Proanthocyanidins and Their Contribution to Sensory Attributes of

May 18, 2015 - Black currant (Ribes nigrum L.) is used for the production of juices, berry wines, nectars, jams, and nutraceutical ingredients. Pectin...
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Proanthocyanidins and Their Contribution to Sensory Attributes of Black Currant Juices Oskar A. Laaksonen,*,† Juha-Pekka Salminen,‡ Leenamaija Mak̈ ila,̈ † Heikki P. Kallio,† and Baoru Yang† †

Food Chemistry and Food Development, Department of Biochemistry, and ‡Laboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of Turku, FI-20014 Turku, Finland S Supporting Information *

ABSTRACT: Black currant juices from five different cultivars were analyzed for composition, content, and mean degree of polymerization (mDP) of proanthocyanidins (PA) by UPLC-MS/MS. Juices contained both procyanidins (PC) and prodelphinidins (PD), but the PC-% varied significantly, from 28 to 82% of the total PA. In addition, high PD-% was related to high mDP and total PA content. Enzyme-assisted processing increased significantly total PA (5−14-fold), PD-% (12−65%), and mDP (1.8−6.2-fold) in the juices of all cultivars. Enzymatic treatment increased the contents of large PAs more than those of small PAs. The contents of PA and mDP were positively associated with the mouth-drying and puckering astringent characteristics. However, the PA content did not contribute to the bitter taste. Juices from the most bitter cultivars had the lowest contents of proanthocyanidins regardless of the processing method. This finding indicates the existence of other bitter compounds in black currants in addition to PA. KEYWORDS: astringency, bitterness, black currant, juice processing, proanthocyanidins

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astringency.5 High galloylation of PA structure may result in a rougher astringent sensation with increased “coarse” attribute.9 Monomeric flavan-3-ols and low molecular weight PAs have been reported to be more bitter than astringent, whereas higher molecular weight PAs are generally more astringent than bitter.14,15 The high (−)-epicatechin concentration was significantly more bitter and astringent than the equal concentration of (+)-catechin.16 In addition to proanthocyanidins, flavonol glycosides and various phenolic acid derivatives have been reported to elicit astringent sensations.17−22 Compounds belonging to the latter two classes have significantly lower sensory threshold levels for astringency than those of the former.17,18,20 Bitter taste is mediated by G protein-coupled receptors of the taste receptor 2 family (TAS2Rs). In general, TAS2Rs are sensitive to several or multiple different bitter compounds. In a recent study, various monomeric flavonoids, including (+)-catechin, (−)-epicatechin, and (−)-epigallocatechin, activated the human bitter taste receptors TAS2R14 and TAS2R39.23 Soares and co-workers24 showed that procyanidin trimer C2 (catechin(4α→8)-catechin-(4α→8)-catechin) and (−)-epicatechin activated TAS2R5 and the latter compound also activated TAS2R4 and TAS2R39. On the other hand, procyanidin dimer B2 (catechin-(4α→8)-catechin) did not activate any receptor in their study. Black currant (Ribes nigrum L.) is used for the production of juices, berry wines, nectars, jams, and nutraceutical ingredients. Pectinolytic enzymes are commonly used in industrial berry processing to increase the juice yield. Simultaneously, the

INTRODUCTION Flavonoids are present especially in plant-derived foods and beverages. Proanthocyanidins (PA), often referred to as condensed tannins, form the most common group of tannins in plants. Proanthocyanidins are oligomeric and polymeric tannins composed of different types of flavan-3-ol units. Proanthocyanidins formed from monomeric (+)-catechin and/or (−)-epicatechin units are called procyanidins (PC), whereas the other common group, prodelphinidins (PD), consists of (+)-gallocatechin and/or (−)-epigallocatechin units. Commonly the monomeric units are linked by B-type (C−C; 4α→8 or 4α→6) or A-type (C−O−C) bonds. Proanthocyanidins are generally linked to astringent and bitter sensory properties in foods. The mechanisms of the astringent sensations have been widely studied, but are not yet well understood.1−4 A commonly accepted hypothesis is that polymeric tannins binding and precipitating salivary proteins are detected as a drying and rough sensation in the mucous membranes. Astringency is considered as a trigeminal sensation and often dependent on oral movement.5 The binding to salivary proteins may be affected by the size and molecular flexibility of the tannins tested.6 Structural characteristics of tannins contribute to different sensory properties. Procyanidins and galloyl glucoses are perceived as more astringent than ellagitannins (hydrolyzable tannins), but the latter have lower thresholds for detection of astringency.7 Additionally, some astringent compounds do not bind to salivary proteins and thus elicit the sensation by different mechanisms.8 The mean degree of polymerization (mDP) of PAs has been associated with astringency.9−11 However, structural characteristics of the polymeric compounds are more important factors.12,13 High mDP and high percentage of galloylation in the subfractions activate trigeminal neurons, resulting in high © 2015 American Chemical Society

Received: Revised: Accepted: Published: 5373

March 12, 2015 May 13, 2015 May 18, 2015 May 18, 2015 DOI: 10.1021/acs.jafc.5b01287 J. Agric. Food Chem. 2015, 63, 5373−5380

Article

Journal of Agricultural and Food Chemistry

Table 1. Mean Degree of Polymerization (mDP) and Total Contents of Proanthocyanidins (Milligrams per 100 mL) in the Juicesa Mortti mDP PC total PD total PA total PA 1−3 PA others PC:PD ratio

2.2 8.66 ± 0.6 1.94 ± 0.1 10.6 4.4 6.2 82:18

mDP* PC total* PD total* PA total PA 1−3 PA others PC:PD ratio

13.7 16.2 ± 1.1 136 ± 5.4 152 24.3 128 17:83

Ola

Breed15

Nonenzymatic Process 7.0 4.7 8.66 ± 0.6 5.58 ± 22.1 ± 0.9 8.33 ± 30.8 13.9 7.7 5.5 23.1 8.4 28:72 40:60 Enzymatic Process 16.2 11.0 31.1 ± 2.2 13.2 ± 151 ± 6.0 70.2 ± 182 83.4 21.6 15.2 160 68.1 16:84 14:86

0.4 0.3

0.9 2.8

Marski

Mikael

4.3 5.08 ± 0.4 5.01 ± 0.2 10.1 3.3 6.8 50:50

7.7 6.63 ± 0.5 11.8 ± 0.5 18.4 4.5 13.9 36:64

10.9 11.6 ± 0.8 76.5 ± 3.1 88.1 18.1 69.9 13:87

14.5 17.4 ± 1.2 109 ± 4.3 126 17.5 109 11:89

a

Total contents of proanthocyanidins (PA total) is the sum of PC (procyanidins) and PD (prodelphinidins) Total and PC:PD ratio are calculated from the total values; PA 1−3 is the sum of monomers, dimers, and trimers (from Supplementary Tables 1 and 2). PA others is the remainder of PA total and PA 1−3 indicating higher level of polymerization. Values after ± show the maximum deviations by the method.33 Statistically significant differences between averaged processes are based on Student’s t test (p < 0.05). Differences are presented as * higher contents in enzymatic process. UPLC-MS/MS, Waters, MA, USA) including a binary solvent manager, a 96-place sample manager, a PDA detector, and a Xevo TQ triple-quadrupole mass spectrometer equipped with an electrospray interface. The column was a Waters Acquity UPLC BEH Phenyl (1.7 μm, 2.1 × 100 mm). Purified proanthocyanidins were used as external standards.33 The standards were obtained from dried black currant leaves with Sephadex LH-20 gel chromatography.34 The Sephadex fractions were further fractionated by semipreparative HPLC using a Waters Delta 600 system equipped with a Fraction Collector III. The column used was Phenomenex Gemini (150 × 21.2 mm, 10 μm, C18, 110 Å). The purified compounds were used with a Waters XEVO triple-quadrupole mass spectrometer to develop compound-specific multiple reaction monitoring (MRM) methods and quantitative calibration curves.33 Sensory Evaluations. The sensory evaluation process using generic descriptive analysis and the sensory characteristics of the juice samples are described by Laaksonen et al.29 The nonenzymatic and enzymatic juice samples were assessed by two separate trained sensory panels (n = 14 and n = 13, respectively) in controlled sensory laboratory conditions (according to ISO8589). The data were collected using Compusense-five software (Compusense Inc., Guelph, Canada). Of the nine attributes examined by Laaksonen et al.,29 only taste (sourness, sweetness, and bitterness) and two astringency (mouth-drying and puckering) attributes were selected for this study to examine their correlations with proanthocyanidins. Statistical Analyses. Differences between the processes were analyzed by Student’s t test (p < 0.05; IBM SPSS Statistics; IBM Corp., Armonk, NY, USA). A partial least-squares regression (PLS) method was applied for standardized data with X-variables (predictors) as chemical compounds and/or their ratios and Y-variables (responses) as the sensory properties. Full cross-validation was used to estimate the number of factors for a statistically reliable model. Multivariate models were conducted using Unscrambler 10.3 (Camo Process AS, Oslo, Norway).

contents of phenolic compounds of juices increases significantly,25−27 as do the astringent and bitter sensory properties.28−30 Moreover, a high content of phenolic compounds in a black currant juice may have a negative impact on liking.30 We have previously examined the roles of sugars, fruit acids, and smaller phenolic compounds, such as anthocyanins, flavonols, and hydroxycinnamic acids, in the sensory profiles of juices produced from various black currant cultivars.29 Especially, various flavonol glycosides and hydroxycinnamic acid derivatives contribute to astringency in black currant juices.28−30 In addition to the aforementioned phenolic compounds, black currants contain also significant amounts of proanthocyanidins.31,32 The aim of the present study was to investigate the proanthocyanidin profiles and contents in black currant juice samples from our previous study.29 Enzyme-aided processing was compared with nonenzymatic juice pressing using five different black currant cultivars produced at laboratory scale. The second aim was to investigate the contribution of the compounds to the sensory properties of black currant juices and especially examine the contribution of flavan-3-ols to astringency and bitterness in the juices.

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MATERIALS AND METHODS

Black Currant Juice Samples. Juice samples were as introduced by Laaksonen et al.29 Berries were harvested when optimally ripe in 2010 (four commercial cultivars, ‘Mortti’, ‘Mikael’, ‘Marski’, and ‘Ola’, and a new breed no. 15, ‘Breed15’) in southern Finland (MTT Piikkiö, Agrifood Research, Finland). Berries were frozen immediately after harvesting and kept at −20 °C before processing. Two juice-processing methods were applied at laboratory scale from berries of each cultivar. The first process was carried out in 2011 without enzymes and the second in 2012 with the aid of commercial enzymes (Pectinase 714L, Biocatalysts Ltd., Cardiff, UK; dosage = 57 mg of enzyme/380 g of berry mash). All juice samples were frozen at −20 °C for further analyses (sensory evaluations in 2011 for nonenzymatic and in 2012 for enzyme-assisted juices; chemical analyses in 2012). Proanthocyanidin Analyses. Qualitative and quantitative analyses of proanthocyanidins were carried out according to method of Enström et al.33 Proanthocyanidins were separated with Sephadex LH20 gel and analyzed with UPLC-MS/MS (Waters Acquity Xevo

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RESULTS AND DISCUSSION Proanthocyanidins in Juice Samples. Mean degree of polymerization (mDP in Table 1) indicates the average number of flavan-3-ol monomeric units present in the numerous oligomeric and polymeric condensed tannins present in the juice samples analyzed. The mDP value was significantly higher 5374

DOI: 10.1021/acs.jafc.5b01287 J. Agric. Food Chem. 2015, 63, 5373−5380

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Journal of Agricultural and Food Chemistry

Figure 1. Differences in proanthocyanidin profiles (%; 100% is the sum of PA 1−3 and PA others, two monomers, four dimers, or eight trimers) of the averaged juices: (A) proportions of the sum of monomers, dimers, and trimers (1−3) in comparison to the sum of tetramers or higher level of polymerization (others); (B) monomer profiles; (C) dimer profiles; (D) trimer profiles. Statistical differences between the juice types are marked with ∗ (t test, p < 0.05).

in the enzyme-aided juices (3.1-fold higher on average) than in the nonenzymatic juices in all cultivars. Cultivar ‘Mortti’ had the notably lowest value of mDP among the nonenzymatic; this cultivar had the highest increase in mDP when treated with enzymes. In general, black currants have been reported to have higher mDP than several other small edible berries.32,35 Freezing of berries can have an impact on their chemical composition. The freezing period between sensory evaluations and the chemical analyses may have had some minor impacts on the mDP values of the juice samples.36 However, the phenolic compounds of black currants have been reported to be more stable than the compounds of other common berries, such as strawberries or bilberries.37 Additionally, black currant procyanidin contents remain relatively stable during storage at −20 °C.38 Condensed tannins in black currant juices consist of procyanidins (PC) and prodelphinidins (PD). The contents of both were significantly higher in the enzymatic than in the nonenzymatic juices (Table 1). Interestingly, the juice of ‘Mortti’ from the nonenzymatic process had notably more PC than PD, whereas it was vice versa in the corresponding products of ‘Ola’, ‘Mikael’, and ‘Breed15’, which had more PD than PC. The nonenzymatic juice process represented the

original berry pulp without seeds or berry skins. On the other hand, the enzyme released various phenolic compounds from the berry skins to the juice. The PC:PD ratio was very similar in all enzyme-aided juices (average 14:86), which indicated the higher PD contents compared to PC in the berry skins across the five cultivars. Currently, only a few studies have focused on the analysis of the proanthocyanidin (PA) composition in black currants. In the study of Wu et al.39 the proanthocyanidins were characterized only by chain length. Liu et al.40 reported PD monomers, dimers, and trimers in black currant leaves and buds. In the current study, we characterized and quantified the whole PA content, including larger oligomers and polymers as well as monomeric, dimeric, and trimeric PAs, by using a novel group-specific MRM technology.33 Enzyme-aided juices contained more PAs than the juices without enzymatic assistance (Table 1). The proportions of monomeric, dimeric, and trimeric PAs (from Supplementary Tables Tables 1 and 2) of the total PAs varied between 24 and 41% and between 12 and 21% in the nonenzymatic and enzyme-aided juices, respectively. Thus, the majority of the PAs found in black currant juices had a polymerization degree >3. The contents of these compounds (PA others in Table 1 and Figure 1) were 6.9−20.4 times 5375

DOI: 10.1021/acs.jafc.5b01287 J. Agric. Food Chem. 2015, 63, 5373−5380

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Journal of Agricultural and Food Chemistry

Figure 2. PLS correlation loadings plot of the interaction between taste and astringencies (Y, n = 5) and proanthocyanidin measures (X, n = 35) in all 10 juice samples from both processes. Samples are in green font, sensory attributes are in red italic font, and compounds are in blue. For abbreviations of compounds refer to Table 1 and Supplementary Tables 1 and 2.

proportion of the most abundant dimer (dimer 3 in PC−PC and dimer 4 in PC−PD and PD−PD dimers) was found in significantly higher proportions in enzyme-treated juices. At the same time, the proportion of one dimer (dimer 2 in PC−PC, 3 in PC−PD, and 1 in PD−PD) was higher in nonenzymatic juices than in the enzyme-treated juices. In addition to significant increases in the contents of PAs, the enzymeassisted juice processing significantly altered the flavan-3-ol composition in the juice by releasing PAs from the berry skins. Trimers made up of prodelphinidin units only (PD−PD− PD) were found in the juices (Supplementary Table 2). One PD−PD−PD trimer (gallocatechin-(4α→8)-gallocatechin(4α→8)-gallocatechin) and one PC−PD−PD trimer (catechin-(4α→8)-gallocatechin-(4α→8)-gallocatechin) have earlier been found in black currant leaves.42,43 The enzyme treatment, again, increased significantly the content of trimeric PAs in the juice. Trimers 3 and 7 were the most abundant in general, and they were significantly more abundant in the juices processed with pectinase. Interestingly, one trimer (PD trimer 8), the most hydrophobic of the PD−PD−PD trimers, was found in the nonenzymatic juice of cultivar ‘Mortti’ but not in the corresponding enzymatic juice or juices from other cultivars. Impact of Juice Processing on Sensory Quality. PLS regression model was used to study the interactions between proanthocyanidins and the sensory attributes (Figure 2). PAs in the model are the compounds or compound ratios from Table 1 and Supplementary Tables 1 and 2 (total of 35 X-variables). Three taste attributes (bitterness, sourness, and sweetness) and two astringent qualities (mouth-drying and puckering astringencies) were selected for the model from Laaksonen et al.29 with a total of five Y-variables. Eighty-eight percent of the variation in X-data explains 55% of the variation in Y with two factors. In the model, proanthocyanidin variation explains 92.4% of the variation (90.0% in validated model) in mouthdrying astringency with one validated factor. Almost all of the X-variables are located on the left side of the plot with the astringency variable. All enzyme-aided juices are located also on the left side of the plot, which indicates that proanthocyanidins have a significant role in the increased mouth-drying astringency in these juices. Especially, a higher degree of polymerization (mDP) and a lower content of PC units in ratio

higher in enzyme-aided juices than in the juices produced without enzymes. The individual PAs found in the samples by the compoundspecific MRM methods are shown in Supplementary Tables 1 and 2 of the Supporting Information in a fixed order of retention in reverse phase liquid chromatography according to their hydrophilic properties. For example, PC−PC dimer 1 elutes earlier than PC−PC dimer 4. The monomeric PC and PD units of PAs both have two diastereoisomeric configurations ((+)-catechin/(−)-epicatechin and (+)-gallocatechin/ (−)-epigallocatechin, respectively) in plants, which results in 12 possible compounds belonging to PC−PC, PC−PD, or PD− PD dimers (Supplementary Table 1). The two PC monomers were identified as (+)-catechin and (−)-epicatechin, respectively. At the same time, PC−PC dimers 1−4 were tentatively identified as catechin-(4α→8)-catechin (trivial name B3), epicatechin-(4α→8)-catechin (B1), catechin-(4α→8)-epicatechin (B4), and epicatechin-(4α→8)-epicatechin (B2), respectively.41 The same order may be applied for PD−PD dimers, the first being gallocatechin-(4α→8)-gallocatechin, the second epigallocatechin-(4α→8)-gallocatechin, the third gallocatechin(4α→8)-epigallocatechin, and the fourth epigallocatechin(4α→8)-epigallocatechin. PD−PD dimers gallocatechin-(4α→ 8)-gallocatechin and gallocatechin-(4α→8)-epigallocatechin have been previously reported from black currant leaves.42,43 Additionally, Tits et al.43 found also a 4α→6 dimer of gallocatechin from the leaves, whereas a 4α→8 bond is more commonly found in edible plants.35 In almost all dimers (excluding two PC−PC dimers), the contents were significantly higher in enzyme-aided juices than in nonenzymatic juices (t test, p < 0.05; Supplementary Table 1). The PC−PC dimers were less abundant (on average 2.9 and 2.0% of all dimers in nonenzymatic and enzymatic juices, respectively) than the PC−PD (27.3 and 23.3%) and PD−PD dimers (69.8 and 74.8%) in all cultivars and in both processes. Figure 1 shows the abundance of individual monomers, dimers, and trimers in the juices and the comparison of the compound profiles between juice processes. Within PC dimers, compounds 1 (dimer B3) and 3 (B4) were more abundant than compound 2 (B1) or 4 (B2), whereas within PD−PD and PC− PD dimers compounds 2 and 4 were more abundant than compounds 1 or 3 (Figure 1). Within all three dimer types, the 5376

DOI: 10.1021/acs.jafc.5b01287 J. Agric. Food Chem. 2015, 63, 5373−5380

Article

Journal of Agricultural and Food Chemistry

Figure 3. PLS correlation loadings plots of the interaction between taste and astringencies (Y, n = 5) and proanthocyanidin measures (X, n = 35) juice samples from different cultivars within processes: (A) juices without enzymatic assistance; (B) juices with enzymatic (E) assistance. Samples are in green font, sensory attributes are in red italic font, and compounds are in blue. For abbreviations of compounds refer to Table 1 and Supplementary Tables 1 and 2.

Impact of Cultivar on Sensory Quality. Two PLS models were created to examine the sensory contribution of proanthocyanidins in the five juices within processes (Figure 3). Figure 3A presents the model for the samples without pectinase treatment. Ninety percent of the variation in the chemical variable data X explained 64% of the variation in sensory data. However, none of the five sensory attributes was well validated in the full cross-validation. Cultivars ‘Mortti’ and ‘Ola’ had the highest contents of proanthocyanidins but were not significantly astringent or bitter in this juice process. Especially nonenzymatic juice from ‘Mortti’ was the sweetest and the most viscose of the five cultivars.29 As discussed earlier, the high viscosity due to the high polysaccharide content in these juices masks the astringency of proanthocyanidins. Some correlation can be found between mDP and mouth-drying astringency on the right side of the plot (Figure 3A), which may indicate the role of highly polymerized proanthocyanidins in astringency despite the masking effect of the juice matrix. A similar model for enzyme-aided juices is shown in Figure 3B with two validated factors. Overall, 86% of the variation in X-data explained 90% of the variation in Y. The best explained Y-variable was mouth-drying astringency with 92.7% (validated 81.1%). Most of the chemical variables, especially variables PA total (total content of proanthocyanidins), PA others (tetramers and larger polymers), and mDP, are located in the

to PD contributed to mouth-drying astringency. This suggests that the protein affinity of PAs is enhanced by the increase in PA size, especially if the increase is brought about by a proportionally larger number of phenolic OH units in the additional monomeric building blocks (PDs have 25% more OH units than PCs). In addition, a significant increase in mouth-drying astringent characteristics is caused by the effective breakage of pectin structures in enzyme-aided juice processing.29 High viscosity and low astringency were observed in our juices without pectinase treatment, which is in agreement with earlier studies.44,45 Other sensory attributes were not well explained in the model with the comparison of processes (Figure 2). The Xvariables explained only 44.3% (27.9% in the validated model) of variation in puckering astringency with the first factor. However, flavan-3-ols, their dimers and trimers, and polymeric phenolic fractions in red wine have also been reported to have puckering astringent properties.17,18,46 Besides astringency, the remaining three attributes were poorly explained in the model with the PA contents: only a little of variation in bitterness (10.1%) was explained with the first factor, whereas the variation in sourness or sweetness was not explained at all. The latter findings were as expected; sourness was not different between processes. 5377

DOI: 10.1021/acs.jafc.5b01287 J. Agric. Food Chem. 2015, 63, 5373−5380

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Journal of Agricultural and Food Chemistry left of the plot with cultivars ‘Ola’ and ‘Mortti’ and the mouthdrying astringency. This supports the earlier finding that especially the size of PAs is important in the protein affinity that causes the mouth-drying astringent sensation in the mucosa. In fact, puckering astringency was the second attribute well explained with the model (96.9% explained, but only 56.9% in validated model) in the upper part of the plot. Interestingly, PC−PC and PC−PD dimers are correlated with mouth-drying astringency and cultivar ‘Ola’, whereas PD dimers and trimers correlate with ‘Mortti’. Simultaneously, the PC:PD ratio correlates with the mouth-drying astringency and puckering astringency on factor 2. These findings indicate that variations in the PC:PD ratio may result in different overall astringent properties. The PC:PD ratio being more closely related to puckering astringency suggests that PC-% is more important in causing the puckering sensation, whereas PD-% might be a more important contributor to the mouth-drying sensation together with the size of PA. A high portion of PD in comparison to PC has been previously reported to result in lower “coarse” attribute of astringency.9 Changes in the PC:PD ratio may result in different overall astringency perception. Bitterness was poorly explained by the chemical variables in the model (Figure 3B). The compounds studied and their contents did not contribute to bitterness of the juices. These compounds were found in the lowest contents in the bitterest cultivars (‘Breed15’ and ‘Marski’). Additionally, neither total contents of proanthocyanidins, total PD, total PC, nor their ratio contributed to bitterness. However, the ratio between PA 1−3 (sum of monomers, dimer,s and trimers; Table 1) and PA others (degree of polymerization ≥ 4; Table 1) correlated with bitterness on factor 1. In other words, there were more smaller and bitter PAs relative to larger and astringent PAs found in the juice of ‘Marski’ than in the less bitter juices. As reported by Laaksonen et al.,29 the flavonols (glycosides or aglycons), hydroxycinnamic acid derivatives, or anthocyanins did not explain the bitterness of the juices. Their contents were the lowest in the bitterest cultivars regardless of the juice production process. All of these compounds, or at least some in each class, activate the human bitter taste receptors.23,24 These findings suggest that the bitterness in juice of the black currant cultivars may be due to complex interactions among multiple sensory-active compounds. In our previous studies,29,30 the sugar-to-acid ratio was a more important factor contributing to sweetness than individual sugar components. Similarly, the bitterness of these juices may be due to the ratios between bitter phenolics and sweet sugars and/or sour acids. In our previous study, the bitterest cultivars had high contents of sugars or nonphenolic fruit acids relative to phenolics. Thus, the sugars or acids may affect and mask the astringent properties of the phenolic compounds, but not the bitterness of the juices. On the other hand, bitterness may have been caused by berry components that were not characterized in this study or our earlier investigations.28−30 The PC monomers, dimers, and trimers in red wine have been reported to be both bitter and astringent, but the thresholds for bitterness are higher than for astringency.17,18 According to Hufnagel and Hofmann,18 the dose-over-threshold (DoT) factors, which indicate the ratio between concentration and taste threshold for the compounds, were