Quantitative studies and sensory analyses on the influence of cultivar, spatial tissue distribution, and industrial processing on the bitter off-taste of carrots (Daucus carota l.) and carrot products

Although various reports pointed to 6-methoxymellein (1) as a key player imparting the bitter taste in carrots, activity-guided fractionation experiments recently gave evidence that not this isocoumarin but bisacetylenic oxylipins contribute mainly to the off-taste. Among these, (Z)-heptadeca-1,9-dien-4,6-diyn-3-ol (2), (Z)-3-acetoxy-heptadeca-1,9-dien-4,6-diyn-8-ol (3), and (Z)-heptadeca-1,9-dien-4,6-diyn-3,8-diol (falcarindiol, 4) have been successfully identified. In the present study, an analytical procedure was developed enabling an accurate quantitation of 1-4 in carrots and carrot products. To achieve this, (E)-heptadeca-1,9-dien-4,6-diyn-3,8-diol was synthesized as a suitable internal standard for the quantitative analysis of the bisacetylenes. On the basis of taste activity values, calculated as the ratio of the concentration and the human sensory threshold of a compound, a close relationship between the concentration of 4 and the intensity of the bitter off-taste in carrots, carrot puree, and carrot juice was demonstrated, thus showing that compound 4 might offer a new analytical measure for an objective evaluation of the quality of carrot products. Quantitative analysis on the intermediate products in industrial carrot processing revealed that removing the peel as well as green parts successfully decreased the concentrations in the final carrot puree by more than 50%.     

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.     

Effects of harvesting date and storage on the amounts of polyacetylenes in carrots, Daucus carota

The amounts of three main polyacetylenes in carrots; falcarinol, falcarindiol, and falcarindiol-3-acetate, were determined by HPLC, during three seasons, in carrots harvested several times per season and at different locations in Sweden. The amounts of falcarindiol first decreased from a relatively high level and then increased later in the harvest season. The amounts of falcarindiol-3-acetate showed similar variations, whereas the amounts of falcarinol did not exhibit any significant variation during the harvest season. During storage the amount of polyacetylenes leveled off, increasing in samples initially low and decreasing in samples initially high in polyacetylenes. The amounts of all polyacetylenes varied significantly due to external factors and between stored and fresh samples. This variation opens up possibilities to achieve a chemical composition of polyacetylenes at harvest that minimizes the risk of bitter off-taste and maximizes the positive health effects reported in connection with polyacetylenes in carrots.     

Determination and distribution of 6-methoxymellein in fresh and processed carrot puree by a rapid spectrophotometric assay.

Isocoumarin or 6-methoxymellein (6-MM) was extracted from carrot tissue using alkali saponification to solubilize the lactone portion of its structure into an aqueous phase. Acidification and subsequent organic solvent extraction allowed isolates to be quantified and verified as 6-MM by spectrophotometric determination. 6-Methoxymellein was analyzed in carrot cross sections, as a function of depth and before and after thermal processing. A natural propensity for 6-MM accumulation was observed in root tip sections exposed to ethylene, and levels increased as a result of wounding. Consecutive layer peeling demonstrated that small-diameter roots accumulated greater amounts of 6-MM in periderm tissue compared to large roots. Processing carrots into a puree resulted in 10-25% greater extraction of 6-MM than grinding fresh carrot samples, whereas steam-cooked and thermally processed purees had 15% greater extraction than unheated purees. This analytical technique will allow carrot processors to accurately estimate raw and processed products for the bitter compound 6-MM.     

Occurrence of 6-methoxymellein in fresh and processed carrots and relevant effect of storage and processing

The occurrence of 6-methoxymellein (6-MM) in fresh and conventionally processed carrot products (for a total of 176 samples) marketed in European locations and the effect of Alternaria spp. infection and storage conditions on 6-MM accumulation were investigated. 6-MM was found in 78% of tested samples with levels ranging from 0.02 to 76.00 microg/g, with only 1 of 79 fresh carrots exceeding the "just noticeable difference" level for 6-MM. Storage of carrots at 1 degree C was suitable to maintain low levels of 6-MM for a period of at least 17 weeks. No effect of Alternaria spp. infection was observed on 6-MM occurrence. The fate of 6-MM during carrot juice processing was also investigated by using different enzyme formulations for maceration and blanching procedures. Levels of 6-MM in blanched carrots obtained by boiling water or steam treatment were reduced by 69 or 33%, respectively, as compared to fresh carrots. No decrease in 6-MM levels was observed after maceration with pectinolytic enzyme preparations (Rapidase Carrot Juice and Ultrazym AFP-L). A reduction of 6-MM by 85 or 94% was obtained after the entire cycle of carrot juice processing, depending on the blanching procedure used.     

Bitter-tasting and kokumi-enhancing molecules in thermally processed avocado (Persea americana Mill.)

Sequential application of solvent extraction and RP-HPLC in combination with taste dilution analyses (TDA) and comparative TDA, followed by LC-MS and 1D/2D NMR experiments, led to the discovery of 10 C(17)-C(21) oxylipins with 1,2,4-trihydroxy-, 1-acetoxy-2,4-dihydroxy-, and 1-acetoxy-2-hydroxy-4-oxo motifs, respectively, besides 1-O-stearoyl-glycerol and 1-O-linoleoyl-glycerol as bitter-tasting compounds in thermally processed avocado (Persea americana Mill.). On the basis of quantitative data, dose-over-threshold (DoT) factors, and taste re-engineering experiments, these phytochemicals, among which 1-acetoxy-2-hydroxy-4-oxo-octadeca-12-ene was found with the highest taste impact, were confirmed to be the key contributors to the bitter off-taste developed upon thermal processing of avocado. For the first time, those C(17)-C(21) oxylipins exhibiting a 1-acetoxy-2,4-dihydroxy- and a 1-acetoxy-2-hydroxy-4-oxo motif, respectively, were discovered to induce a mouthfulness (kokumi)-enhancing activity in sub-bitter threshold concentrations.     

Volatile components and aroma active compounds in aqueous essence and fresh pink guava fruit puree (Psidium guajava L.) by GC-MS and multidimensional GC/GC-O

Characterization of the aromatic profile in commercial guava essence and fresh fruit puree by GC-MS yielded a total of 51 components quantified. Commercial essence was characterized to present a volatile profile rich in components with low molecular weight, especially alcohols, esters, and aldehydes, whereas in the fresh fruit puree terpenic hydrocarbons and 3-hydroxy-2-butanone were the most abundant components. In the olfactometric analyses totals of 43 and 48 aroma active components were detected by the panelists in commercial essence and fruit puree, respectively. New components were described for the first time as active aromatic constituents in pink guava fruit (3-penten-2-ol and 2-butenyl acetate). Principal differences between the aroma of the commercial guava essence and the fresh fruit puree could be related to acetic acid, 3-hydroxy-2-butanone, 3-methyl-1-butanol, 2,3-butanediol, 3-methylbutanoic acid, (Z)-3-hexen-1-ol, 6-methyl-5-hepten-2-one, limonene, octanol, ethyl octanoate, 3-phenylpropanol, cinnamyl alcohol, alpha-copaene, and an unknown component. (E)-2-Hexenal seems to be more significant to the aroma of the commercial essence than of the fresh fruit puree.     

Identification of bitter off-taste compounds in the stored cold pressed linseed oil

Whereas freshly pressed linseed oil provides a delicate nutty flavor, a lingering bitter off-taste is developing upon storage at room temperature. Using a sensory guided fractionation approach, the key bitter compound was identified in stored linseed oil, and its structure was determined as the methionine sulfoxide-containing, cyclic octapeptide cyclo(PLFIM OLVF) by means of FTIR, LC-MS, NMR spectroscopy, and amino acid analysis. Although this peptide is known in the literature as cyclolinopeptide E, the bitter taste activity of this compound has not previously been described. Sensory evaluation revealed a recognition threshold concentration of 12.3 micromol/L water.     

Chemical and sensory quality of processed carrot puree as influenced by stress-induced phenolic compounds

Physicochemical analysis of processed strained product was performed on 10 carrot genotypes grown in Texas (TX) and Georgia (GA). Carrots from GA experienced hail damage during growth, resulting in damage to their tops. Measurements included pH, moisture, soluble phenolics, total carotenoids, sugars, organic acids, and isocoumarin (6-MM). Sensory analysis was conducted using a trained panel to evaluate relationships between chemical and sensory attributes of the genotypes and in carrots spiked with increasing levels of 6-MM. Preharvest stress conditions in GA carrots seemed to elicit a phytoalexic response, producing compounds that impacted the perception of bitter and sour flavors. Spiking 6-MM into strained carrots demonstrated the role bitter compounds have in lowering sweetness scores while increasing the perception of sour flavor. Screening fresh carrots for the phytoalexin 6-MM has the potential to significantly improve the sensory quality of processed products.     

Detection of changes in taste of japonica and indica brown and milled rice (Oryza sativa L.) during storage using physicochemical analyses and a taste sensing system

Changes in the taste of japonica, hybrid, and indica brown and milled rice, stored for 10 months at low (5 degrees C, 65-70% relative humidity) and room temperatures were observed by physicochemical analyses and a novel method using a taste sensing system. During storage, some properties increased or decreased while others were fairly constant. The main taste components of cooked rice such as sweetness (sucrose) and umami tastes (glutamic acid and aspartic acid) were reduced during storage, whereas glucose and fructose increased. The increase of fat acidity and consequent decrease of the pH value of the cooking solution may contribute to the off-taste of cooked stored rice. A taste sensing system with 10 lipid membrane sensors was also used to classify new and old rice samples using principal component analysis. Fresh and room temperature stored japonica and indica rice could be clearly distinguished; however, it was not possible to differentiate the samples stored at low temperature.     

Cytotoxic phenylpropanoids from carrot

Carrot is widely used as a foodstuff. The active components such as beta-carotene and panaxynol have been studied by many researchers. In this investigation of nonpolar active components from carrot, a new phenylpropanoid, epilaserine oxide ( 3), was isolated along with six known compounds, laserine ( 1), 2-epilaserine ( 2), panaxynol ( 4), ginsenoyne K ( 5), (8 E)-1,8-heptadecadiene-4,6-diyne-3,10-diol ( 6), and vaginatin ( 7). Their structures were deduced on the basis of spectroscopic methods. Significant cytotoxicity of 2-epilaserine against HL-60 cells was observed, which implied that phenylpropanoids were cytotoxic compounds in carrot. Laserine and 2-epilaserine in carrots from diverse locations in China were quantified by HPLC.     

Changes in volatile compounds of carrots (Daucus carota L.) during refrigerated and frozen storage

Carrots (Daucus carota L.) of cv. Bolero and cv. Carlo were processed into shreds and stored for up to 4 months at -24 degrees C (frozen storage), or the roots were stored for up to 4 months at 1 degrees C (refrigerated storage) followed by processing into shreds. Volatiles from the carrot shreds were collected by dynamic headspace technique and analyzed by GC-FID, GC-MS, GC-MS/MS, and GC-O to determine the volatile composition and aroma active components of carrots stored under different temperature conditions. A total of 52 compounds were quantified, of which mono- and sesquiterpenes accounted for approximately 99% of the total volatile mass. Major volatile compounds were (-)-alpha-pinene, beta-myrcene, (-)-limonene, (+)-limonene, (+)-sabinene, gamma-terpinene, p-cymene, terpinolene, beta-caryophyllene, alpha-humulene, and (E)- and (Z)-gamma-bisabolene. A considerable increase in the concentration of mono- and sesquiterpenes was observed during refrigerated storage, whereas the concentration of terpenoids was around the same level during frozen storage. GC-O revealed that the major volatiles together with (+)-alpha-pinene, (-)-beta-pinene, (+)-beta-pinene, 6-methyl-5-hepten-2-one, (-)-beta-bisabolene, beta-ionone, and myristicin had an odor sensation, which included notes of "carrot top", "terpene-like", "green", "earthy", "fruity", "citrus-like", "spicy", "woody", and "sweet".     

Influence of L-cysteine on the formation of bitter-tasting aminohexose reductones from glucose and L-proline: identification of a novel furo[2,3-b]thiazine.

Thermal treatment of a 1 + 1 mixture of glucose and L-proline led to the development of an intense bitter taste being reflected in high amounts of the bitter-tasting bispyrrolidino- (1) and pyrrolidinohexose reductones (2) formed. Heating the reaction mixture in the presence of L-cysteine drastically reduced the amounts of these aminohexose reductones and, thereby, the intensity of the bitter taste. Studies on the mechanism of the cysteine-induced reduction of the bitter taste revealed that the precursor of the aminohexose reductones, the hexose-derived acetylformoin (3), reacted more easily with L-cysteine to form the 7-hydroxy-4a,6-dimethyl-2H,3H,4aH-furo[2,3-b]thiazine (4), a previousely unknown Maillard reaction product, than with L-proline to the aminohexose reductones 1 and 2, thereby blocking the formation of bitter-tasting compounds.     

Purple carrot (Daucus carota L.) polyacetylenes decrease lipopolysaccharide-induced expression of inflammatory proteins in macrophage and endothelial cells

Carrots ( Daucus carota L.) contain phytochemicals including carotenoids, phenolics, polyacetylenes, isocoumarins, and sesquiterpenes. Purple carrots also contain anthocyanins. The anti-inflammatory activity of extracts and phytochemicals from purple carrots was investigated by determining attenuation of the response to lipopolysaccharide (LPS). A bioactive chromatographic fraction (Sephadex LH-20) reduced LPS inflammatory response. There was a dose-dependent reduction in nitric oxide production and mRNA of pro-inflammatory cytokines (IL-6, IL-1beta, TNF-alpha) and iNOS in macrophage cells. Protein secretions of IL-6 and TNF-alpha were reduced 77 and 66% in porcine aortic endothelial cells treated with 6.6 and 13.3 microg/mL of the LH-20 fraction, respectively. Preparative liquid chromatography resulted in a bioactive subfraction enriched in the polyacetylene compounds falcarindiol, falcarindiol 3-acetate, and falcarinol. The polyacetylenes were isolated and reduced nitric oxide production in macrophage cells by as much as 65% without cytotoxicity. These results suggest that polyacetylenes, not anthocyanins, in purple carrots are responsible for anti-inflammatory bioactivity.     

Comparison of volatiles, phenolics, sugars, antioxidant vitamins, and sensory quality of different colored carrot varieties

Four different colored carrots, orange, purple with orange core, yellow, and white, were examined for their content of phenolics, antioxidant vitamins, and sugars as well as their volatiles and sensory responses. A total of 35 volatiles were identified in all carrots, 27 positively. White carrot contained the highest content of volatiles, followed by orange, purple, and yellow. In total, 11, 16, 10, and 9 phenolic compounds were determined for the first time in orange, purple, yellow, and white carrots, respectively. Of these, chlorogenic acid was the most predominant phenolic compound in all carrot varieties. Differences (p < 0.05) in relative sweetness, the contents of vitamin C and alpha- and beta-carotenes, and certain flavor characteristics were observed among the colored carrot varieties examined. Purple carrots contained 2.2 and 2.3 times more alpha- and beta-carotenes (trace in yellow; not detected in white) than orange carrots, respectively. Purple carrot may be used in place of other carrot varieties to take advantage of its nutraceutical components.     

Orosensory-directed identification of astringent mouthfeel and bitter-tasting compounds in red wine

Application of sequential solvent extraction, followed by HPLC combined with the taste dilution analysis, enabled the localization of the most intense velvety astringent, drying, and puckering astringent, as well as bitter-tasting, compounds in red wine, respectively. Isolation of the taste components involving gel adsorption chromatography, ultrafiltration, and synthesis revealed the identification of 26 sensory-active nonvolatiles, among which several hydroxybenzoic acids, hydroxycinnamic acids, flavon-3-ol glycosides, and dihydroflavon-3-ol rhamnosides as well as a structurally undefined polymeric fraction (>5 kDa) were identified as the key astringent components. In contradiction to literature suggestions, flavan-3-ols were found to be not of major importance for astringency and bitter taste, respectively. Surprisingly, a series of hydroxybenzoic acid ethyl esters and hydroxycinnamic acid ethyl esters were identified as bitter compounds in wine. Taste qualities and taste threshold concentrations of the individual wine components were determined by means of a three-alternative forced-choice test and the half-mouth test, respectively.     

Application of hydrophilic interaction liquid chromatography/comparative taste dilution analysis for identification of a bitter inhibitor by a combinatorial approach based on Maillard reaction chemistry

Activity-directed fractionation of heated carbohydrate/alanine solutions recently led to the discovery of (+)-(S)-1-(1-carboxyethyl)-5-hydroxy-2-(hydroxymethyl)pyridinium inner salt (1, alapyridaine), and it has been shown that this compound lowers the detection thresholds of sugars, glutamate, and NaCl solutions, whereas no influence on bitter perception was observed. As this class of Maillard-derived pyridinium betaines seemed to be promising targets for further research on their taste modulatory activity, the objective of the present investigation was to screen for bitter taste-suppressing target molecules in combinatorial libraries of pyridinium betaines prepared from 5-(hydroxymethyl)furan-2-aldehyde and amino acid mixtures by use of Maillard-type reaction chemistry instead of synthesizing and purifying each derivative individually. By application of hydrophilic interaction liquid chromatography in combination with the recently developed comparative taste dilution analysis, followed by structure determination, synthesis, and sensory studies, we have now succeeded in identifying 1-carboxymethyl-5-hydroxy-2-hydroxymethylpyridinium inner salt (2) as a potential bitter-suppressing candidate. While tasteless on its own, 2 was found to reduce the bitterness of various bitter tastants such as the amino acid L-phenylalanine, the peptide Gly-Leu, the alkaloid caffeine, and the bitter glycosides salicin and naringin.     

Identification of enterodiol as a masker for caffeine bitterness by using a pharmacophore model based on structural analogues of homoeriodictyol

Starting from previous structure-activity relationship studies of taste modifiers based on homoeriodictyol, dihydrochalcones, deoxybenzoins, and trans-3-hydroxyflavones as obvious analogues were investigated for their masking effect against caffeine. The most active compounds of the newly investigated taste modifiers were phloretin, the related dihydrochalcones 3-methoxy-2',4,4'-trihydroxydihydrochalcone and 2',4-dihydroxy-3-methoxydihydrochalcone, and the deoxybenzoin 2-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)ethanone. Starting with the whole set of compounds showing activity >22%, a (Q)SAR pharmacophore model for maskers of caffeine bitterness was calculated to explain the structural requirements. After docking of the pharmacophore into a structural model of the broadly tuned bitter receptor hTAS2R10 and docking of enterolactone and enterodiol as only very weakly related structures, it was possible to predict qualitatively their modulating activity. Enterodiol (25 mg L(-1)) reduced the bitterness of the 500 mg L(-1) caffeine solution by about 30%, whereas enterolactone showed no masking but a slight bitter-enhancing effect.     

Investigation of bound aroma constituents of yellow-fleshed nectarines (Prunus persica L. Cv. Springbright). changes in bound aroma profile during maturation

Glycosidically bound volatile constituents of yellow-fleshed clingstone nectarines (cv. Springbright) were identified and quantified at three stages of maturity. Glycoconjugates were isolated by LC on a C(18) reversed phase column with methanol elution followed by hydrolysis with a commercial pectinase enzyme. Forty-five bound aglycons were identified for the first time in yellow-fleshed nectarine. Thirty were terpene derivatives, and the most abundant ones were (E)- and (Z)-furan linalool oxides, linalool, alpha-terpineol, (E)-pyran linalool oxide, 3,7-dimethylocta-1,5-diene-3,7-diol, linalool hydrate, 8-hydroxy-6,7-dihydrolinalool, (E)- and (Z)-8-hydroxylinalools, and (E)- and (Z)-8-hydroxygeraniols. The group of C(13) norisoprenoids included 3-hydroxy-beta-damascone, 3-hydroxy-7,8-dihydro-beta-ionone, 3-oxo-alpha-ionol, 3-hydroxy-7,8-dihydro-beta-ionol, 3-hydroxy-beta-ionone, 3-oxo-7,8-dihydro-alpha-ionol, 3-hydroxy-5,6-epoxy-beta-ionone, 3-oxo-retro-alpha-ionol (isomers I and II), 3-hydroxy-7,8-dehydro-beta-ionol, 4,5-dihydrovomifoliol, and vomifoliol. Generally, levels of bound compounds, in particular monoterpenols and C(13) norisoprenoids, increased significantly with maturation. delta-Decalactone was the only lactone found in the enzymatic hydrolysate of yellow-fleshed nectarine, but its level was much lower than that of its free form.     

Aroma active components in aqueous kiwi fruit essence and kiwi fruit puree by GC-MS and multidimensional GC/GC-O

Gas chromatography-mass spectrometry (GC-MS) and multidimensional gas chromatography olfactometry (GC/GC-O) were utilized to study the aroma profile and the aroma active components of commercial kiwi essence and the initial fresh fruit puree. Totals of 29 and 33 components were identified and quantified in the essence and the puree, respectively. Ten components were quantified for the first time as constituents of the kiwi fruit including 3-penten-2-ol, 3-hydroxy-2-butanone, 3-methyl-2-butenal, 2-hexanol, nonanal, 3-methyl-1-butanol, 2-methyl-1-butanol, 3-methyl-2-butanone, 3-methyl 3-buten-2-one, and octane. Analysis of these samples by multidimensional gas chromatography-olfactometry (GC-O) allowed for the identification of >80% of the aroma active components present at level traces in this fruit. A total of 35 components appear to contribute to the aroma of kiwi fresh puree and its aqueous essence. Components described for the first time as constituents of the aroma profile in this fruit were 2-ethylfuran, 3-methyl-1-butanol, 2-cyclohexen-1-one, (E,E)-2,6-nonadienal, diethyl succinate, and hexyl hexanoate.