Anthocyanins Contents, Profiles, and Color Characteristics of Red

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Anthocyanins Contents, Profiles, and Color Characteristics of Red Cabbage Extracts from Different Cultivars and Maturity Stages Neda Ahmadiani,† Rebecca J. Robbins,§ Thomas M. Collins,§ and M. Monica Giusti*,† †

Department of Food Science and Technology, The Ohio State University, 2015 Fyffe Road, Columbus, Ohio 43210, United States Analytical and Applied Sciences Group, Mars Inc., 800 High Street, Hackettstown, New Jersey 07840, United States

§

ABSTRACT: Red cabbage (Brassica oleracea L.) is an excellent source of food colorant. This study aimed to evaluate the anthocyanin pigment contents and profiles from seven red cabbage cultivars at two maturity stages (8 weeks apart) and evaluate their color characteristics and behavior under acidic and neutral pH. Anthocyanin concentrations ranged from 1111 to 1780 mg Cy3G/100 g DM and did not increase with time. Cultivar and maturation affected pigment profile. Some varieties accumulated ≥30% of diacylated pigments, and proportions of monoacylated pigments decreased with time. Extracts from selected varieties at first harvesting time produced colors similar (λmax = 520 nm and ΔE = 6.1−8.8) to FD&C Red No. 3 at pH 3.5. At pH 7, extracts from the second harvest with s higher proportion of diacylation produced λmax ≃ 610 nm, similar to FD&C Blue No. 2. Cultivar selection and maturation affected color and stability of red cabbage extracts at different pH values. KEYWORDS: anthocyanins, red cabbage (Brassica oleracea L.), cultivar, harvesting time, color

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INTRODUCTION Anthocyanins are water-soluble pigments with potential application in coloring of different food products.1,2 Colorants made of these pigments are currently manufactured for food use from horticultural crops and processing wastes.3 Fruit and vegetable juices containing anthocyanins such as concentrated red cabbage, black carrot, purple sweet potato, radish, bilberry, and elderberry are being used as approved food color additives in most countries.2,4 In addition, anthocyanins are proven to be good antioxidant compounds due to their effective free radical scavenging properties and have shown numerous potential health benefits in in vitro and vivo studies.5−9 Red cabbage (Brassica oleracea L.) is an edible source with high content and high potential yield per unit area of anthocyanins.10 Red cabbage anthocyanin extract is known to have considerable amounts of mono- or diacylated cyanidin anthocyanins.11,12 Type and acylation of anthocyanins are two important factors that determine their color characteristics at certain pH values.13−17 Due to its anthocyanin compositions, red cabbage anthocyanin extracts can exhibit a wide spectrum of color, ranging from orange through red to purple and blue based upon the pH of the environment.18 Acylation of anthocyanins also influences their antioxidant properties and stability in the food matrix.15,19,20 Diacylated anthocyanins are linked to higher antioxidant activity compared to the other nonand monoacylated ones.12 Anthocyanin pigments with higher number of acylation have also shown good stabilities to light and processing temperature.20 There are several known intrinsic and extrinsic factors that affect anthocyanin content and composition in plants.21−23 Plants cultivar and maturation time are among the essential factors that can influence the phytochemical content, including anthocyanins.24−26 The objectives of this study were to evaluate the anthocyanins content and profile from different red cabbage cultivars at two maturity stages and evaluate their color characteristics and behavior under acidic and neutral pH. © 2014 American Chemical Society

Knowing the anthocyanin composition of red cabbage cultivar and maturation time would help us to select the cultivar and maturation that could provide the desired characteristics for a specific application.

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MATERIALS AND METHODS

Plant Materials. Seven red cabbage cultivars, Cairo, Kosaro, Integro, Buscaro, Azurro, Primero, and Bandolero (three heads from each cultivar), at two maturity stages (harvested 13 and 21 weeks after transplanting) were donated by Bejo Seeds Inc. (Geneva, NY, USA). Cabbages were grown side-by-side during the summer season. Samples were shipped immediately after harvest and refrigerated until analyzed (within a week). The water content in each sample was determined by placing 4−5 g of sample in a mechanical convection incubator (Precision Scientific, Buffalo, NY, USA) at 37 °C for 2 days to dry. Extraction and Purification. Cabbage heads were sliced, and ≃30 g was frozen with liquid nitrogen and kept frozen until analyzed the following day. The frozen materials were ground using a stainless steel Waring Commercial Blender (New Hartford, CT, USA) coupled with a 0.95 L container.27 The acetone/chloroform extraction procedure was adopted from that of Giusti and Wrolstad.28 Frozen plant powder was mixed with 30 mL of acetone. The mixture was filtered through a Whatman no. 1 filter (Whatman Inc., Florham, NJ, USA), and the residual cake was washed with 70% aqueous acetone acidified with 0.1% formic acid (≃250 mL) until the powder was white and the filtrate was clear. The filtrates were combined, transferred to a separatory funnel, and mixed with 1 volume of chloroform. The phases were allowed to separate for 4−5 h. The aqueous phase was collected, and the residual acetone was evaporated using a Büchi rotavapor (Brinkmann Instruments, Inc., Westbury, NY, USA). The aqueous extract was purified using a Sep-Pak C18 cartridge (Waters Corp., Milford, MA, USA). The cartridge was activated with methanol and washed with acidified water before the sample was loaded. The cartridge was further washed with acidified water (0.1% formic acid), Received: Revised: Accepted: Published: 7524

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Figure 1. HPLC chromatograms of two representative red cabbage anthocyanin extracts: second-harvested Cairo (top) and first-harvested Integro (bottom) red cabbage at 510−540 nm. Refer to Table 1 for major peak identifications. HPLC conditions: solvent A, 4.5% formic acid in LC-MS grade water; solvent B, LC-MS acetonitrile; gradient, 0−50 min, 0−30% B. The major peaks (1−8) were found in all other red cabbage samples. and the anthocyanins were recovered with acidified methanol (0.1% formic acid). The methanol was removed using the rotavapor, and the volume was taken to 25 mL with acidified water (0.1% formic acid). Anthocyanins Content. The total monomeric anthocyanin was determined by using the pH differential method according to Giusti and Wrolstad.29 The extract was diluted using pH 1 (0.025 M potassium chloride) and pH 4.5 (0.4 M sodium acetated) buffers with a dilution factor of 100. The solutions were allowed to equilibrate for 15 min in the dark. Absorbance was read on 1 cm path length cuvettes at 520 and 700 nm using a Shimadzu UV−visible spectrophotometer (Shimadzu, Columbia, MD, USA). The total monomeric anthocyanin was calculated on the basis of the dry matter (DM) and fresh matter (FM) and reported as milligrams of cyanidin-3-glucoside (Cy3G) per 100 g of sample using the equation

monitored at 510−540 nm. Peak areas at this region were then integrated and normalized. The proportion of the total peak area of each individual anthocyanin was calculated and reported as percentage of total peak area at 510−540 nm. For MS analyses, a 0.2 mL/min volume was diverted into the MS and ionized under positive ion condition using an electrospray probe. Data were monitored using total ion scan (SCAN) (m/z 200−1200) and selected ion monitoring at m/z 271 (pelargonidin), m/z 287 (cyanidin), m/z 301 (peonidin), m/z 303 (delphinidin), m/z 317 (petunidin), and m/z 331 (malvidin). Color and Spectrophotometric Analyses. A ColorQuest XE colorimeter (HunterLab, Hunter Associates Laboratories Inc., Reston, VA, USA) was used to measure the color characteristics (Hunter CIE LCh) of the samples. The equipment was set for transmittance with specular included, and D65/10° was used for the measurements. Samples were placed in a 1 cm path length plastic cuvette and CIE L*, a*, b*, chroma (c*), and hue angle (h°) were measured. To measure the color in acidic condition, the extract was mixed (in triplicate) with distilled (DI) water (1:20 v/v). The pH of the solutions were measured after 30 min of equilibration and was 3.5. To measure the color in neutral conditions, the extract was diluted with water (1:2 v/v, in triplicate). The diluted solutions were then mixed with 0.1 M potassium phosphate buffer (pH 7) with a dilution factor (DF) of 20. The maximum absorbances (λmax) in the visible range of the neutral solutions were recorded using a Shimadzu UV−visible spectrophotometer 2450. FD&C Red No. 3, FD&C No. 40, and FD&C Blue No. 2 (Noveon Hilton Davis, Inc., Cincinnati, OH, USA) were also dissolved in DI water to concentrations that most closely matched the lightness (L*) and chroma (c*) of the samples. Statistical Analysis. Seven different cultivars at two maturity stages (three heads each) for a total of 42 samples were analyzed using principal component analysis (PCA). PCAs for total monomeric anthocyanin, nonacylated, monoacylated, and diacylated pigments (based on the proportional peak areas shown in Table 2) were performed. Autoscaling was used to normalize each variable before the analysis. To compare the anthocyanin contents and profile, normality of the variables were first checked using the Kolmogorov−Smirnov test (α = 0.05). Analysis of variance (ANOVA) was then used to analyze the total monomeric anthocyanin and the proportion of each group of pigments separately using the following model: Yijk = μ + vi + tj + vtij +

total monomeric anthocyanin (mg/L) = [((A520 − A 700)pH1 − (A520 − A 700)pH4.5 ) × DF × 1000 × MW]/(ε × P) where DF is the dilution factor, MW is the molecular weight (449.2 for Cy3G), ε is the molar absorptivity coefficient (26900 cm−1 mg−1 for Cy3G), and P is the cuvette path length. Alkaline Hydrolysis of Anthocyanins. Alkaline hydrolysis (saponification) was adopted from the method of Giusti and Wrolstad.28 Purified red cabbage anthocyanin extract (10 mL) was mixed with 10 mL of 10% KOH in a capped test tube and set aside for 15 min at room temperature in the dark. The solution was neutralized using 2 N HCl until the color turned pink. The neutralized sample was then purified using a Sep-Pak C18 cartridge (Waters Corp., Milford, MA, USA) and prepared for HPLC analysis. Chromatographic Analysis. A Shimadzu Prominence reverse phase high-pressure LC-MS was coupled to an SPD-M20-A photodiode array and a single-quadrupole electrospray ionization (ESI) mass spectrophotometer (Shimadzu Scientific, Inc.). For data analysis, LCMS Solution software was used (Shimadzu Scientific, Inc.). The column was a 100 × 4.5 mm Kinetex PFP 2.6 μm (Phenomenex Inc., Torrance, CA, USA). The solvents were phase A, 4.5% formic acid in LC-MS grade water, and phase B, LC-MS acetonitrile (Fisher Scientific Inc., Fair Lawn, NJ, USA), and the gradient was 0−50 min, 0−30% B. Injection volume was 20 μL. Spectral data were obtained from 250 to 700 nm, and elution of anthocyanins was 7525

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αk + εijk, where Y is the individual variable, μ is the grand mean, vi is the cultivar effect, tj is the harvesting time effect, vtij is the interaction between the main factors, αk is the heads effect defined as random factor, and εijk is the random error of the model. When a significant difference was obtained (P value < 0.05), the Tukey meanscomparison test was used to compare each pair of means. All statistical analyses were done on the basis of at least three independent replicate samples from each individual head. Results were analyzed by using Minitab 16 statistical software (Minitab Inc.).

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RESULTS AND DISCUSSION HPLC-PDA-MS and Identification of Major Pigments. According to the HPLC-PDA data obtained at 510−540 nm, up to 23 peaks were observed. In previous studies, up to 36 different anthocyanins have been detected in red cabbage.29−31 Figure 1 shows examples of anthocyanin profiles (Cairo and Integro extracts) obtained by HPLC-PDA. The eight major peaks, representing ≃90% of the total anthocyanins and common to all seven cultivars at both maturity stages, were selected for further identification and analyses. The λmax at the UV and vis ranges, molecular ions, and fragments along with tentative identification of each peak are presented in Table 1. The pigments were identified using HPLC-PDA and HPLCMS and compared with data reported in the literature.11,12

Figure 2. Correlation between the first two principal components and the variables as well as score plot with respect to cultivars and harvesting time for seven different red cabbage cultivars: a, total monomeric anthocyanin; b, diacylated pigments; c, monoacylated pigments; d, nonacylated pigments. AZ, Azurro; Ba, Bandolero; Bu, Buscaro; Ca, Cairo; In, Integro; Ko, Kosaro; Pr, Primero. Symbols in black and gray represent harvest times after 13 or 21 weeks, respectively.

Table 1. PDA Absorbance and MS Data for Red Cabbage Anthocyaninsa peak

RTb (min)

λvisc (nm)

λacyld (nm)

M+ e

1 2 3

12.13 16.95 27.41

513 528 523

334 313

773 (287) 979 (287) 919 (287)

4 5 6

28.33 28.85 30.53

523 524 536

326 329 319

949 (287) 979 (287) 1125 (287)

7

31.55

536

330

1155 (287)

8

32.31

536

331

1185 (287)

According to this analysis, the samples were clearly separated diagonally on the basis of their maturation time. With maturation the percentage of diacylation increased for most samples, so for most of the second-harvested samples PC1 is negative. Also, because the early mature samples (week 13) had slightly higher amounts of total monomeric anthocyanin, PC2 tend to be more positive (Figure 2). PCA is a proper way to obtain relevant information from the original variables into fewer new latent variables (PCs). According to our results, this analysis was helpful in classification of the samples at two maturity stages. Most samples with different maturity times were separated on the basis of the first two principal components. Azurro and Primero cultivars, however, were exceptions because their pigment profiles were not significantly changed with maturation. Anthocyanin Content. The average anthocyanin contents for the 13- and 21-week-harvested plants were ≃1442 and 1269 mg Cy3G/100 g DM respectively, and these values for the fresh matter were ≃150 and 145 mg Cy3G/100 g FM, respectively. Piccaglia et al. also reported the anthocyanin content of three red cabbage cultivars in Italy to be >1000 mg/100 g DM.10 The anthocyanin content for the fresh weight red cabbage reported by Oregon State University database and Wu et al. were, however, 25 and 322 ± 40.8 mg/100 g FW, respectively.35,36 According to our findings, cultivar made a difference in anthocyanin contents at both maturity stages. As shown in Table 2, Buscaro and Integro harvested after 13 weeks had the highest anthocyanin contents. Anthocyanin contents, however, did not change significantly from the first to the second harvest except for the Buscaro (DM) cultivar, which showed a lower anthocyanin content when plants were left longer on the ground (Table 2). Accumulation of anthocyanins can be explained by developmental factors, which could be different in different varieties.23 Proportion of Major Pigments. Although red cabbage is known to have more than 20 different anthocyanins, relative proportions of 8 of them (Figure 1 and Table 1) represented ≃90% of the total anthocyanins and were measured and

identification Cy-3diG-5G Cy-3diG-5G + Cy-3diG-5G + coumaric Cy-3diG-5G + Cy-3diG-5G + Cy-3diG-5G + and ferulic Cy-3diG-5G + and ferulic Cy-3diG-5G + and sinapic

sinapicf pferulic sinapic ferulic sinapic sinapic

a

m/z 287 was the major fragment in all eight peaks. Cy-3diG-5G, cyanidin-3-diglucoside-5-glucoside. bRetention time. cλ vis−max. dλ of acylation. eMass ion. fTentative identification.

As shown in Table 1, m/z 287 was the fragment in all eight anthocyanins indicating that cyanidin derivatives were the major aglycon as reported in previous studies.12,30,32−34 All of the pigments were nonacylated, monoacylated, and diacylated derivatives of cyanidin-3-diglucoside-5-glucoside (Cy-3diG5G), which was also confirmed by saponification (results are not shown). The acylating groups were aromatic acids: sinapic, ferulic, and p-coumaric acids (Table 1). Anthocyanin Content and Proportion of the Pigments. Principal Component Analysis of Red Cabbage Anthocyanin Extracts. The analysis of the objects (i.e., red cabbage cultivars at two maturity stages) is performed visually using the scores plot (Figure 2), where the objects are represented as a function of the principal components (PCs). As shown in Figure 2, PC1 correlated positively with monoacylated pigments as opposed to the diacylated pigments. PC2, on the other hand, was more affected by the nonacylated pigments and the total monomeric anthocyanin. PC1 and PC2 extracted 83.7 and 12.8% of the total variances, respectively. 7526

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Table 2. Anthocyanin Contents (Total Monomeric Anthocyanin) and Percentage of Major Pigments (Percent Total Peak Area at 510−540 nm) in Seven Red Cabbage Cultivars at Two Different Harvesting Timesa total monomeric anthocyanin (mg Cy3G/100 g) cultivar

harvest time (week)

DMb

FMc

nonacylated pigmentsd (%)

monoacylated pigmentse (%)

diacylated pigmentsf (%)

Primero

13 21

1111 e 1026 e

109 gh 104 h

21.24 bc 26.84 a

67.63 a 63.71 ab

4.56 g 4.09 g

Integro

13 21

1660 ab 1637 ab

185 a 188 a

18.17 cde 25.59 ab

65.22 ab 53.32 cde

7.23 fg 12.28 ef

Azurro

13 21

1392 bcd 1217 de

144 bcdef 137 cdef

15.85 def 19.33 cde

69.33 a 65.01 ab

9.19 fg 9.21 fg

Kosaro

13 21

1247 cde 1001 e

128 efgh 115 fgh

15.51 ef 20.25 cd

55.43 cd 46.74 ef

11.42 f 19.41 cd

Cairo

13 21

1389 bcd 1256 cde

153 bcde 168 ab

20.08 cde 28.5 a

58.23 bc 43.96 fg

12.87 ef 17.74 de

Bandolero

13 21

1517 abc 1512 abc

165 abcd 165 abcd

13.1 f 19.07 cde

48.67 def 36.75 gh

24.54 bc 29.1 b

Buscaro

13 21

1780 a 1236 de

170 abcd 137 defg

13.36 f 17.04 cdef

52.1 cde 35.48 h

23.59 bcd 35.45 a

a

Different letters in the same column indicate significant differences (p < 0.05). bDry matter. cFresh matter. dPeak 1 (Table 1). ePeaks 2, 3, 4, and 5 (Table 1). fPeak 6, 7, and 8 (Table 1).

Table 3. CIE L*a*b*, Chroma (c*), Hue (h°), and λmax of Seven Red Cabbage Cultivar Extracts at Two Different Harvesting Times at pH 3.5 and Their Comparison with Two FD&C Synthetic Red Dyes HTa (week)

L*

a*

b*

c*

h°

λmax (nm)

ΔE1b

ΔE2c

Primero

13 21

76.2 ± 0.8 77.1 ± 2

45.3 ± 0.6 42.1 ± 4

−3.5 ± 0.7 −2.7 ± 0.7

47.4 ± 3.5 42.2 ± 4

359.2 ± 4.4 356.3 ± 0.9

520 ± 1.2 520 ± 1.4

15.9 ± 2 18.5 ± 4.2

18 ± 0.7 17.9 ± 1

Integro

13 21

67.4 ± 2.6 66.2 ± 1.8

59.5 ± 1.7 61 ± 2.5

−0.5 ± 0.3 −1.9 ± 1.2

59.6 ± 4.6 61 ± 2.5

357.5 ± 2.6 358.2 ± 1.2

520 ± 0.8 520 ± 1.1

8.8 ± 3.1 9.3 ± 2.2

23.4 ± 1.3 24.8 ± 1.4

Azurro

13 21

69.6 ± 3 71.2 ± 2.9

56.1 ± 1 53.2 ± 4.7

−2.8 ± 2 −2.9 ± 1.4

56.6 ± 5.3 53.3 ± 4.6

357.7 ± 2.7 356.7 ± 1.9

520 ± 0.6 520 ± 0.5

6.8 ± 2.9 9.1 ± 2.2

20.7 ± 2 20.2 ± 1.4

Kosaro

13 21

69 ± 3.2 69.5 ± 1.5

57 ± 3.5 55.5 ± 2.4

−5.9 ± 1.2 −7.1 ± 0.9

57.3 ± 5 56 ± 2.4

354.1 ± 2.6 352.7 ± 1

520 ± 0.3 520 ± 0.8

6.1 ± 2.7 7 ± 0.8

23.7 ± 3.7 24.8 ± 1.6

Cairo

13 21

68.9 ± 3.5 64.9 ± 1.5

57.1 ± 1.6 63 ± 2

−4.5 ± 0.6 −4.3 ± 0.4

55.9 ± 3.6 63.2 ± 2

357 ± 1.4 356.1 ± 0.5

520 ± 1.4 520 ± 0.6

6.6 ± 3.2 10.1 ± 2

23 ± 1.3 28.1 ± 1.6

Bandolero

13 21

62.5 ± 1.6 60.7 ± 1.8

67.3 ± 4.3 68.2 ± 2.1

−4.7 ± 1.3 −4.8 ± 1.5

68.4 ± 1.5 68.4 ± 2

356.8 ± 4.4 356 ± 1.4

520 ± 0.8 530 ± 1.2

14.8 ± 3 15.9 ± 2.7

32 ± 1.5 33.6 ± 1.3

Buscaro

13 21

62.9 ± 4.1 61.7 ± 4.2

66.9 ± 2.3 66 ± 4.5

−3.7 ± 1 −6.4 ± 2.9

64.5 ± 1.2 66.4 ± 4.2

357.9 ± 1.4 354.4 ± 2.8

520 ± 0.9 530 ± 1.5

12.8 ± 3 14.2 ± 5.7

29.9 ± 4.4 33 ± 3

cultivar

FD&C Red No. 3 FD&C Red No. 40 a

74.2 75.64

59.99 45.11

−6.1 14.48

60.3 47.38

354.19 17.79

520 500

Harvesting time. bColor difference with FD&C Red No. 3. cColor difference with FD&C Red No. 40.

According to our results, nonacylated pigments represented an average of ≃19.6% (±4.5) of the major pigments (Table 2). Charron et al. also found the percentage of nonacylated pigment in red cabbage to be 21.3%.31 Among the major pigments identified, the amounts of mono- and diacylated

compared among cultivars and maturation stages as they are more likely to affect the color and stability of the extract. The pigments were also grouped into nonacylated, monoacylated, and diacylated. 7527

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Bandolero and Buscaro extracts as opposed to Primero. The wavelengths of maximum absorbances (λmax) of the samples were similar to that of Red No. 3 (≃520 nm) except for lateharvested Bandolero and Buscaro cultivars (λmax ≃ 530 nm). Generally, samples showed more similar color characteristics to FD&C Red No. 3 than to FD&C Red No. 40 synthetic dyes (ΔE1 < ΔE2). Under acidic pH, maturation of the plants did not seem to significantly affect the color of the solutions. Earlyharvested Azurro, Kosaro, Cairo, and Integro at the tested concentrations were the samples that produced colors most similar to FD&C Red No. 3 with similar λmax and ΔE between 6.1 and 8.8 (Table 3). Stability in Neutral pH. The spectral characteristics of the samples at pH 7 were monitored over 72 h of refrigerated storage. Figure 3 shows the changes in λmax for three

anthocyanins were on average 54.4 (±12.6) and 15.8% (±8.7), respectively (Table 2). Red cabbage has been identified as a highly acylated anthocyanin source according to previous research.30,31 Before the percentages of each group of pigments were compared using ANOVA, the normality test was done to verify the validity of the test, and none of the variables had a p value smaller than 0.05. Cultivar had a significant (p value < 0.05) impact on pigment profile. The proportions of monoacylated and diacylated pigments were the major differences found among the cultivars. Primero and Azurro had the highest proportions of monoacylated pigments and the lowest proportions of diacylated pigments, whereas Buscaro and Bandolero varieties had the lowest proportions of monoacyltaed pigments and the highest proportion of diacylated pigments. The effect of maturity on pigment profile was largely dependent on the cultivar for monoacylated and diacylated pigments (p value < 0.05); this interaction was not observed for the nonacylated pigments. As shown in Table 2, the proportion of nonacylated pigments increased significantly in the 21-week-mature plants except for Azurro and Buscaro cultivars, which did not show a significant increase. More mature Primero, Cairo, and Integro had highest amounts of nonacylated pigments. The proportions of monoacylated anthocyanins were, however, decreased with longer maturation time, except for Primero and Azurro cultivars, which did not show a significant decrease on monoacylated pigments. The proportion of monoacylated pigments was highest (≃67%) for cultivar Primero, Integro, and Azurro first harvests as compared to the other cultivars (≃54%) harvested at the same time (Table 2). The proportion of diacylated pigments varied remarkably among cultivars. They increased significantly in Kosaro and Buscaro due to maturation. Buscaro and Bandolero, harvested after 21 weeks, had the highest (≃30%) proportion of diacylated pigments, whereas Primero (≃4%) had the lowest at both maturity stages (Table 2). Because the plants were grown side by side, the main reason for the profile differences could arise from intrinsic factors such as plant genetics and enzymes and their activities throughout the maturation, which influence the anthocyanin synthesis within the plant. Variation in anthocyanin structures can be correlated with alteration of single genes, which influence the enzymatic step of anthocyanin synthesis pathways.37 The reason for accumulation of certain anthocyanins in certain cultivars could be due to the difference in the activities of different genes. Also, during the maturation, the activity of the genes controlling the synthesis of monoacylated pigments could have slowed, whereas those responsible for the synthesis of nonacylated anthocyanins or for the addition of a second acyl group may have remained active. Color Characteristics and Stability. Color in Acidic pH. Table 3 shows the color characteristics of solutions colored with red cabbage anthocyanin extracts from the different cultivars at pH 3.5 and their color comparisons to synthetic colorants FD&C Red No. 3 and No. 40. The lightness of the samples was approximately between 60 and 77 (Table 3). The synthetic colorant solutions were also adjusted to have a similar lightness. Extracts from all seven cultivars produced a deep pink color at pH 3.5 with a hue angle close to 350°. Chroma values of the samples were higher for solutions prepared with

Figure 3. Change in λmax for the second-harvested Buscaro, Bandolero, and Primero at pH 7 over 72 h of refrigeration. Most color changes happened during the first 6 h of storage.

representative samples. Solutions colored with Buscaro and Bandolero red cabbage extracts showed the highest variations in the λmax, whereas solutions colored with Primero extract showed the least changes over the 72 h. Most of the changes observed happened during the first 5−6 h (Figure 3). Table 4 shows the λmax of all seven varieties 30 min after the extracts were mixed with buffer pH 7 and 6 h afterward (refrigerated storage). The λmax for samples containing significantly higher amounts of diacylated anthocyanins (Buscaro and Bandolero) at this pH seemed to be the highest (≃600 nm) compared to the other samples. Torskangerpoll and Andersen also investigated the absorbance and color change in cyanidin 3(2″-(2‴-sinapoylglucosyl)-6″-sinapoylglucoside)-5-glucoside (a diacylated pigment isolated from red cabbage) at different pH values. They also found out that at pH 7.2 the pigment had λmax of ≃605 nm.38 A bathochromic effect of up to 7 nm was observed for most samples after 6 h of refrigerated storage. More mature Buscaro and Bandolero varieties produced a λmax (≃610 nm) similar to that of FD&C Blue No. 2. For other samples such as Primero and Integro, however, minute bathochromic effects were observed (Table 4). Anthocyanins tend to have lower stabilities at neutral to alkaline pH values.14,39 After mixing with buffer pH 7 followed by 6 h of refrigeration, the color degradation was highest (≃53%) and lowest (≃19%) for the second-harvested Primero and Buscaro, respectively (Table 4). Higher stabilities of the Buscaro and Bandolero cultivars at the tested pH values could be explained by the larger number of diacylated anthocyanins, which have higher stabilities due to the pigments intramolecular and/or intermolecular copigmentation and self-association reactions.15 Torskangerpoll and Andersen also demonstrated 7528

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that diacylation of anthocyainins increases their color stabilities.38 The color characteristics of pH 7 buffer solutions colored with the different red cabbage extracts after 6 h of refrigeration compared to FD&C Blue No. 2 are shown in Table 5. The lightness of the samples ranged between ≃60 and 85. Chroma values were higher for Bandolero and Buscaro cultivars at the tested pH value (Table 5). The synthetic FD&C Blue No. 2 was diluted until the lightness was similar to that of the evaluated samples. ΔE values of the samples when compared to this synthetic dye were between 17.6 and 26.4 (Table 5). The color difference with FD&C Blue No. 2 at the tested concentration for the late-harvested Kosaro sample was the smallest; however, the λmax values for the second-harvested Buscaro and Bandolero cultivars were closer to this value for FD&C Blue No. 2 (Tables 4 and 5). Acylation of anthocyanins with aromatic acids (e.g., cinnamic acid) has proven to increase the λmax and shift the hue angle to purple color under acidic conditions.16,17 As shown in Table 1, late-harvested Buscaro and Bandolero cultivars, with the highest percentages of diacylated pigments, also exhibited the highest λmax under neutral conditions. In conclusion, anthocyanin content varied among red cabbage cultivars, and leaving the cabbages in the ground for additional time did not increase the pigment content. The pigment profiles changed among the cultivars and maturation stages. For most cultivars, the amount of nonacylated and diacylated pigments increased as opposed to the monoacylated pigments. Cultivars with lower proportions of diacylated pigments better reproduced the color of FD&C Red No. 3 under acidic conditions, whereas cultivars with higher proportions of diacylated pigments better matched the colors of FD&C Blue No. 2 at neutral pH. Pigment profile also affected the color stability at neutral pH. For future studies,

Table 4. Maximum Absorbance (λmax) of Red Cabbage Extracts at Two Different Harvesting Times Measured after 30 min and 6 h of Refrigeration Storage in Buffer pH 7 Compared to FD&C Synthetic Blue No. 2 and Stabilities (Percent Degradation) during This Timea λmax (nm) HTb (weeks)

30 min

6h

% degradation

Primero

13 21

591.4 ± 0.6 591.3 ± 0.4

591.6 ± 0.8 591.9 ± 1

50.1 ± 5.5 53.3 ± 8.9

Integro

13 21

591.4 ± 1.1 598.6 ± 2.3

592.1 ± 0.1 599.7 ± 2

38.9 ± 5.4 44 ± 9.9

Azurro

13 21

590.8 ± 0.8 595.9 ± 0.7

593.5 ± 0.5 597.8 ± 2.1

41.5 ± 4.9 49.3 ± 7.9

Kosaro

13 21

596.3 ± 1 602 ± 0.3

599.9 ± 1.4 604.8 ± 0.8

31 ± 3.3 39.5 ± 5.2

Cairo

13 21

593.6 ± 0.2 599.7 ± 2.4

595.3 ± 4.2 602.9 ± 0.5

42.1 ± 8.4 36.7 ± 8.2

Bandolero

13 21

601.8 ± 1.2 599.7 ± 2.3

609.4 ± 1.2 610.1 ± 1.6

27.1 ± 4 22.5 ± 3.8

Buscaro

13 21

600.1 ± 0.7 601.8 ± 0.4

606.7 ± 1.9 610.1 ± 1.8

28.6 ± 2.3 19.1 ± 3.8

cultivar

FD&C Blue No. 2

610

610

Percent degradation was calculated by dividing the absorbance at λmax after 6 h by the absorbance at λmax after 30 min in buffer pH 7 × 100. b Harvesting time. a

Table 5. CIE L*a*b*, Chroma (c*), and Hue (h°) of Seven Red Cabbage Extracts at Two Different Harvesting Times after 6 h of Refrigeration Storage at pH 7 Compared to FD&C Blue No. 2 c*

h°

ΔEb

−9.9 ± 0.6 −8.7 ± 2.2

10.2 ± 2.4 9 ± 2.2

284.6 ± 1.5 284.5 ± 5.1

25.7 ± 0.5 26.4 ± 2

1.7 ± 2.2 −0.2 ± 0.6

−22.4 ± 2.8 −23.5 ± 2.6

22.5 ± 2.7 23.5 ± 2.6

274.7 ± 5.8 269.5 ± 1.3

21.4 ± 1 19.8 ± 0.7

77.7 ± 1.5 78.5 ± 3.7

1.4 ± 0.2 1.2 ± 0.4

−15.8 ± 1.5 −15.2 ± 3.1

15.9 ± 1.4 15.3 ± 3.1

275.3 ± 1.3 274.7 ± 1.9

21.2 ± 1.3 21.5 ± 1.3

13 21

72.1 ± 1.5 71.9 ± 2.5

−1.3 ± 0.7 −1.8 ± 1.8

−22.1 ± 1.9 −22.5 ± 2

22.1 ± 2.4 22.6 ± 2.2

266.7 ± 1.3 265.6 ± 4.2

17.9 ± 2.3 17.6 ± 1.1

Cairo

13 21

69.3 ± 4.4 66.2 ± 3.4

−0.5 ± 1.7 −2.3 ± 0.6

−24.7 ± 4.2 −27.9 ± 3

24.7 ± 4.2 28 ± 3

269.2 ± 3.5 265.3 ± 0.8

20.2 ± 1.1 20.2 ± 1.8

Bandolero

13 21

60.8 ± 1.8 59.6 ± 3.1

−4.9 ± 0.7 −5.7 ± 0.6

−31.9 ± 2.7 −33.2 ± 2.6

32.3 ± 4.3 33.7 ± 2.5

261.2 ± 1.4 260.2 ± 1.6

22.9 ± 1.2 23.9 ± 3.6

Buscaro

13 21

63.4 ± 3 63.4 ± 1.4

−3.8 ± 1.5 −5.4 ± 0.2

−29.9 ± 2.9 −30.6 ± 0.8

30.2 ± 2.1 31 ± 0.8

262.9 ± 2.9 260 ± 0.7

21.2 ± 1.6 20.3 ± 1.4

HTa (weeks)

L*

Primero

13 21

83.6 ± 0.6 84.8 ± 2

2.6 ± 0.2 2.1 ± 0.6

Integro

13 21

70.5 ± 2.9 69.9 ± 3.2

Azurro

13 21

Kosaro

cultivar

FD&C Blue No. 2 a

78.06

a*

b*

−17.95

−24.28

30.19

233.52

Harvesting time. bColor difference with FD&C Blue No. 2. 7529

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however, year-to-year variability of these cultivars should be investigated.

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AUTHOR INFORMATION

Corresponding Author

*(M.M.G.) Phone: (614) 247-8016. Fax: (614) 292-0218. Email: [email protected]. Funding

We are thankful to MARS Global Chocolate, Hackettstown, NJ, USA, for providing funding for the project. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Ken McCammon and Bejo Seeds, Inc., for providing the plant materials. Also, we especially thank Marçal Plans Pujolras from Universitat Politècnica de Catalunya for his help with data statistical analysis.

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ABBREVIATIONS USED DM, dry matter; FM, fresh matter; AZ, Azurro; Ba, Bandolero; Bu, Buscaro; Ca, Cairo; In, Integro; Ko, Kosaro; Pr, Primero REFERENCES

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