Effects of Growth Temperature and Postharvest Cooling on

Article pubs.acs.org/JAFC

Effects of Growth Temperature and Postharvest Cooling on Anthocyanin Profiles in Juvenile and Mature Brassica oleracea Didier Socquet-Juglard,† Alexandra A. Bennett,† David C. Manns,‡ Anna Katharine Mansfield,‡ Rebecca J. Robbins,§ Thomas M. Collins,§ and Phillip D. Griffiths*,† †

Department of Horticulture, and ‡Department of Food Science, Cornell University, New York State Agricultural Experiment Station, Geneva, New York 14456, United States § Analytical and Applied Sciences Group, Mars, Incorporated, 800 High Street, Hackettstown, New Jersey 07840, United States ABSTRACT: The effects of growth temperatures on anthocyanin content and profile were tested on juvenile cabbage and kale plants. The effects of cold storage time were evaluated on both juvenile and mature plants. The anthocyanin content in juvenile plants ranged from 3.82 mg of cyanidin-3,5-diglucoside equivalent (Cy equiv)/g of dry matter (dm) at 25 °C to 10.00 mg of Cy equiv/g of dm at 16 °C, with up to 76% diacylated anthocyanins. Cold storage of juvenile plants decreased the total amount of anthocyanins but increased the diacylated anthocyanin content by 3−5%. In mature plants, cold storage reduced the total anthocyanin content from 22 to 12.23 mg/g after 5 weeks of storage in red cabbage, while the total anthocyanin content increased after 2 weeks of storage from 2.34 to 3.66 mg of Cy equiv/g of dm in kale without having any effect on acylation in either morphotype. The results obtained in this study will be useful for optimizing anthocyanin production. KEYWORDS: Brassica oleracea, red cabbage, kale, HPLC, acylated anthocyanins, low temperature

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INTRODUCTION Anthocyanins are a nearly ubiquitous group of water-soluble pigments in the plant kingdom that belong to the flavonoid family. They are responsible for a diverse range of colors, including blue, purple, violet, and bright red, and exist in fruits, flowers, leaves, grains, and roots.1−3 Anthocyanins play a role in the protection of plant tissues against cold, drought, biotic stresses, and ultraviolet (UV) irradiation and have a role in the regulation of auxin biosynthesis.4−7 Both in vivo and in vitro studies have shown that a diet rich in anthocyanins can reduce the risks of cardiovascular disease, degenerative diseases, and cancer.8−14 Red cabbage (Brassica oleracea L.) and red kale (Brassica oleracea var. acephala) have high anthocyanin content and yield potential, with up to 90 kg of anthocyanin per hectare in the case of cabbage.15 Recently, an increased interest in red cabbage and kale natural colors has developed, because they contain anthocyanins that are primarily cyanidin-3-O-diglucoside-5-Oglucoside, which can be non-, mono-, or diacylated, with an important amount of acylated anthocyanin content.15−18 Acylation gives an increased stability to heat and light,19−21 with diacylated anthocyanins having higher antioxidant properties than the monoacylated types.22 With increased consumer awareness concerning food composition, anthocyanins from red cabbage have become an ideal natural pigment that can be used as an alternative to artificial food colorants.23 Anthocyanin biosynthesis and accumulation is influenced by many factors, including genotype, light, temperature, nutrient availability, and plant maturity stage.4,15,16,24,25 Elevated temperature treatments have a negative impact on grapes, decreasing anthocyanin accumulation in the skin.26−28 Similar observations have been made in purple kale.24 To our knowledge, no report has been made on temperature-related treatments specifically affecting the acylation of anthocyanins in Brassica plants. © XXXX American Chemical Society

This current research focused on the red cabbage cultivar ‘Futurima’ and the red kale cultivar ‘Redbor’ for total and diacylated anthocyanin content. The objectives included the evaluation of the anthocyanin content of these two cultivars under different growth temperatures as juvenile plants and the evaluation of the cooling effects on the anthocyanin content. Additional evaluations measured the effects of cold storage on field grown mature plants of the same cultivars. The results obtained provide a better understanding of the optimal growth conditions needed for a large-scale production of anthocyanins derived from B. oleracea.

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MATERIALS AND METHODS

Plant Material and Growth Conditions. Seeds of red cabbage cultivar ‘Futurima’ and red kale cultivar ‘Redbor’ were obtained from Reeds Seeds (Cortland, NY) and Bejo Seeds (Warmenhuizen, Netherlands), respectively. Each cultivar was sown in 72-cell Styrofoam trays (Speedling, Plant City, FL) and filled with “Cornell mix” as a growing medium.29 The plants were grown in five identical growth chambers (Conviron, Canada) with a 16 h photoperiod and a light intensity averaging 350 μmol m−2 s−1. Each chamber was configured with a specific temperature range, with temperatures of 13, 16, 19, 22, and 25 °C during the light period and 10, 13, 16, 19, and 22 °C during the dark period, respectively. Half of the plants were harvested 28 days after sowing, and the other half were transferred to a cold chamber maintained at 4 °C for 10 days. All plants were frozen immediately after harvest until lyophilization. In addition, ‘Futurima’ and ‘Redbor’ plants were sown in a greenhouse in Ithaca, NY, and transplanted to a field in Freeville, NY, in June 2014 on raised plastic beds, where they were spaced at 50 cm Received: November 5, 2015 Revised: January 14, 2016 Accepted: February 1, 2016

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DOI: 10.1021/acs.jafc.5b05309 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Chromatographic Properties of Anthocyanins from Red Cabbage and Kale Futurima

Redbor

peaka

RTb

λmax (nm)

RTb

λmax (nm)

[M]+ (m/z)

assignmentc

compounds, tentative identification

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

17.82 18.82 23.26 23.51 24.36 26.68 27.57 28.29 30.48 31.36 32.04 33.27 34.14 34.65 36.00 36.96 37.95 38.60 39.30

514 514 528 528 520 522 524 524 534 534 532 522 522 524 522 534 536 536 534

18.54 NA NA 23.89 NA NA 27.71 NA NA 31.46 NA 33.40 34.29 34.78 NA 37.07 37.86 38.52 39.27

514 NA NA 528 NA NA 524 NA NA 534 NA 526 522 524 NA 536 536 536 534

773 611 1141 979 979 1081 1111 1141 1287 1317 1347 919 949 979 1111 1125 1155 1185 1155

N N D M M D D D D D D M M M D D D D D

cyanidin-3-diglucoside-5-glucoside cyanidin 3,5-diglucoside cyanidin-3-(glycopyranosyl-sinapoyl)-diglucoside-5-glucoside cyanidin-3-(sinapoyl)-diglucoside-5-glucoside cyanidin-3-(sinapoyl)-diglucoside-5-glucoside cyanidin-3-(caffeoyl)(p-coumaroyl)-diglucoside-5-glucoside cyanidin-3-(glycopyranosyl-feruloyl)-diglucoside-5-glucoside cyanidin-3-(glycopyranosyl-sinapoyl)-diglucoside-5-glucoside cyanidin-3-(feruloyl)(feruloyl)-triglucoside-5-glucoside cyanidin- 3-(sinapoyl)(feruloyl)-triglucoside-5-glucoside cyanidin-3-(sinapoyl)(sinapoyl)-triglucoside-5-glucoside cyanidin-3-(p-coumaroyl)-diglucoside-5-glucoside cyanidin-3-(feruloyl)-diglucoside-5-glucoside cyanidin-3-(sinapoyl)-diglucoside-5-glucoside cyanidin-3-(glycopyranosyl-feruloyl)-diglucoside-5-glucoside cyanidin-3-(sinapoyl)(p-coumaroyl)-diglucoside-5-glucoside cyanidin-3-(sinapoyl)(feruloyl)-diglucoside-5-glucoside cyanidin-3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside cyanidin-3-(sinapoyl)(feruloyl)-diglucoside-5-glucoside

a

Peak numbering refers to peaks in Figures 1 and 2. bRT = retention time. cPeak assignment is based on the elution time, absorbance spectrum, and literature. Abbreviations: N, non-acylated; M, monoacylated; D, diacylated; and NA, not available. Numbers refer to the text. and resolved over a 40 min period at a flow rate of 0.2 mL/min. For HPLC work, mobile phases A and B were made up in water and methanol, respectively, each containing 0.5% (v/v) o-phosphoric acid, while for HPLC−MS/MS work, 3% formic acid was used in place of phosphoric acid. The gradient profile for mobile phase B was 0 min, 15%; 10 min, 30%; 15 min, 35%; 38 min, 50%; 40 min, 15%; followed by a 10 min equilibration before subsequent injection. A diode array detector fitted with a 10 mm path, 1 μL volume Max-Light cartridge flow cell was set to monitor the eluent at 520 nm (630 nm reference). The method resulted in a relative standard deviation (RSD) < 0.5%, and triplicate standard curves using cyanindin-3,5-diglucosde were validated for quantification (r2 > 0.9999). As such, all measurements are reported as cyanidin-3,5-diglucoside equivalent (Cy equiv). Anthocyanin quantification was performed using Agilent Chemstation software version B.04.03, service pack 2 with the spectral software module. Initial speciation and screening between acylated and non-acylated anthocyanins was judged on the basis of elution time, absorbance spectrum, and previously published literature16,30−32 (Table 1). Anthocyanin identifications based on intact and fragment ion masses were performed via mass spectrometry using an Agilent 6410 triple quadrupole MS/MS operated in positive-ion mode using full-scan acquisition [total ion chromatogram (TIC)]. Statistical Analysis. For both cultivars, five replicates each containing six pooled juvenile plants were removed from each of the five growth chambers 28 days post-seeding, while a second batch of five replicates was submitted to an extra 10 day cooling period. Four replicates of mature plants for each time point and cultivar were included for analysis of variance (ANOVA) using statistical software JMP (SAS Institute). The Tukey t test was used, and p values under 0.05 were considered as showing significant differences.

with 2 m row centers and grown to maturity. The whole plants were harvested in October before the first frost and placed in a refrigerated cold storage room at 4 °C immediately after harvest. Average maximum daily temperatures for October at harvest time was 20 °C, and the average minimum temperature was 10.5 °C (data from Ithaca Cornell orchards weather station). Four plants were removed on a daily basis the first week and every 7 days for 8 weeks thereafter. Segments of cabbage heads representing the total head content were taken as samples, and kale leaves were removed and frozen prior to freeze-drying. Reagents and Standards. Anthocyanin extraction, quantification, and speciation were performed with High-Performance Liquid Chromatography (HPLC)-grade organic solvents and pH modifiers obtained from Fisher Scientific (Pittsburgh, PA). The primary reference standard cyanidin-3,5-diglucoside was acquired from Extrasynthese (Genay, France). A Milli-Q integral water purification system (Millipore Corporation, Bedford, MA) provided all 0.22 μm filtered ultrapure water used throughout this study. Sample Preparation. Samples were lyophilized over 24 h using a MX53 Magnum series freeze-drying unit (Millrock Technology, Kingston, NY). Subsequently, each sample was ground to a fine powder using a mortar and pestle and finally stored at room temperature protected from light until further analysis. A 50 mg portion of each sample was extracted 3 times with 5 mL aliquots of 0.01 M hydrochloric acid (HCl) in methanol (MeOH). During each extraction, samples were sonicated for 1 min before centrifugation at 3000g using an Eppendorf 5810R centrifuge (Eppendorf, North America, Hauppauge, NY). The extracts were pooled and evaporated in a 35 °C water bath under a gentle stream of nitrogen using a Parker Balston model N2-04 nitrogen generator (Parker Hannifin Corp., Haverhill, MA) until the samples were dried. The residue was reconstituted in 1 mL of 0.01 M HCl in water, filtered through a 0.22 μm polyethersulfone (PES) membrane (Krackeler Scientific, Inc., Albany, NY), and immediately analyzed via HPLC. Anthocyanin Speciation via HPLC−Tandem Mass Spectrometry (MS/MS). A novel method to separate acylated cyanidin anthocyanins in Brassica was developed. Anthocyanin extracts were resolved using a Kinetex core−shell pentafluorophenyl (PFP) column (100 × 2.1 mm, 2.6 μm diameter particle size with a 100 Å pore size) fitted with an inline Krudkatcher column filter (Phenomenex, Torrance, CA) connected to an Agilent 1260 Infinity series HPLC system. A total of 5 μL of each sample was injected into the column heated to 35 °C

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RESULTS AND DISCUSSION Identification of Pigments in Juvenile and Mature Brassica Plants. Juvenile red cabbage and kale plants were evaluated for anthocyanin accumulation under five different temperature regimes in growth chambers. Field grown mature plants of the same cultivars were also evaluated for anthocyanin content changes following postharvest cooling for different time periods. A total of 19 and 11 anthocyanins were identified and investigated for red cabbage and kale, respectively B


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Journal of Agricultural and Food Chemistry

Figure 1. Representative HPLC chromatograms of red cabbage cultivar ‘Futurima’ at (A) juvenile stage and (C) mature stage and kale ‘Redbor’ at (B) juvenile stage and (D) mature stage. Names of anthocyanin compounds correspond to the numbers given in Table 1.

Figure 2. Temperature effects on the plant size and color of (A−E) cabbage ‘Futurima’ and (F−J) kale ‘Redbor’ grown at (A and F) 13 °C, (B and G) 16 °C, (C and H) 19 °C, (D and I) 22 °C, and (E and J) 25 °C.

anthocyanins (peaks 16−18). Juvenile and mature red kale chromatograms were very similar, with five major peaks, including one non-acylated cyanidin anthocyanin (peak 1), two monoacylated anthocyanins (peaks 4 and 13), and two diacylated anthocyanins (peaks 17 and 18). Both juvenile and mature red kale chromatograms were also very similar to the red cabbage juvenile profiles (Figure 1). This similarity to juvenile red cabbage profiles could be explained by the very similar plant morphology among juvenile Brassica plants and mature kale plants. Effect of the Growth Temperature on Juvenile Plants. A range of growth temperatures from 13 to 25 °C was used to better understand the effects of temperatures on anthocyanin accumulation in red cabbage and kale juvenile plants. Samples were evaluated 28 days after sowing, and growth temperature parameters were observed to have a large effect on plant vigor, size, and color intensity in both red cabbage and red kale (Figure 2). At the two lowest temperatures, 13 and 16 °C, both

(Table 1 and Figure 1). The 11 anthocyanins identified in ‘Redbor’ were in common to both kale and cabbage (Table 1). The method that was developed to separate acylated cyanidin anthocyanins in Brassica led to results in accordance with what has been previously observed in red cabbage16,18,22,30 and kale.35 On the basis of the elution time, absorbance spectrum, MS/MS identification, and previous literature, we could classify anthocyanins into three subcategories: non-, mono-, and diacylated anthocyanins (Table 1). Juvenile red cabbage plants were characterized by chromatograms comprised of six major anthocyanins that each accounted for at least 10% of the total anthocyanins in our experiments: one non-acylated (peak 1), three monoacylated (peaks 4, 13, and 14), and two diacylated (peaks 17 and 18). Mature ‘Futurima’ plants were characterized by seven major anthocyanins but showed very different profiles compared to the juvenile plants, with one non-acylated (peak 1), three monoacylated (peaks 12−14), and three diacylated C


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between 55 and 57% at temperatures ranging from 13 to 19 °C, this ratio decreased rapidly to 47% at 22 °C and 37% at 25 °C (Figure 3). This decrease was compensated by an increase in monoacylated anthocyanins from 33% at 19 °C to 42% at 25 °C. To our knowledge this is the first time that such high ratios of diacylated anthocyanins have been reported in red cabbage. The significant decrease in diacylated anthocyanin ratios was observed for both major diacylated peaks (17 and 18) and for two minor peaks (10 and 16), while a significant increase in the ratio of the monoacylated peaks 12−14 as well as the non-acylated peak 1 was obtained (Table 3). A different pattern was observed in ‘Redbor’, where the total anthocyanin content continuously decreased with increasing temperatures, ranging from 6.29 mg of Cy equiv/g of dm at 13 °C to 3.82 mg of Cy equiv/g of dm at 25 °C (Figure 3). Those results are in accordance with previous work in mature purple kale, with anthocyanin accumulation increasing with lowtemperature stress.24 However, the ratio of diacylated anthocyanins increased with increasing temperatures, ranging from 59% at 13 °C to 75% at 25 °C, while the ratio of monoacylated anthocyanins decreased from 36% at 13 °C to 22% at 25 °C. This indicates that the dark purple color observed in plants grown at low temperatures was not linked to acylation of the anthocyanins but rather to the total amount of anthocyanins. Single-peak analysis showed a significant decrease of the ratios of monoacylated peaks 13 and 14 between 16 and 19 °C (Table 3). The ratio of non-acylated peak 1 significantly decreased with increasing temperatures. Concerning diacylated peaks, the two major peaks showed a different response to the increasing temperatures, the ratio of peak 17 increased significantly at 19 °C and then plateaued, while the ratio of peak 18 only significantly increased at 25 °C (Table 3). Effects of Cooling on Juvenile Plants. To investigate the impact cooling may have on acylation and total anthocyanin content, we submitted 28-day-old juvenile plants to 4 °C cooling for 10 days to contrast with juvenile plants evaluated without 4 °C cooling. This resulted in a decrease in the total amount of

cultivars showed an intensified color, with the extent of green coloration increasing gradually with higher temperatures. Plant size and weight showed a continuous increment with increasing temperatures for ‘Redbor’, while the fresh weight of ‘Futurima’ doubled between 16 and 22 °C temperatures and then plateaued (Table 2 and Figure 2). Table 2. Effects of Growth Temperature and 10 Days of Cooling on the Fresh Weight of ‘Futurima’ and ‘Redbor’ Juvenile Plantsa no cooling temperature (°C) 13 16 19 22 25

Futurima 2.20 2.32 3.24 5.52 5.48

a ab b c c

cooling

Redbor 1.40 1.68 2.04 2.88 3.36

a a b c d

Futurima 2.56 2.56 3.64 5.36 5.28

a a b c c

Redbor 1.40 1.88 2.20 2.68 2.28

a ab b bc bc

a Fresh weight was measured right after cutting the plants. Different letters in the same column show a significant difference among the different growth temperatures (p < 0.05).

Optimum growth temperatures for the total anthocyanin content differed between ‘Futurima’ and ‘Redbor’. Total anthocyanin accumulation was highest in the 16 °C chamber for ‘Futurima’, with 10.00 mg of Cy equiv/g of dry matter (dm), and was lowest in the 25 °C chamber, with 4.53 mg of Cy equiv/g of dm (Figure 3). The total anthocyanin content was lower than that reported in mature red cabbage plants, which can be as high as 19 mg of Cy equiv/g of dm.15 The difference in the anthocyanin concentration between juvenile and mature plants could be due to chlorophyll concentrations, which can be found in high quantities in juvenile tissues and in adult kale plants, while chlorophyll is minimal or absent in cabbage heads (data not shown). The percentage of total acylated anthocyanins continuously decreased from 90% at 13 °C to 79% at 25 °C. While the diacylated anthocyanin content was

Figure 3. Anthocyanin content (mg of Cy equiv/g of dm) in cabbage cultivar ‘Futurima’ and kale cultivar ‘Redbor’ juvenile plants grown under five different temperature conditions 4 weeks post-sowing. Error bars show the standard deviation of the mean of five independent replicates, and values having the same letters were not significantly different at p < 0.05. D


E

C

8.62 0.43 0.03 9.09 1.03 0.10 0.63 0.38 1.34 4.49 0.25 2.25 9.74 7.99 1.14 7.28 24.39 18.71 2.10

c

N

9.92 0.56 0.03 9.51 0.69 0.11 0.54 0.52 0.98 3.96 0.26 2.52 10.74 9.64 1.29 6.53 21.64 18.71 1.85

b a a b a a a a a a b a a a a b ab a a

16 °C C

10.24 0.51 0.04 10.9 0.97 0.07 0.53 0.37 1.32 4.50 0.29 1.68 8.40 6.70 0.85 6.24 23.26 21.42 1.71

N 10.65 0.84 0.03 8.41 1.09 0.17 0.64 0.43 0.77 2.71 0.30 2.91 11.31 10.89 2.13 6.13 20.68 17.54 2.36

b b a a ab b b a b b b a a a b c b a a

19 °C C 10.02 0.64 0.08 8.52 1.25 0.08 0.63 0.39 0.77 2.84 0.21 2.76 10.43 9.10 1.66 6.47 23.07 18.58 2.51

Futurima

N 14.32 0.97 0.05 7.54 1.81 0.17 0.63 0.44 0.65 2.14 0.35 5.03 13.02 13.02 2.17 5.52 15.57 14.31 2.29

c b a a b b b a b c b b ab b b d c b a

22 °C C 14.37 0.83 0.04 9.10 1.46 0.11 0.57 0.40 0.65 2.41 0.28 3.64 11.55 10.52 1.56 5.96 18.32 16.23 2.00

N 19.12 0.98 0.04 7.33 1.55 0.28 0.75 0.86 0.71 1.89 0.18 6.12 15.46 13.65 1.48 4.46 12.38 10.81 1.96

d b a a b c c b b c a b c b a e d c a

C 17.51 0.90 0.06 7.61 1.70 0.36 0.69 0.61 0.65 1.85 0.23 6.64 15.27 11.32 1.35 5.36 14.6 11.21 2.08

25 °C N 4.75 a N/A N/A 4.5 a N/A N/A 1.00 a N/A N/A 3.47 a N/A 2.41 a 18.47 a 10.84 a N/A 4.69 a 27.53 a 20.18 a 2.15 a

C 5.23 N/A N/A 4.68 N/A N/A 0.94 N/A N/A 3.57 N/A 1.65 17.91 10.51 N/A 4.41 27.85 21.22 2.03

13 °C N 4.66 a N/A N/A 5.33 b N/A N/A 0.96 a N/A N/A 3.56 a N/A 2.23 a 15.96 b 9.38 b N/A 4.49 a 27.79 a 23.2 ab 2.44 a


16 °C 3.28 b N/A N/A 6.07 b N/A N/A 0.99 a N/A N/A 3.92 a N/A 1.29 b 12.67 c 6.46 c N/A 5.45 bc 35.12 c 21.56 a 3.2 b

N


Redbor 19 °C N 3.37 b N/A N/A 5.89 b N/A N/A 1.26 ab N/A N/A 4.29 ab N/A 1.41 b 13.1 c 7.88 c N/A 5.65 c 31.23 b 22.64 a 3.29 b

22 °C C 3.02 N/A N/A 5.58 N/A N/A 1.02 N/A N/A 4.38 N/A 1.35 13.36 7.77 N/A 4.91 31.37 24.17 3.08

N 2.41 c N/A N/A 5.97 b N/A N/A 0.85 a N/A N/A 4.98 b N/A 0.94 b 8.23 d 7.15 c N/A 6.31 d 31.77 b 27.38 b 4.01 c

25 °C C 2.04 N/A N/A 5.83 N/A N/A 0.67 N/A N/A 4.62 N/A 1.14 9.20 6.43 N/A 4.87 32.92 28.49 3.80

Different letters in the same row show a significant difference among the different growth temperatures (p < 0.05). Values in bold show a significant difference between plants that spent an extra 10 days at 4 °C and those that did not. bPlants harvested directly after 28 days. cPlants with an extra 10 days at 4 °C.

a

N

8.49 a 0.46 a 0.03 a 8.17 a 0.74 a 0.11 a 0.54 a 0.46 a 1.04 a 3.89 a 0.18 a 2.87 a 11.93 a 10.61 a 1.35 a 7.36 a 22.22 a 17.5 a 2.06 a

peak

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

b

13 °C

Table 3. Effects of Growth Temperature and 10 Days of Cooling on Each Anthocyanin Compound in Cabbage ‘Futurima’ and Kale ‘Redbor’a

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Figure 4. Anthocyanin content (mg of Cy equiv/g of dm) in cabbage cultivar ‘Futurima’ and kale cultivar ‘Redbor’ juvenile plants grown under five different temperature conditions for 4 weeks and then submitted to a temperature of 4 °C for 10 days. Error bars show the standard deviation of the mean of five independent replicates, and values having the same letters were not significantly different at p < 0.05.

of pigments per plant) to 25 °C (1.57 mg of pigments per plant) (Figure 5). Very similar observations were made with plants that were cooled, with an optimum temperature of 22 °C for ‘Futurima’ and with no significant differences observed between temperatures ranging from 16 to 25 °C in ‘Redbor’ (Figure 6). Plants that were grown under lower temperatures (between 13 and 19 °C) continued to grow during the cooling period, because an increase in the fresh weight was observed for both ‘Futurima’ and ‘Redbor’ plants (Table 2). However, the fresh weight of plants that were grown at 22 and 25 °C decreased after 10 days of cooling, probably as a result of cold stress. Cooling may have an economic penalty for anthocyanin production as a result of the reduction of both the fresh weight at optimal temperatures and in total pigment content, along with the high expenses for facilities that would be required to store the plants at 4 °C. Postharvest Storage Effects on the Anthocyanin Content in Mature Plants. Mature plants were stored at 4 °C, and samples were taken daily for the first week and then every 7 days for a period of 8 weeks. A low temperature has been shown to induce anthocyanin accumulation through upregulation of anthocyanin biosynthetic genes in Arabidopsis33,34 and kale,24,35 but not much is known about the effects of postharvest storage in Brassica plants or the potential influence on the quantity and quality of anthocyanins. For ‘Futurima’, plant samples that were evaluated without postharvest cooling had an average anthocyanin concentration of 22 mg of Cy equiv/g of dm (Figure 7), which is higher than previously reported in red cabbage. Piccaglia et al. reported yields between 10 and 19 mg of Cy/g of dm;15 Ahmadiani et al. observed an average of 14.4 and 12.6 mg of Cy/g of dm for 13 and 21 week harvested plants;16 and Wiczkowski et al. reported an average from 1.13 to 6.29 mg of Cy/g of dm.22 This could be due to the growth environment, plant age at harvest, or genotype; however, major differences can also be attributed to light, plant turgidity relative to field moisture, and biotic stress.22 Because the yield consistency and quality of

anthocyanins in ‘Futurima’, which had a mean of 4.03 mg of Cy equiv/g of dm for plants that had been grown in 25 °C chambers to 8.42 mg of Cy equiv/g of dm for the samples grown in 16 °C chambers (Figure 4). Total diacylated anthocyanins showed an increase of 3−5% compared to untreated plants, and a decrease in monoacylated anthocyanins was also observed, leading to similar percentages of total acylated anthocyanins to those observed in plants that had not been cooled. Single-peak analysis revealed that cooling significantly decreased ratios of monoacylated peak 14 for all growth temperatures, while ratios of monoacylated peak 4 increased significantly for plants that had been grown at 13, 16, and 22 °C (Table 3). Ratios of diacylated peaks 17 and 18 were significantly increased by the 10 day cooling period, regardless of the growth temperature, while a decrease in ratios of diacylated peak 15 was observed at all temperatures, except 25 °C. The cooling period also resulted in a decrease in the total anthocyanin content in ‘Redbor’, which ranged from 2.90 mg of Cy equiv/g of dm for the plants that had been grown in 25 °C chambers to 5.63 mg of Cy equiv/g of dm for the samples grown in 16 °C chambers. The only significant increase in the percentage of diacylated anthocyanins was observed for the plants previously grown in the 16 °C chambers (Figure 4). Single-peak analysis showed that cooling Redbor plants only had a minor impact on peak ratios, with most significant effects obtained for plants grown at 16 °C that showed significant decreases in ratios of peaks 1, 4, 12, and 19 and a significant increase in the ratio of peak 17 (Table 3). Because higher plant biomass can be obtained with higher temperature growing conditions, we also investigated which temperature would be more optimal for the most efficient production of anthocyanins per plant. For ‘Futurima’, the 22 °C chamber was more optimal with an average of 4.52 mg of anthocyanins produced per plant; this was significantly higher than samples taken from plants grown under the other temperatures (Figure 5). For ‘Redbor’, no significant difference was obtained between temperatures ranging from 19 °C (1.29 mg F


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Figure 5. Average anthocyanin content per plant (in milligrams of dm) of cabbage cultivar ‘Futurima’ and kale cultivar ‘Redbor’ juvenile plants grown under five different temperature conditions 4 weeks post-sowing. Error bars show the standard deviation of the mean of five independent replicates, and values having the same letters were not significantly different at p < 0.05.

Figure 6. Average anthocyanin content per plant (in milligrams of dm) of cabbage cultivar ‘Futurima’ and kale cultivar ‘Redbor’ juvenile plants grown under five different temperature conditions for 4 weeks and then submitted to a temperature of 4 °C for 10 days. Error bars show the standard deviation of the mean of five independent replicates, and values having the same letters were not significantly different at p < 0.05.

‘Futurima’ can vary depending upon the growing season and location, further investigation would be required to determine consistency of anthocyanin yields. Non-acylated anthocyanins in mature plants were between 10 and 15% in our study (Figure 7), which is less than previously reported by Ahmadiani et al.16 and Charron et al.17 Non-acylated anthocyanin levels observed in samples from our study could be due to the age of plants at harvest (120 days after transplant) and the cold/light stress of harvesting plants in the fall, which corresponds to the accumulation obtained by Wiczkowski et al.,22 with similar vegetation time. Wiczkowski et al.22 further noted a higher non-acylated

anthocyanin accumulation in later maturing red cabbage cultivars than in the early maturing red cabbage cultivars; however, this could be due to climatic stress at the time of harvest or genotype. Ahmadiani et al.16 showed that cabbage cultivars had higher non-acylated pigment content when they were harvested late in the season, which also indicates climatic stress effects from shorter days and cooler temperatures. It is likely that ‘Futurima’ would have had a higher non-acylated anthocyanin concentration if plants had been harvested later in the season. Total anthocyanin means in mature red cabbage ranged between 17.32 mg of Cy equiv/g of dm after 3 weeks of cooling G


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Figure 7. Anthocyanin content (mg of Cy equiv/g of dm) of cabbage cultivar ‘Futurima’ mature heads submitted to a temperature of 4 °C for up to 8 weeks and kale cultivar ‘Redbor’ mature plant submitted to a temperature of 4 °C for up to 2 weeks. Error bars show the standard deviation of the mean of five independent replicates, and values having the same letters were not significantly different at p < 0.05.

Figure 8. Time series of ratios of anthocyanin peak compounds (in percentage) for (A) cabbage cultivar ‘Futurima’ and (B) kale cultivar ‘Redbor’. Lines represent linear regressions. Only peaks that significantly changed over time are shown.

(Figure 7). Anthocyanin levels from samples removed before 2 weeks of postharvest storage were not significantly different. Similar to ‘Futurima’, the percentage of diacylated anthocyanin did not change significantly over time, with a concentration of diacylated pigments between 60 and 65% and a concentration of monoacylated pigments between 28 and 33%. Only four individual peaks significantly changed over storage time in a pattern similar to that for ‘Futurima’: ratio of monoacylated peak 14 significantly decreased over time, while ratios of non-acylated peak 1 and diacylated peak 16 increased (Figure 8). This is the first report of the effects of cool storage on the acylation of anthocyanins in red kale. Plants are able to respond to environmental stress, such as pathogen attacks, light, nutrient depletion, and low temperature, by synthesizing anthocyanins.4 The effect of stress as a result of low temperatures on anthocyanin biosynthesis has been reported in Arabidopsis, grape, maize, and apple.26,36−38 In grapes, high temperatures decreased the concentration of anthocyanins in the skin26,28 as did abscisic acid38 but the level of anthocyanin accumulation could be restored by spraying abscisic acid.39 Low temperatures do not enhance anthocyanin accumulation without visible light40 or ultraviolet light B (UVB).41 When considering the effects of cold on anthocyanin accumulation, it has been

and 25.85 mg of Cy equiv/g of dm after 2 weeks of cooling, which are similar amounts to what has been previously reported in purple corn and blueberry.36 This amount significantly decreased after 5 weeks of cooling to means ranging between 12.23 and 13.47 mg of Cy equiv/g of dm. Means of diacylated anthocyanins ranged from 28 to 36%, but storage time did not have any significant impact on the ratios (Figure 7). The ratios were in accordance with maximum diacylated anthocyanin producing cultivars investigated by Ahmadiani et al.16 and slightly lower than the means observed by Wiczkowski et al.22 Although anthocyanin ratios remained stable over storage time, a regression analysis performed only with plants harvested weekly showed that individual monoacylated peaks 12−14 significantly decreased over time, while a significant increase was observed for non-acylated peak 1 and diacylated peaks 16−18 (Figure 8). ‘Redbor’ kale plants without postharvest cooling had a total amount of anthocyanin of 2.34 mg of Cy equiv/g of dm, which is lower than the 5.1 mg of Cy equiv/g of dm previously reported for this cultivar on the basis of spectrophotometric estimations.37 A period of 2 weeks of cooling significantly increased the total amount of pigments in comparison to plants that were not cooled, with a total amount of 3.66 mg of Cy equiv/g of dm H


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(10) He, J.; Giusti, M. M. Anthocyanins: Natural Colorants with Health-Promoting Properties. Annu. Rev. Food Sci. Technol. 2010, 1 (1), 163−187. (11) Kong, J. Analysis and Biological Activities of Anthocyanins. Phytochemistry 2003, 64 (5), 923−933. (12) Paredes-López, O.; Cervantes-Ceja, M. L.; Vigna-Pérez, M.; Hernández-Pérez, T. Berries: Improving Human Health and Healthy Aging, and Promoting Quality LifeA Review. Plant Foods Hum. Nutr. 2010, 65 (3), 299−308. (13) Chen, P.-N.; Chu, S.-C.; Chiou, H.-L.; Kuo, W.-H.; Chiang, C.L.; Hsieh, Y.-S. Mulberry Anthocyanins, Cyanidin 3-Rutinoside and Cyanidin 3-Glucoside, Exhibited an Inhibitory Effect on the Migration and Invasion of a Human Lung Cancer Cell Line. Cancer Lett. 2006, 235 (2), 248−259. (14) Lila, M. A. Anthocyanins and Human Health: An in Vitro Investigative Approach. J. Biomed. Biotechnol. 2004, 2004 (5), 306− 313. (15) Piccaglia, R.; Marotti, M.; Baldoni, G. Factors Influencing Anthocyanin Content in Red Cabbage (Brassica oleracea Var capitata L F Rubra (L) Thell). J. Sci. Food Agric. 2002, 82 (13), 1504−1509. (16) Ahmadiani, N.; Robbins, R. J.; Collins, T. M.; Giusti, M. M. Anthocyanins Contents, Profiles, and Color Characteristics of Red Cabbage Extracts from Different Cultivars and Maturity Stages. J. Agric. Food Chem. 2014, 62 (30), 7524−7531. (17) Charron, C. S.; Clevidence, B. A.; Britz, S. J.; Novotny, J. A. Effect of Dose Size on Bioavailability of Acylated and Nonacylated Anthocyanins from Red Cabbage (Brassica oleracea L. Var. capitata). J. Agric. Food Chem. 2007, 55 (13), 5354−5362. (18) Park, S.; Arasu, M. V.; Lee, M.-K.; Chun, J.-H.; Seo, J. M.; AlDhabi, N. A.; Kim, S.-J. Analysis and Metabolite Profiling of Glucosinolates, Anthocyanins and Free Amino Acids in Inbred Lines of Green and Red Cabbage (Brassica oleracea L.). LWT - Food Sci. Technol. 2014, 58 (1), 203−213. (19) Dyrby, M.; Westergaard, N.; Stapelfeldt, H. Light and Heat Sensitivity of Red Cabbage Extract in Soft Drink Model Systems. Food Chem. 2001, 72 (4), 431−437. (20) Markakis, P. Stability of Anthocyanins in Foods. In Anthocyanins as Food Colors; Markakis, P., Ed.; Academic Press: New York, 1982; pp 163−180, DOI: 10.1016/B978-0-12-472550-8.50010-X. (21) Malien-Aubert, C.; Dangles, O.; Amiot, M. J. Color Stability of Commercial Anthocyanin-Based Extracts in Relation to the Phenolic Composition. Protective Effects by Intra- and Intermolecular Copigmentation. J. Agric. Food Chem. 2001, 49 (1), 170−176. (22) Wiczkowski, W.; Topolska, J.; Honke, J. Anthocyanins Profile and Antioxidant Capacity of Red Cabbages Are Influenced by Genotype and Vegetation Period. J. Funct. Foods 2014, 7, 201−211. (23) Bridle, P.; Timberlake, C. F. Anthocyanins as Natural Food Coloursselected Aspects. Food Chem. 1997, 58 (1), 103−109. (24) Zhang, B.; Hu, Z.; Zhang, Y.; Li, Y.; Zhou, S.; Chen, G. A Putative Functional MYB Transcription Factor Induced by Low Temperature Regulates Anthocyanin Biosynthesis in Purple Kale (Brassica oleracea Var. acephala F. Tricolor). Plant Cell Rep. 2012, 31 (2), 281−289. (25) Xie, Q.; Hu, Z.; Zhang, Y.; Tian, S.; Wang, Z.; Zhao, Z.; Yang, Y.; Chen, G. Accumulation and Molecular Regulation of Anthocyanin in Purple Tumorous Stem Mustard (Brassica juncea Var. tumida Tsen et Lee). J. Agric. Food Chem. 2014, 62 (31), 7813−7821. (26) Mori, K.; Sugaya, S.; Gemma, H. Decreased Anthocyanin Biosynthesis in Grape Berries Grown under Elevated Night Temperature Condition. Sci. Hortic. 2005, 105 (3), 319−330. (27) Mori, K.; Goto-Yamamoto, N.; Kitayama, M.; Hashizume, K. Loss of Anthocyanins in Red-Wine Grape under High Temperature. J. Exp. Bot. 2007, 58 (8), 1935−1945. (28) Yamane, T.; Jeong, S. T.; Goto-Yamamoto, N.; Koshita, Y.; Kobayashi, S. Effects of Temperature on Anthocyanin Biosynthesis in Grape Berry Skins. Am. J. Enol. Vitic. 2006, 57 (1), 54−59. (29) Boodley, J. W.; Sheldrake, R., Jr. Cornell Peat-Lite Mixes for Commercial Growing; New York State College of Agriculture and Life

shown recently that anthocyanin biosynthetic genes responded to cold stresses in both Chinese cabbage and purple kale.24,35 In our study, the effects of growth and storage temperatures on the anthocyanin content and acylation in juvenile and mature plants of red cabbage ‘Futurima’ and red kale ‘Redbor’ were measured. We showed that, in juvenile plants, anthocyanins could be nearly all acylated (over 90%) and that ‘Futurima’ had higher diacylated ratios at 16 °C, while ‘Redbor’ showed higher diacylated anthocyanin ratios at 25 °C. More importantly, for commercial production of anthocyanins, which requires high biomass, higher anthocyanin levels can be obtained by optimizing daily temperatures, for example, 22 °C for ‘Futurima’ and 25 °C for ‘Redbor’, if grown in growth chambers. Although a 10 day cooling during growth increased the diacylated anthocyanin ratio for ‘Futurima’, it does not necessarily mean that commercial production would be improved using plant materials from cold storage. Postharvest cooling of mature plants did not affect this ratio in this investigation but did cause a decrease in the total anthocyanin content in ‘Futurima’ and an increase in the total anthocyanin content in ‘Redbor’. Further studies will be needed to investigate other environmental effects, such as light and soil, on those cultivars.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +1-315-787-2222. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Matt Wavrick, Traci Hoogland, Sarah Durkee, and Kristin Marino for technical support.

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REFERENCES

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Effects of Growth Temperature and Postharvest Cooling on

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