressed as HPLC % area). The antioxidant activity of GSE and other reference compounds was investigated by measuring their ability to scavenge the ABTS(*)(+) radical cation (TEAC). The most effective compounds were, in order: GSE > rutin > (+)-catechin > diosmin >/= ascorbic acid. The radioprotective effects of GSE and other reference compounds were determined by using the micronucleus test for anticlastogenic activity, any reduction of the frequency of micronucleated polychromatic erythrocytes (MnPCEs) being evaluated in the bone marrow of mouse exposed to X-rays. The most effective compounds were, in order: GSE > rutin > dimethyl sulfoxide (DMSO) > ascorbic acid > 6-n-propyl-2-thiouracil-6c (PTU) > diosmin. The higher ABTS(*)(+) scavenging capacity and anticlastogenic activity of GSE can be explained, structurally, by the high number of conjugated structures between the catechol groups in the B-rings and the 3-OH free groups of the polymeric polyphenolic skeleton and, in addition, by the stability of the aroxyl flavonoid radical generated in the above processes.     

Reinvestigation of the bitter compounds in carrots (Daucus carota L.) by using a molecular sensory science approach

In order to reinvestigate the key molecules inducing bitter off-taste of carrots ( Daucus carota L.), a sensory-guided fractionation approach was applied to bitter carrot extracts. Besides the previously reported bitter compounds, 6-methoxymellein (1), falcarindiol (2), falcarinol (3), and falcarindiol-3-acetate (4), the following compounds were identified for the first time as bitter compounds in carrots with low bitter recognition thresholds between 8 and 47 micromol/L: vaginatin (5), isovaginatin (6), 2-epilaserine oxide (7), laserine oxide (8), laserine (14), 2-epilaserine (15), 6,8-O-ditigloyl- (9), 6-O-angeloyl-, 8-O-tigloyl- (10), 6-O-tigloyl-, 8-O-angeloyl- (11), and 6-, 8-O-diangeloyl-6 ss,8alpha,11-trihydroxygermacra-1(10) E,4 E-diene (12), as well as 8-O-angeloyl-tovarol (13) and alpha-angeloyloxy-latifolone (16). Among these bitter molecules, compounds 9, 10, 13, and 16 were not previously identified in carrots and compounds 6, 11, and 12 were yet not reported in the literature.     

Genetic diversity and parentage in farmer varieties of cacao (Theobroma cacao L.) from Honduras and Nicaragua as revealed by single nucleotide polymorphism (SNP) markers

Cacao (Theobroma cacao L.) is the main source for chocolate with an annual production of four million tons worldwide. This Neotropical tree crop was domesticated in Mesoamerica as far back as 3,000 years ago. Knowledge of genetic diversity and population structure in farmer varieties of cacao in the center of domestication is essential for sustainable production of fine-flavored cacao beans and contributes to in situ/on-farm conservation of farmer varieties. Based on 70 single nucleotide polymorphism markers, we analyzed 84 fine-flavored farmer varieties collected from traditional cacao farms in Honduras and Nicaragua. The study also included 31 clones from the international cacao collections to serve as references. The SNP based multilocus matching identified six synonymous groups, including 14 Criollo and two Amelonado varieties. A moderately high level of genetic diversity was observed in these farmer varieties, indicating the possibility to further explore intra-population variation and breed for fine-flavored cocoa. Multivariate analysis showed clustering of the 84 farmer accessions in five genetic groups: ancient Criollo, Amelonado, Trinitario (including Nicaragua Trinitario and Honduras Trinitario) and Upper Amazon Forastero (only one accession). The Honduras Trinitario differed from the Nicaragua Trinitario group. The clustering results largely supported the perceived classification of cacao by local farmers and researchers, which was mainly based on morphological traits. However, the well known traditional variety “Indio” in this region was identified as synonymous with Amelonado. Parentage analysis showed that the variety “Indio” (or Amelonado) contributed more to the Trinitario type farmer varieties, whereas ancient Criollo had less influence. The present study demonstrates the efficacy of using a small set of SNP makers for cacao germplasm characterization, and further depicts the diverse origins and parentage in farmer varieties from Mesoamerica. This information thus will be highly useful for conservation and utilization of cacao germplasm from this region.     

Isolation, structure determination, and sensory activity of mouth-drying and astringent nitrogen-containing phytochemicals isolated from red currants (Ribes rubrum)

Application of chromatographic separation and taste dilution analyses recently revealed, besides a series of flavon-3-ol glycosides and (E)/(Z)-aconitic acid, four nitrogen-containing phytochemicals as the key astringent and mouth-drying compounds in red currants (Ribes rubrum). The isolation and structure determination of the astringent indoles 3-carboxymethyl-indole-1-N-beta-D-glucopyranoside (1) and 3-methylcarboxymethyl-indole-1-N-beta-D-glucopyranoside (2), as well as the astringent, noncyanogenic nitriles 2-(4-hydroxybenzoyloxymethyl)-4-beta-D-glucopyranosyloxy-2(E)-butenenitrile (3) and 2-(4-hydroxy-3-methoxybenzoyloxymethyl)-4-beta-D-glucopyranosyloxy-2(E)-butenenitrile (4) by means of 1D/2D NMR, LC-MS/MS, and UV-vis spectroscopy are reported. The structures of compounds 1 and 2 were confirmed by synthesis. Using the recently developed half-tongue test, human recognition thresholds for the astringent and mouth-drying nitrogen compounds were determined to be between 0.0003 and 5.9 micromol/L (water). In particular, the extraordinarily low threshold of 0.0003 micromol/L evaluated for the indole 1 represents the lowest recognition threshold of any astringent phytochemical reported to date.     

Biodegradation of oak (Quercus alba) wood during growth of the shiitake mushroom (Lentinula edodes): a molecular approach

The chemical transformations that occur during growth of the shiitake mushroom (Lentinula edodes) on oak (Quercus alba) were investigated to improve mushroom cultivation and utilization of the spent substrate. Oak logs were decayed by L. edodes over 8 years, during which time they were sampled at six intervals (30, 40, 66, 76, 77, and 101 months). Fresh and decayed oak samples were analyzed using solid-state (13)C NMR and pyrolysis-gas chromatography-mass spectrometry as well as off-line thermochemolysis with tetramethylammonium hydroxide. Degraded oak exhibited lower carbon contents and increased oxygen content compared to the control. Solid-state (13)C NMR analysis revealed that polysaccharides were the major component of both fresh and decayed oak but that L. edodes mediated the preferential loss of cellulose and xylans as compared to lignin, which remained in an altered form. Several trends point toward the degradation of lignin, including a decrease in the proportion of syringyl units as compared to guaiacyl units and a reduction in side-chain length. An increase in guaiacyl and syringyl acid-to-aldehyde ratios occurred with growth, which suggested that the fungus had caused oxidation of Calpha-Cbeta bonds. The overall effect of L. edodes on oak is similar to that of many white-rot fungi, which simultaneously degrade all cell wall components.     

Characterization of the key odorants in raw Italian hazelnuts ( Corylus avellana L. var. Tonda Romana) and roasted hazelnut paste by means of molecular sensory science

The concentrations of 19 odorants, recently characterized by GC-olfactometry and aroma extract dilution analysis as the most odor-active compounds in raw hazelnuts, were quantitated by stable isotope dilution assays (SIDA). Calculation of odor activity values (OAV) on the basis of odor thresholds in oil revealed high OAVs, in particular for linalool, 5-methyl-4-heptanone, 2-methoxy-3,5-dimethylpyrazine, and 4-methylphenol. A model mixture in sunflower oil containing the 13 odorants showing OAVs above 1 in their natural concentrations resulted in a good similarity compared to the overall nut-like, fruity aroma of the raw hazelnuts. Quantitation of the 25 most odor-active compounds in roasted hazelnut paste by SIDA showed clear changes in the concentrations of most odorants, and formation of new odor-active compounds induced by the roasting process was observed. The highest OAVs were calculated for 3-methylbutanal (malty), 2,3-pentanedione (buttery), 2-acetyl-1-pyrroline (popcorn), and (Z)-2-nonenal (fatty), followed by dimethyl trisulfide, 2-furfurylthiol, 2,3-butanedione, and 4-hydroxy-2,5-dimethyl-3(2H)-furanone. The aroma of a model mixture containing the 19 odorants with OAVs above 1 in their actual concentrations in the roasted nut material was judged to elicit a very good similarity to the popcorn-like, coffee-like, and sweet-smoky aroma of the roasted hazelnut paste. New SIDAs were developed for the quantitation of 5-methyl-4-heptanone, 5-methyl-(E)-2-hepten-4-one, 2-thenylthiol, and 3,5,5-trimethyl-2(5H)-furanone.     

Structural basis for antioxidant activity of trans-resveratrol: ab initio calculations and crystal and molecular structure

From the experimental crystal structure and ab initio calculations on resveratrol and its derivatives, structural features of mechanistic importance are described. The molecular structure reveals the relative coplanarity of the trans-stilbene skeleton, and the molecular packing in the solid state shows an extensive hydrogen bond network that elucidates the flip-flop motion of the three hydroxyl groups that alternately form and break H bonds with each of the neighboring phenolic oxygens. The dynamic behavior provoked by the alternation of hydrogen bond formation and breaking can result in the ready mobility of up to three hydrogen atoms per resveratrol molecule that can be transferred to reactive oxidants that are rich in electron density. In addition, theoretical studies confirm the planarity of resveratrol as well as for half of the molecule of a condensation dimeric derivative of resveratrol, trans-sigma-viniferin. Furthermore, these studies show the p-4'-OH group to be more acidic compared to the other two m-OH groups. These features correlate with the biological activity of resveratrol as an antioxidant and support earlier studies showing H-atom transfer to be the dominant mechanism by which phenolic antioxidants intercept free radicals.     

Binding of phthalate plasticizers to human serum albumin in vitro: a multispectroscopic approach and molecular modeling

As endocrine-disrupting chemicals, a few frequently used phthalate plasticizers were banned or restricted for use as additives in food in some countries. The interaction mechanisms between three phthalate plasticizers with human serum albumin (HSA) were studied by fluorescence (quenching, synchronous, and three-dimensional), UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy, in combination with molecular modeling under simulative physiological conditions, respectively. The results obtained from fluorescence quenching data revealed that the plasticizers-HSA interaction altered the conformational strcture of HSA. Meanwhile, the alterations of HSA secondary structure in the presence of phthalate plasticizers were investigated. The binding distances for the plasticizers-HSA system were provided by the efficiency of fluorescence resonance energy transfer. Furthermore, the thermodynamic analysis implied that hydrophobic forces were the main interaction for the plasticizers-HSA system, which agreed well with the results from the molecular modeling study.