Stabilization of anthocyanins in blackberry juice by glutathione fortification

Nathan B. Stebbins a, Luke R. Howard *a, Ronald L. Prior a, Cindi Brownmiller a and Andy Mauromoustakos b
aUniversity of Arkansas, Department of Food Science, Fayetteville, Arkansas 72704, USA. E-mail:; Fax: +1 479 575 6936; Tel: +1 479 575 2978
bUniversity of Arkansas, Agricultural Statistics Lab, Fayetteville, Arkansas 72701, USA

Received 1st June 2017 , Accepted 29th August 2017

First published on 30th August 2017

Blackberry anthocyanins provide attractive color and antioxidant activity. However, anthocyanins degrade during juice processing and storage, so maintaining high anthocyanin concentrations in berry juices may lead to greater antioxidant and health benefits for the consumer. This study evaluated potential additives to stabilize anthocyanins during blackberry juice storage. The anthocyanin stabilizing agents used were: glutathione, galacturonic acid, diethylenetriaminepentaacetic acid and tannic acid, which were added at a level of 500 mg L−1. Juice anthocyanin, flavonol, and ellagitannin content and percent polymeric color were measured over five weeks of accelerated storage at 30 °C. Glutathione had the greatest protective effect on total anthocyanins and polymeric color. Therefore a second study was performed with glutathione in combination with lipoic and ascorbic acids in an effort to use antioxidant recycling to achieve a synergistic effect. However, the antioxidant recycling system had no protective effect relative to glutathione alone. Glutathione appears to be a promising blackberry juice additive to protect against anthocyanin degradation during storage.

1. Introduction

Blackberries are a popular fruit with high concentrations of antioxidants, particularly anthocyanins and ellagitannins that possess positive health effects.1–3 Fresh berries have a limited shelf-life and thus need to be processed into jams, juice, or frozen to limit postharvest loss. Anthocyanin content decreases over processing and storage, which probably has an adverse effect on health benefits. Various additives have been used in berry juices to stabilize anthocyanins, including β-cyclodextrin,4 pectins and other polysaccharides,5 and copigments such as phenolic acids.6,7 Cyclodextrin's effectiveness may be due to inclusion of the anthocyanin within the ring shape of the molecule and/or hydrophobic interactions, both of which could restrict the hydration reaction responsible for anthocyanin bleaching.4 Pectin can interact with anthocyanins through ionic and hydrogen bonding potentially limiting the hydration reaction as well.8 Galacturonic acid, the monomeric unit of pectin, may protect anthocyanins by electrostatic interactions.9 Protection via copigments is frequently described by π–π stacking of phenolic compounds above and below the anthocyanin in a “sandwich” effect.10

Often very high additive concentrations are needed to limit anthocyanin degradation, therefore a recycling mechanism of multiple additives could be effective at lower concentrations. An antioxidant recycling approach may be advantageous since both enzymatic and non-enzymatic oxidation are reported to play a role in anthocyanin degradation.11,12 Antioxidant recycling occurs in both in vitro and in vivo cellular systems;13 however, to the best of our knowledge this mechanism has not been tested in a juice product. Combinations of glutathione, lipoic acid, ascorbic acid, and vitamin E have been linked in recycling mechanisms.14–17 Recycling mechanisms involve equilibria between each component in an effort to keep all antioxidants active for greater periods of time. The concentration of each component in the system must be balanced in order to prevent a pro-oxidant environment.18 The objective of this study was to evaluate the effect of anthocyanin stabilizing agents with different mechanistic tactics on blackberry juice color and an antioxidant recycling mechanism on anthocyanin, flavonol, and ellagitannin content in blackberry juice over accelerated storage.

2. Materials and methods

2.1. Materials

Cyanidin-3-O-glucoside was obtained from Chromadex (Irvine, CA, USA), whereas other standards: ellagic acid, ascorbic acid, lipoic acid, glutathione, galacturonic acid, diethylenetriaminepentaacetic acid (DTPA), tannic acid, potassium metabisulfite were purchased from Sigma Aldrich (St Louis, MO, USA). HPLC-grade methanol was purchased from EMD Millipore (Billerica, MA, USA) and formic acid from Fischer-Scientific (Fair Lawn, NJ, USA). Frozen blackberries (Mexican origin) were purchased from a local supermarket.

2.2. Juice production

Frozen blackberries were thawed by heating in a steam kettle until boiling and then held for three minutes. The berries were cooled and centrifuged at 9447g for 10 min. Since we were not interested in improving juice yield, no commercial enzyme cocktail was used to macerate cell wall polysaccharides prior to centrifugation. The supernatant was pooled and 500 mg L−1 of each stabilizing agent was added to the respective volume of blackberry juice. We selected this concentration of stabilizing agents based on the range of maximum concentrations (2–1000 mg L−1) allowed by the FDA for the synthetic antioxidants BHA and BHT in foods.19 Two separate studies were performed. In stage one, the stabilizing agents glutathione, galacturonic acid, tannic acid and diethylenetriaminepentacetic acid (DTPA) were studied and in stage two glutathione, lipoic acid, ascorbic acid alone and the combinations (500 mg L−1 of each) of glutathione + lipoic acid and glutathione + lipoic acid + ascorbic acid (GLA) were studied. A different lot of frozen blackberries was used for each study. Juice samples in triplicate stored in 8 mL glass tubes were pasteurized in a water bath at 90 °C for 1.5 min. The pasteurization conditions were comparable to those used in previous studies on blackberry juice.20–22 Although we did not measure polyphenol oxidase activity in the juices following processing, we believe the two thermal treatments used during processing were sufficient to inactivate the enzyme. Pasteurized juices were stored in a 30 °C oven for accelerated storage until analysis. New samples were removed from the oven each week and analyzed for five weeks.

2.3. HPLC-PDA analysis of anthocyanins

Anthocyanin analysis followed the method of Cho et al. 2004.23 Juice samples were passed through 0.45 μm nylon syringe filters prior to HPLC injection. A Waters HPLC system (Waters Corp., Milford, MA) comprised of dual 515 pumps, a 717plus autosampler, and a 996 photodiode array detector was used for chromatographic analyses. A Waters Symmetry C18 column (4.6 × 250 mm, 5 μm) was used for separation of anthocyanins with a 1 mL min−1 flow rate and solvent A as 5% formic acid and solvent B as methanol. Elution started with 5% B, increased to 15% B in 10 min, from 15% to 35% in 35 min, then 100% in 5 min, with an isocratic wash at 100% B for 10 min, finishing with 5% B for a 10 min re-equilibration. UV-visible spectra were monitored from 250–600 nm and peak areas were integrated at 510 nm. Anthocyanins were quantified as cyanidin-3-glucoside equivalents using external calibration curves ranging from 1–200 μg mL−1.

2.4. HPLC-PDA analysis of ellagitannins and flavonols

Ellagitannin and flavonol analysis followed the method of Hager et al. 2008[thin space (1/6-em)]24 using the same HPLC system described above for anthocyanins. Juice samples were passed through 0.45 μm nylon syringe filters prior to HPLC injection. Separation was accomplished on a 250 × 4.6 mm Phenomenex Aqua 5 μm C18 column (Torrance, CA) with solvent A as 2% acetic acid and solvent B as 0.5% acetic acid in water/acetonitrile (1[thin space (1/6-em)]:[thin space (1/6-em)]1 v/v) at 1 mL min−1. A gradient was run from 10% to 55% B (0–50 min), from 55% to 100% B (50–60) min, decreasing to 10% B (60–65 min), finishing with a 15 min re-equilibration. UV-visible spectra were monitored from 250–600 nm, with peak areas integrated at 255 nm for ellagitannins and 360 nm for flavonols. Ellagitannins were quantified as ellagic acid equivalents and flavonols were quantified as rutin equivalents using external calibration of authentic standards ranging from 1–100 μg mL−1.

2.5. HPLC-electrospray ionization tandem mass spectrometry (LC-ESI-MS) analysis of anthocyanins, flavonols and ellagitannins

LC-ESI-MS analysis was conducted using an HP 1000 series HPLC and a Bruker Esquire 2000 quadrapole ion trap mass spectrometer. Anthocyanins were separated using a Waters Symmetry (250 × 4.6 mm; 5 μm) column and ellagitannins and flavonols on a Phenomenex Aqua (250 × 4.6 mm; 5 μm) column with gradients as described above. The mass spectrometry analysis was performed in positive ion mode for anthocyanins and negative ion mode for ellagitannins and flavonols under the following conditions: capillary voltage at 4 kV with polarity [+] for negative ion mode and [−] for positive ion mode analysis, nebulizer gas pressure 32 psi, dry gas flow 12 L min−1, and skim voltage at 53.7 V and again polarity switched for respective mode. Ions were isolated and fragmented in quadrapole ion trap with excitation amplitude of 1.2 volts.

2.6. Polymeric color analysis

Percent polymeric color was measured according to the method of Giusti and Wrolstad 2001.25 The juice was diluted to have an absorbance reading between 0.5 to 1.0 at 510 nm, detected by an 8452A diode array spectrophotometer (Hewlett-Packard, Palo Alto, CA). A control, unbleached sample was prepared with 0.2 mL of DI water and 2.8 mL of the diluted juice and a bleached sample consisted of 2.8 mL of diluted juice and 0.2 mL of 0.9 M potassium metabisulfite. The samples were allowed to equilibrate for 15 min and absorbance readings were recorded at λ = 420, 510, and 700 nm. Color density was calculated from the control sample and polymeric color from the bleached sample using the following formulas:
color density = [(A420 nmA700 nm) + (A510 nmA700 nm)] × dilution factor

polymeric color = [(A420 nmA700 nm) + (A510 nmA700 nm)] × dilution factor

Percent polymeric color was calculated from the result of these formulas:

% polymeric color = (polymeric color/color density) × 100.

2.7. Color monitoring

A Konica Minolta Chroma meter CR-400 and data processor DP-400 was used to measure lightness (L), chroma (C), and hue (h) in the L*C*h color scheme (Konica Minolta, Japan). The chroma meter was calibrated with a white tile: x = 92.41, y = 0.3145, z = 0.3200, observer = 2° prior to each use. Color measurements were taken at the end of each five-week study. Juice samples were frozen (−20 °C) until the time of analysis.

2.8. Statistical analysis

Three samples stored in different individual tubes per treatment were analyzed and graphed at each storage time using JMP Pro 13. The Fit Model platform in JMP was used to analyze all continuous responses for both experiments as a two factor (treatments and storage TIME) factorial design with 5 and 7 treatments respectively and time (week with 6 levels: 0_PAST, 1, 2, 3, 4, and 5 weeks) and their interaction. Due to the highly significant interactions for the most important responses such as the anthocyanins and polymeric color, the treatment × week combination LSmeans (there were few missing values) were sliced and simple effect comparisons among the treatment LSmeans at week 5 were performed. Comparisons among any differences of the interaction LSmeans were assessed using Tukey's HSD test (α = 0.05).

Nonlinear regression modeling was performed using the nonlinear curve fitting platform of JMP Pro 13 to model and compare the decay rates of the total anthocyanins and percent polymeric color of the various treatments for each experiment. Several linear and nonlinear curves were fitted to the data varying for 2–4 parameters and the best fit was in every case the three parameter exponential decay model a + b × e(c×week) (where a was the asymptote, b the scale and c the rate). The analysis provided ANOM type of comparisons of the three individual treatment nonlinear parameter estimates to their overall mean for each experiment. In addition, useful inverse predictions allowed us to estimate and compare the amount of time it will take on average for each treatment in both experiments starting with the similar initial amounts of total anthocyanins to reduce to 1000 mg L−1 (>50% of the starting anthocyanins total for each experiment).

All analysis presented in this paper used SAS/Stat®, Version 9.4 and the Fit Model and Nonlinear univariate platforms of JMP Pro®, Version 13.1.

3. Results & discussion

3.1. Stabilizer effect on monomeric anthocyanins and percent polymeric color throughout accelerated storage

Blackberry juice with the addition of DTPA had the highest total anthocyanin content following pasteurization relative to other stabilizing agents in Stage 1 (Fig. 1). Blackberries have 0.62 mg iron and 0.17 mg copper per 100 g berry.26 Therefore, it is possible for small quantities of iron or copper to generate hydroxyl radicals that may degrade anthocyanins.27,28 DTPA is a strong chelator and likely protects anthocyanins by chelating the free iron and copper in the blackberry juice to limit hydroxyl radical formation. The control in Stage 1 had the lowest content of total anthocyanins following pasteurization showing that all additives were protective. However in Stage 2, the control and lipoic acid treatments had lower total anthocyanin content after pasteurization than all other treatments (Fig. 2). All glutathione-containing juices had a protective effect on total anthocyanin content as well as the juice with only ascorbic acid added. There was no notable pH change with the addition of ascorbic acid or any other additives. Glutathione and other thiols are reported to protect semi-purified extracts of bilberry and black currant anthocyanins (35–37% concentration) and double their absorption in Caco-2 cells and human plasma. Glutathione is described as having superior functionality relative to other thiols due to two carboxylic acid moieties. The thiols were postulated to be tightly associated with the anthocyanidin at position 4 or the sugar moiety, which is the basis for thiol stabilization.29 The author included a structure showing a thiol bound to C-4 on delphinidin; however, we did not detect any new anthocyanin peaks over the course of storage.
image file: c7fo00801e-f1.tif
Fig. 1 Total anthocyanin concentrations and percent polymeric color in blackberry juice fortified with stabilizing agents through pasteurization and accelerated storage in Stage 1. LSmeans treatment × time ± one standard error (n = 3) based on the fitted model. Dashed horizontal line represents mean value for prepasteurized juice.

image file: c7fo00801e-f2.tif
Fig. 2 Total anthocyanin concentrations and percent polymeric color in blackberry juice fortified with stabilizing agents through pasteurization and accelerated storage in Stage 2. LSmeans treatment × time ± one standard error (n = 3) based on the fitted model. Dashed horizontal line represents mean value for prepasteurized juice.

Glutathione treated juices had 13% more total anthocyanins than control juices following five weeks of storage (P = 0.0477), while all other treatments were similar to the control (Fig. 1). Galacturonic acid treatment was nearly identical to the control (no stabilizing agent added) with regard to total anthocyanin content. Galacturonic acid was chosen for this study because of the potential for anthocyanin stabilization by molecular electrostatic interactions between the molecules.9 Co-pigments and self-association are believed to protect anthocyanins from the hydration reaction by sterically excluding water from the vicinity of the flavylium ion.30 However, our results indicate galacturonic acid and tannic acid at 500 mg L−1 concentration had no protective or degradative effect on anthocyanins over five weeks of storage. The structure of tannic acid has a multitude of phenolic moieties that could physically protect the flavylium cation from hydration via a sandwich effect. Previous studies have shown tannic acid's potential to stabilize anthocyanins in other fruit juices.31–33 Other researchers have used tannic acid in a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 ratio to anthocyanin concentration in model system using grape skins with significant stabilizing effects.33 A copigmentation review of literature using model systems and fruit juices depicted copigment[thin space (1/6-em)]:[thin space (1/6-em)]anthocyanin ratios ranging from 0.1 to 200 with the addition of various copigments, including flavonoids, cinnamic acids, or phenolic acids.7 The majority of research used very high ratios that are unrealistic for industrial application.

Due to the significant stabilizing effect of glutathione in Stage 1, combinations of glutathione with lipoic acid, and glutathione with both lipoic and ascorbic acids were added at 500 mg L−1 of each component in an attempt to stabilize anthocyanins via antioxidant recycling in blackberry juice. Control and juice samples containing solely lipoic acid or ascorbic acid were also included in Stage 2. All combinations of glutathione protected against anthocyanin loss over storage. Following five weeks of accelerated storage, juices supplemented with GLA, glutathione + lipoic acid, and glutathione alone contained 28%, 23% and 22% higher levels of total anthocyanins than control juices, respectively (P < 0.001 for all three treatments). Cyanidin-3-xyloside decreased below the limit of quantification (5 mg L−1) following week four and showed a nonlinear decline. As in Stage 1, cyandin-3-glucoside was the prevalent anthocyanin in blackberry juice, accounting for 92% of total anthocyanins after five weeks of storage, but showed a nonlinear decline over accelerated storage in Stage 2. Following five weeks of storage, all treatment combinations with glutathione were not different than glutathione alone.

The percent polymeric color data is shown in Fig. 1 and 2 for Stages 1 and 2, respectively. The assay measures the ratio of bleachable monomeric anthocyanins to non-bleachable anthocyanins (presumably anthocyanin–tannin polymers) in a sample, which typically negatively correlates with total anthocyanin content. An increase in percent polymeric color may indicate anthocyanin–tannin polymers forming over storage or the same concentration of anthocyanin–tannin polymers accompanied by a decreasing concentration of monomeric anthocyanins. Pyranoanthocyanins are also resistant to bleaching in the presence of SO2,34 but we saw no evidence of these compounds in our HPLC chromatograms. Our findings reflect the anthocyanin quantification by HPLC, indicating a greater likelihood that the percent polymeric color values are caused by a decrease in monomeric anthocyanins and not an increase in anthocyanin–tannin polymers. A strong inverse relationship between percent polymeric color and total anthocyanins was observed in Stage 1 (R2 = 0.937) and Stage 2 (R2 = 0.941) indicating that the low amount of anthocyanins remaining following storage are resistant to SO2 bleaching and may be polymeric compounds.

The changes in the major blackberry anthocyanins, cyanidin-3-glucoside, cyanidin-3-rutinoside, and cyanidin-3-xyloside throughout accelerated storage are shown in Fig. 3 for Stage 1 and Fig. 4 for Stage 2. In Fig. 3, it is clear that glutathione provided the greatest stabilizing effect on each cyanidin glycoside compared to other additives. Following five weeks of accelerated storage (Stage 1) glutathione treated juices had 13% and 12% higher levels of cyanidin-3-glucoside and cyanidin-3-rutinoside than the control (P = 0.05 and 0.003). Glutathione did not significantly preserve cyanidin-3-xyloside during the final two weeks of storage. In Stage 2, all glutathione treatments preserved anthocyanins throughout accelerated storage compared with control juices. Juices supplemented with GLA, glutathione + lipoic acid and glutathione alone had 29%, 24% and 23% higher levels of cyanidin 3-glucoside, and 21%, 20%, and 16% higher levels of cyanidin 3-rutinoside than control juices following five weeks of storage (P < 0.001 for all). However, all glutathione treatments had similar levels of the minor anthocyanin cyanidin 3-xyloside as the control following five weeks of storage.

image file: c7fo00801e-f3.tif
Fig. 3 Individual anthocyanin concentrations in blackberry juice fortified with stabilizing agents over accelerated storage in Stage 1. LSmeans treatment × time ± one standard error (n = 3) based on the fitted model. Dashed horizontal line represents mean value for prepasteurized juice.

image file: c7fo00801e-f4.tif
Fig. 4 Individual anthocyanin concentrations in blackberry juice fortified with stabilizing agents over accelerated storage in Stage 2. LSmeans treatment × time ± one standard error (n = 3) based on the fitted model. Dashed horizontal line represents mean value for prepasteurized juice.

Due to the marked changes in total anthocyanins and polymeric color throughout storage in both stages it was important to calculate degradation kinetics of the compounds. Total anthocyanin content and percent polymeric color best fit a three parameter exponential decay model with a correlation coefficient of at least 0.967 in both stages, while other goodness of fit measures are listed in Table 1. The degradation rate of each treatment is depicted as the exponent in Table 2, with Stage 1 glutathione decaying at 512 + 1662e(−0.20×week) and the control at 888 + 1220e(−0.48×week). The inverse predictions in Table 3 display this rate in more practical terms by listing the time to reach 1000 mg L−1, which was selected to show over 50% loss of anthocyanin content compared to starting conditions. The predicted week for glutathione is 1.2 weeks longer than the control in both stages. All other treatments in Stage 1 are similar to the control, while in Stage 2 all glutathione-containing treatments has similar predicted weeks: 4.09, 3.86, and 3.95 for glutathione, glutathione + lipoic acid, and GLA, respectively. This is visually displayed in the modeled curves in Fig. 5 for Stage 1 and Fig. 6 for Stage 2. The time for each treatment to reach 1000 mg L−1 is noted on Fig. 5 & 6 and the predicted week represented in Table 3. The accelerated storage at 30 °C allows this time to be approximately doubled if juices were stored at ambient temperature (∼20 °C) due to the increase reaction rate with elevated temperatures. Modeled curves of percent polymeric color for both Stage 1 and 2 can be seen in Fig. S1 and S2. The treatment trends of the modeled data match the polymeric color data described above. To summarize, supplementing blackberry juice with glutathione will stabilize anthocyanins approximately two weeks longer at ambient storage than juice without any supplementation.

image file: c7fo00801e-f5.tif
Fig. 5 Three parameter exponential decay model with inverse predictions of total anthocyanins in Stage 1.

image file: c7fo00801e-f6.tif
Fig. 6 Three parameter exponential decay model with inverse predictions of total anthocyanins in Stage 2.
Table 1 Goodness of fit measures for the fitted three parameter exponential decay model
Stage Variable AICc BIC SSE MSE RMSE R 2
1 Total ACY 1044.559 1076.821 417[thin space (1/6-em)]873.66 5646.941 75.146 0.972
1 Polymeric color 328.596 360.859 134.077 1.811 1.346 0.973
2 Total ACY 12[thin space (1/6-em)]380.013 1280.337 390[thin space (1/6-em)]633.281 4340.369 65.881 0.987
2 Polymeric color 443.907 486.231 250.307 2.781 1.667 0.967

Table 2 Three parameter exponential decay model
Stage Treatment Total ACY Percent polymeric color
1 Control 888 + 1220e(−0.48×week) 47–38e(−0.20×week)
1 Glutathione 512 + 1662e(−0.20×week) 102–92e(−0.04×week)
1 DTPA 898 + 1344e(−0.47×week) 34–25e(−0.30×week)
1 Galacturonic acid 770 + 1387e(−0.42×week) 49–40e(−0.16×week)
1 Tannic acid 819 + 1371e(−0.46×week) 62–52e(−0.12×week)
2 Control 557 + 1569e(−0.45×week) 51–26e(−0.47×week)
2 Glutathione 745 + 1627e(−0.45×week) 42–22e(−0.67×week)
2 Glutathione + lipoic 691 + 1757e(−0.45×week) 47–26e(−0.37×week)
2 GLA 759 + 1670e(−0.49×week) 43–20e(−0.40×week)
2 Lipoic acid 664 + 1540e(−0.50×week) 49–26e(−0.49×week)
2 Ascorbic acid 631 + 1708e(−0.68×week) 53–30e(−0.40×week)

Table 3 Inverse prediction of total anthocyanin retention based on nonlinear regression model
Stage Treatment Specified Total ACY (mg L−1) Predicted week Std error Lower 95% Upper 95%
1 Control 1000 5.04 0.66 3.75 6.33
1 Glutathione 1000 6.21 0.69 4.87 7.56
1 DTPA 1000 5.46 0.82 3.85 7.06
1 Galacturonic acid 1000 4.28 0.28 3.73 4.82
1 Tannic acid 1000 4.4 0.35 3.71 5.09
2 Control 1000 2.84 0.1 2.64 3.04
2 Glutathione 1000 4.09 0.19 3.72 4.47
2 Glutathione + lipoic 1000 3.86 0.15 3.57 4.15
2 GLA 1000 3.95 0.18 3.6 4.31
2 Lipoic acid 1000 3.04 0.11 2.81 3.26
2 Ascorbic acid 1000 2.25 0.09 2.08 2.42

3.2. Stabilizer effect on ellagitannins throughout accelerated storage

The three predominant ellagitannins in blackberries were previously identified by mass spectrometry and two of the compounds had identical m/z values: sanguiin H-6 and lambertianin A (m/z = 1868.7).35 These two compound totals were summed in Fig. S5 and S6 for Stage 1 and 2, respectively, due to the lack of differentiation among the isomers. Lambertianin C was identified with m/z of 1401.1 with doubly charged ion peaks (MW = 2802.2). Collectively, these compounds were fairly stable during blackberry juice accelerated storage independent of stabilizer treatment.

Ellagitannin concentration increased slightly after pasteurization (Fig. S5 and S6) and this may be caused by either the depolymerization of larger ellagitannins we detected or the release of bound ellagitannins from soluble cell wall polysaccharides by the thermal treatment. Gancel et al.22 also found an increase in ellagitannin content after pasteurization; however they used blackberry puree and attributed the ellagitannin increase to extraction from seeds. This author performed a second juice pasteurization on the juice and found a decline in ellagitannins accompanied by an increase in ellagic acid. We found the ellagitannins to be stable over accelerated storage at 30 °C, which is in contrast to the linear decline of lambertianin C and sanguiin H6 found at the same temperature in tropical highland blackberry juice storage over 35 days.22

The concentration of blackberry juice ellagitannins in this study were nearly 10-fold lower than values reported in a previous study using identical HPLC methodology.22,35 In the previous studies cv. Apache and tropical highland blackberries were used, while in the present study berries of Mexican origin, likely cv. Tupy, were used so the concentration difference could be due to different cultivars or varying environmental growing conditions. Stage 2 displayed higher total sanguiin H-6/lambertianin A levels with nearly 20 mg L−1 than Stage 1, which only had 8.6 mg L−1 in the sample with greatest concentration. This concentration difference is likely due to the use of two different lots of frozen blackberries used to process juice in stage one and two. The lower ellagitannin content found in our juice samples may be due to ellagitannin loss in the pomace since the majority of ellagitannins are found in the seeds.24,36

3.3. Stabilizer effect on flavonols throughout accelerated storage

The HPLC profile of flavonols in the blackberry juice is shown in Fig. 7 and are exclusively quercetin derivatives. The flavonols were identified by mass spectrometry as quercetin-3-rutinoside (peak 1), quercetin-3-pentosyl-glucuronide (peak 2), quercetin-3-galactoside (peak 3), quercetin-3-glucoside (peak 4), and quercetin-3-glucuronide (peak 5) in Table 4. The sixth peak is also a quercetin moiety based on the mass fragment of m/z 301; however, the substituents were not identified. The mass spectra for each flavonol are depicted in Fig. S9–S14. This is the first evidence in blackberries of quercetin-3-glucuronide (m/z 477, 301), which was previously identified in strawberries,37 northern highbush blueberries,38 and raspberries39 and quercetin-3-pentosyl-glucuronide (m/z 609, 433, 301), which was previously identified in eastern teaberries.40
image file: c7fo00801e-f7.tif
Fig. 7 HPLC chromatogram (360 nm) of flavonols in blackberry juice.
Table 4 Flavonol identification depicted in Fig. 7
Peak Tentative identification m/z
M Fragments
1 Quercetin-3-rutinoside 609 301
2 Quercetin-3-pentosyl-glucuronide 609 433, 301
3 Quercetin-3-galactoside 463 301
4 Quercetin-3-glucoside 463 301
5 Quercetin-3-glucuronide 477 301
6 Unidentified 505 465, 301

Quercetin derivatives, regardless of substituent, were stable after pasteurization and five weeks of storage in Stage 1 (Fig. S7). In Stage 2, fluctuation in concentration occurred between weeks, but remained consistent throughout storage (Fig. S8). None of the additives had a stabilizing effect on flavonol content in Stage 1 or Stage 2. Other researchers found similar stability of quercetin derivatives in blackberry juice stored at temperatures ranging from 5–45 °C, with no effect of temperature on flavonol stability.22 Gancel et al. measured total quercetin derivatives as gallic acid equivalents and reported a concentration of 6.7 mg L−1 total quercetin derivatives in blackberry juice, while we used rutin equivalents and found 69.7 mg L−1 total quercetin derivatives in the pasteurized blackberry juice.22

3.4. Changes in blackberry juice color

Color was measured using L*C*h and there was a significant increase in lightness values in all samples over accelerated storage in both Stages 1 and 2 (data not shown). This was likely caused by the decline in anthocyanin content measured by HPLC. A similar trend of increasing lightness value was shown in blood orange juice stored over seven weeks at 4.5 °C.41 Elderberries contain high quantities of cyanidin-3-glucoside, which is comparable to blackberries, and the lightness of elderberry juice and concentrate at pH 3.5 increased significantly after thermal processing at 95 °C.42 Anthocyanins are sensitive to elevated temperature; therefore pasteurization must be performed quickly. Marchese43 demonstrated fluctuations in anthocyanin content and color change over a range of pasteurization temperatures and times, recommending pasteurization at 80 °C.

There were no major changes in hue during storage with all Stage 1 samples averaging 17.20 ± 5.77, while Stage 2 samples had an average hue value of 9.54 ± 2.89. Tannic acid produced the most notable effect on chroma with a significant hyperchromic effect following pasteurization and storage. The same observation was made in model systems of pure anthocyanins after addition of tannic acid.9 Tannic acid was shown to improve color stability, but not necessarily chroma, in blood orange juice.31 Several studies have reported on the degradative effect of ascorbic acid on anthocyanins;11,28,44,45 however, ascorbic acid at 500 mg L−1 did not significantly affect any color measurements in the present study. After extreme thermal treatment, elderberry juice hue increased from 10.79 to 40.75 and the chroma decreased from 24.77 to 10.61.42 The change in chroma is related to a drastic decline in anthocyanins after heating, while the hue change is indicative of more yellow color. These authors compared strawberry, elderberry, and black carrot juice, which are all pigmented due to anthocyanins, finding food matrix critical to color stability.42

3.5. Antioxidant recycling and redox potentials

The relative redox potentials of each component in our recycling mechanism allow a comparison of the reduction probability over time. The greater the potential, the greater the likelihood of that compound being reduced. The anthocyanin profile of blackberries is composed primarily of cyanidin-3-glucoside and is one of the simplest anthocyanin profiles among berries. This allows for easier analysis, especially with redox potentials. At pH 3.5 of blackberry juice, cyanidin-3-glucoside is reported to have a redox potential of 490 mV by differential pulse voltammetry,46 which aligns with 584 mV in the aglycone, cyanidin.47 Glycosylation lowers redox potential. Other researchers have measured redox potential in blackberries finding 468 mV and 400 mV in Romanian and Croatian blackberries, respectively.48,49

The other components of the antioxidant recycling mechanism in Stage 2 are shown in Fig. 8, where lipoic acid has a redox potential of 1100 mV,50 ascorbic acid is 282 mV, and glutathione disulfide is −1500 mV.51 This illustrates that lipoic acid will be the first to be reduced, while glutathione disulfide will be the first to be oxidized. These two compounds can potentially recycle each other and then assist in anthocyanin recycling. Cyanidin-3-glucoside and ascorbic acid will be intermediates in the process due to their median redox potentials. It is thermodynamically favorable to exchange electrons between ascorbic acid/C3G and lipoic acid radical cation in order to regenerate lipoic acid. Ascorbic acid radicals are easily recycled and are “relatively harmless, being neither strongly oxidizing, nor strongly reducing”.51 The recycling mechanism allows lipoic acid to function as an antioxidant for ROS (reactive oxygen species), but would not recycle any oxidized anthocyanins due to the difference in redox potentials. On the other hand, reduced glutathione has a redox potential of −258 mV (ref. 52) and could serve as the reducing agent for any anthocyanin oxidation in the samples containing glutathione. This is likely why the addition of lipoic acid or lipoic and ascorbic acids did not improve anthocyanin content over storage of blackberry juice more than glutathione alone.

image file: c7fo00801e-f8.tif
Fig. 8 Proposed antioxidant recycling mechanism (adapted from Lu & Liu 200250).

4. Conclusion

The addition of glutathione to blackberry juice resulted in greater retention of anthocyanins over accelerated storage compared to other stabilizing agents. Color was not visibly different over the course of storage, but lightness values increased presumably in response to anthocyanin degradation. Glutathione in combination with lipoic acid and ascorbic acid was tested to determine if an antioxidant recycling mechanism would reduce the concentration of stabilizing agents needed and stabilize anthocyanins more successfully; however, the combination was not more effective than glutathione alone. Anthocyanin degradation fit an exponential decay model and visually demonstrated that glutathione supplementation can postpone anthocyanin degradation to a specified concentration of 1000 mg L−1 by at least a week compared to the control according to our accelerated storage at 30 °C. Glutathione did not bind to the anthocyanins, so further research is needed to determine the mechanism of stabilization.

Conflicts of interest

There are no conflicts to declare.


  1. D. X. Hou, Potential mechanisms of cancer chemoprevention by anthocyanins, Curr. Mol. Med., 2003, 3, 149–159 CrossRef CAS PubMed.
  2. C. A. Rice-Evans and N. J. Miller, Antioxidant activities of flavonoids as bioactive components of food, Biochem. Soc. Trans., 1996, 24, 790–794 CrossRef CAS PubMed.
  3. L. Kaume, L. R. Howard and L. Devareddy, The blackberry fruit: A review on its composition and chemistry, metabolism and bioavailability, and health benefits, J. Agric. Food Chem., 2012, 60, 5716–5727 CrossRef CAS PubMed.
  4. L. R. Howard, C. Brownmiller, R. L. Prior and A. Mauromoustakos, Improved stability of chokeberry juice anthocyanins by β-cyclodextrin addition and refrigeration, J. Agric. Food Chem., 2013, 61, 693–699 CrossRef CAS PubMed.
  5. D. S. Ferreira, A. F. Faria, C. R. F. Grosso and A. Z. Mercadante, Encapsulation of blackberry anthocyanins by thermal gelation of curdlan, J. Braz. Chem. Soc., 2009, 20, 1908–1915 CrossRef CAS.
  6. M. J. Rein and M. Heinonen, Stability and enhancement of berry juice color, J. Agric. Food Chem., 2004, 52, 3106–3114 CrossRef CAS PubMed.
  7. R. Boulton, The copigmentation of anthocyanins and its role in the color of red wine: A critical review, Am. J. Enol. Vitic., 2001, 52, 67–87 CAS.
  8. A. Fernandes, N. F. Bras, N. Mateus and V. de Freitas, Understanding the molecular mechanism of anthocyanin binding to pectin, Langmuir, 2014, 30, 8516–8527 CrossRef CAS PubMed.
  9. P. Mazzaracchio, P. Pifferi, M. Kindt, A. Munyaneza and G. Barbiroli, Interactions between anthocyanins and organic food molecules in model systems, Int. J. Food Sci. Technol., 2004, 39, 53–59 CrossRef CAS.
  10. N. Teixeira, L. Cruz, N. F. Bras, N. Mateus, M. J. Ramos and V. de Freitas, Structural features of copigmentation of oenin with different polyphenol copigments, J. Agric. Food Chem., 2013, 61, 6942–6948 CrossRef CAS PubMed.
  11. M. S. Poei-Langston and R. E. Wrolstad, Color degradation in an ascorbic acid-anthocyanin-flavanol model system, J. Food Sci., 1981, 46, 1218–1222 CrossRef CAS.
  12. A. Patras, N. P. Brunton, C. O'Donnell and B. K. Tiwari, Effect of thermal processing of anthocyanin stability in foods; mechanisms and kinetics of degradation, Trends Food Sci. Technol., 2010, 21, 3–11 CrossRef CAS.
  13. R. A. Jacob and G. Sotoudeh, Vitamin C function and status in chronic disease, Nutr. Clin. Care, 2002, 5, 66–74 CrossRef PubMed.
  14. L. Packer, E. H. Witt and H. J. Tritschler, Alpha-lipoic acid as a biological antioxidant, Free Radicals Biol. Med., 1995, 19, 227–250 CrossRef CAS PubMed.
  15. V. E. Kagan and Y. Y. Tyurina, Recycling and redox cycling of phenolic antioxidants, Ann. N. Y. Acad. Sci., 1998, 854, 425–434 CrossRef CAS PubMed.
  16. B. S. Winkler, S. M. Orselli and T. S. Rex, The redox couple between glutathione and ascorbic acid: A chemical and physiological perspective, Free Radicals Biol. Med., 1994, 17, 333–349 CrossRef CAS PubMed.
  17. K. Rahman, Studies on free radicals, antioxidants, and co-factors, Clin. Interventions Aging, 2007, 2, 219–236 CAS.
  18. D. C. Liebler, D. S. Kling and D. J. Reed, Antioxidant protection of phospholipid bilayers by α-tocopherol, J. Biol. Chem., 1986, 261, 12114–12119 CAS.
  19. FDA Food for Human Consumption 21 CFR § 172.110 and 172.115. (2016).
  20. G. Azofeifa, S. Quesada, A. M. Pérez, F. Vaillant and A. Michel, Pasteurization of blackberry juice preserves polyphenol-dependent inhibition for lipid peroxidation and intracellular radicals, J. Food Compos. Anal., 2015, 42, 56–62 CrossRef CAS.
  21. M. Kopjar, K. Jakšić and V. Piližota, Influence of sugars and chlorogenic acid addition on anthocyanin content, antioxidant activity and color of blackberry juice during storage, J. Food Process. Preserv., 2012, 36, 545–552 CrossRef.
  22. A. Gancel, A. Feneuil, O. Acosta, A. M. Perezm and F. Vaillant, Impact of industrial processing and storage on major polyphenols and the antioxidant capacity of tropical highland blackberry (Rubus adenotrichus), Food Res. Int., 2011, 44, 2243–2251 CrossRef CAS.
  23. M. J. Cho, L. R. Howard, R. L. Prior and J. R. Clark, Flavonoid glycosides and antioxidant capacity of various blackberry, blueberry and red grape genotypes determined by high-performance liquid chromatography/mass spectrometry, J. Sci. Food Agric., 2004, 84, 1771–1782 CrossRef.
  24. T. J. Hager, L. R. Howard, R. Liyanage, J. O. Lay and R. L. Prior, Ellagitannin composition of blackberry as determined by HPLC-ESI-MS and MALDI-TOF-MS, J. Agric. Food Chem., 2008, 56, 661–669 CrossRef CAS PubMed.
  25. M. M. Giusti and R. E. Wrolstad, Characterization and measurement of anthocyanins by UV-visible spectroscopy, in Current Protocols in Food Analytical Chemistry, ed. R. E. Wrolstad, T. E. Acree, H. An, et al., Wiley, New York, 2001, pp. F1.2.1–F1.2.9 Search PubMed.
  26. U.S. Department of Agriculture, USDA National Nutrient Database for Standard Reference, release 28, May 2016, Search PubMed.
  27. A. E. Martell, Chelates of ascorbic acid: Formation and catalytic properties, in Advances in Chemistry: Ascorbic acid: Chemistry, metabolism, and uses, 1982, pp. 153–178,  DOI:10.1021/ba-1982-0200.ch007.
  28. N. B. Stebbins, L. R. Howard, R. L. Prior, C. Brownmiller, R. Liyanage, J. O. Lay, X. Yang and S. Y. Qian, Ascorbic acid-catalyzed degradation of cyanidin-3-O-β-glucoside: Proposed mechanism and identification of a novel hydroxylated product, J. Berry Res., 2016, 6, 175–187 CrossRef CAS.
  29. T. Eidenberger, Stabilized anthocyanin compositions, US Patent8623429, 2014 Search PubMed.
  30. P. Trouillas, J. C. Sancho-Garcia, V. De Freitas, J. Gierschner, M. Otyepka and O. Dangles, Stabilizing and modulating color by copigmentation: Insight from theory and experiment, Chem. Rev., 2016, 116, 4937–4982 CrossRef CAS PubMed.
  31. E. Maccarone, A. Maccarrone and P. Rapisarda, Technical note: Colour stabilization of orange fruit juice by tannic acid, Int. J. Food Sci. Technol., 1987, 22, 159–162 CrossRef CAS.
  32. F. O. Bobbio, M. T. Varella and P. A. Bobbio, Effect of light and tannic acid on the stability of anthocyanins in DMSO and in water, Food Chem., 1994, 51, 183–185 CrossRef CAS.
  33. L. D. Falcao, C. Gauche, D. M. Barros, E. S. Prudencio, E. F. Gris, E. S. Sant'Anna, P. J. Ogliari and M. T. B. Luiz, Stability of anthocyanins from grape (Vitis vinifera L.) skins with tannic acid in a model system, Ital. J. Food Sci., 2004, 16, 323–332 CAS.
  34. K. A. Bindon, M. G. McCarthy and P. A. Smith, Development of wine colour and non-bleachable pigments during the fermentation and ageing of (Vitis vinifera L. cv.) Cabernet Sauvignon wines differing in anthocyanin and tannin concentration, LWT-Food Sci. Technol., 2014, 59, 923–932 CrossRef CAS.
  35. T. F. Hager, L. R. Howard and R. L. Prior, Processing and storage effects on the ellagitannin composition of processed blackberry products, J. Agric. Food Chem., 2010, 58, 11749–11754 CrossRef CAS PubMed.
  36. T. Siriwoharn and R. E. Wrolstad, Polyphenolic composition of Marion and Evergreen blackberries, J. Food Sci., 2004, 69, 233–240 CrossRef.
  37. K. Aaby, S. Mazur, A. Nes and G. Skrede, Phenolic compounds in strawberry (Fragaria x ananassa Duch.) fruits: Composition in 27 cultivars and changes during ripening, Food Chem., 2012, 132, 86–97 CrossRef CAS PubMed.
  38. M. J. Cho, L. R. Howard, R. L. Prior and J. R. Clark, Flavonol glycosides and antioxidant capacity of various blackberry and blueberry genotypes determined by high-performance liquid chromatography/mass spectrometry, J. Sci. Food Agric., 2005, 85, 2149–2158 CrossRef CAS.
  39. M. Mikulic-Petkovsek, A. Slatnar, F. Stampar and R. Veberic, HPLC-MSn identification and quantification of flavonol glycosides in 28 wild and cultivated berry species, Food Chem., 2012, 135, 2138–2146 CrossRef CAS PubMed.
  40. P. Michel, A. Dobrowolska, A. Kicel, A. Owczarek, A. Bazylko, S. Granica, J. P. Piwowarski and M. A. Olszewska, Polyphenolic profile, antioxidant and anti-inflammatory activity of eastern teaberry (Gaultheria procumbens L.) leaf extracts, Molecules, 2014, 19, 20498–20520 CrossRef PubMed.
  41. M. H. Choi, G. H. Kim and H. S. Lee, Effects of ascorbic acid retention on juice color and pigment stability in blood orange (Citrus sinensis) juice during refrigerated storage, Food Res. Int., 2002, 35, 753–759 CrossRef CAS.
  42. E. Sadilova, F. C. Stintzing, D. R. Kammerer and R. Carle, Matrix dependent impact of sugar and ascorbic acid addition on color and anthocyanin stability of black carrot, elderberry and strawberry single strength and concentrate juices upon thermal treatment, Food Res. Int., 2009, 42, 1023–1033 CrossRef CAS.
  43. D. Marchese, Citrus consumers trend in Europe. New tastes sensation: The blood orange juice case, in Citrus processing short course proceedings, University of Florida, Gainesville, FL, 1995, pp. 19–39 Search PubMed.
  44. P. Markakis, Stability of anthocyanins in foods, in Anthocyanins as food colorants, Academic Press, New York, 1982, pp. 163–180 Search PubMed.
  45. L. Jurd, Some advances in the chemistry of anthocyanin-type plant pigments, in The chemistry of plant pigments, ed. C. O. Chichester, Academic Press, New York, 1972, pp. 123–142 Search PubMed.
  46. P. Janeiro and A. M. O. Brett, Redox behavior of anthocyanins present in Vitis vinifera L., Electroanalysis, 2007, 17, 1779–1786 CrossRef.
  47. M. Andoni, M. Medeleanu, M. Stefanut, A. Cata, I. Ienascu, C. Tanasie and R. Pop, Theoretical determination of the redox electrode potential of cyanidin, J. Serb. Chem. Soc., 2016, 81, 177–186 CrossRef.
  48. A. Cata, M. N. Stefanut, R. Pop, C. Tanasie, C. Mosoarca and A. D. Zamfir, Evaluation of antioxidant activities of some small fruits containing anthocyanins using electrochemical and chemical methods, Croat. Chem. Acta, 2016, 89, 37–48 CrossRef CAS.
  49. S. Komorsky-Lovric and I. Novak, Abrasive stripping square-wave volammetry of blackberry, raspberry, strawberry, pomegranate, and sweet and blue potatoes, J. Food Sci., 2011, 76, 916–920 CrossRef PubMed.
  50. C. Lu and Y. Liu, Interactions of lipoic acid radical cations with vitamin C and E analogue and hydroxycinnamic acid derivatives, Arch. Biochem. Biophys., 2002, 406, 78–84 CrossRef CAS PubMed.
  51. G. R. Buettner, The pecking order of free radicals and antioxidants: Lipid peroxidation, α-tocopherol, and ascorbate, Arch. Biochem. Biophys., 1993, 300, 535–543 CrossRef CAS PubMed.
  52. W. G. Kirlin, J. Cai, S. A. Thompson, D. Diaz, T. J. Kavanagh and D. P. Jones, Glutathione redox potential in response to differentiation and enzyme inducers, Free Radicals Biol. Med., 1999, 27, 1208–1218 CrossRef CAS PubMed.


Electronic supplementary information (ESI) available: Fig. S1–S14. See DOI: 10.1039/c7fo00801e

This journal is © The Royal Society of Chemistry 2017