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Changes in the content and composition of anthocyanins in red cabbage

and its antioxidant capacity during fermentation, storage and stewing

Wieslaw Wiczkowski , Dorota Szawara-Nowak, Joanna Topolska

Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences in Olsztyn, Tuwima 10, 10-748 Olsztyn, Poland

a r t i c l e i n f o

Article history:

Received 29 July 2013Received in revised form 7 February 2014

Accepted 21 June 2014

Available online 28 June 2014

Keywords:

Red cabbage

Nonacylated and acylated anthocyanins

HPLC-DAD-MS/MS

Fermentation

Storage

Stewing

Antioxidant capacity

a b s t r a c t

The effect of fermentation, storage and stewing on the content and composition of anthocyanins as well

as antioxidant capacity of red cabbage was studied. The observation of anthocyanins profile by HPLC-

DAD-MS/MS was conducted. Red cabbage products contained twenty different nonacylated and acylated

anthocyanins with main structure of cyanidin-3-diglucoside-5-glucoside. Treatments applied affected

concentration and profile of red cabbage anthocyanins. Anthocyanins content was reduced by 24%,

25% and 34% in fermented and stewed (30 and 60-min) red cabbage, respectively. The intensity of

anthocyanins degradation during storage depended on the process length. Derivatives of cyanidin-3-

diglucoside-5-glucoside acylated with sinapic acid were characterised by the highest losses. Five assays

were used to analysis of antioxidant capacity. Fresh red cabbage had stronger antioxidant capacity in

comparison to fermented, stored and stewed red cabbage. The study has shown that red cabbage prod-

ucts are valuable vegetables for daily consumption in fresh, fermented, stored as well as stewed form.

2014 Elsevier Ltd. All rights reserved.

1. Introduction

Anthocyanins content and profile as well as antioxidant capac-

ity of both fruits and vegetables strongly depend on genetic and

environmental conditions, however food processing and storage

conditions also constitute major influential factors. Fruits and

vegetables are often subjected to various types of processing in

order to obtain more suitable and attractive food products, as well

as to achieve longer and stable storage capacity. It has to be noted

however that during treatment the stability of anthocyanins is

dependent on their structure, plants matrix and the process envi-

ronment. Temperature, length of the process, presence of oxygen,

light, plant enzymes and microorganism activities, as well as

accompanying substances and pH value affect the half-life of

anthocyanins (Clifford, 2000).

Anthocyanins are characterised by complex patterns of hydrox-

ylation, methoxylation, glycosylation, and acylation (Wu & Prior,

2005). These factors are linked to plant species to form a character-

istic pattern of anthocyanins. Glycosylation and acylation of

anthocyanins raise their stability through intra-molecular and/or

inter-molecular co-pigmentation, and self-association reactions

(Bakowska-Barczak, 2005). Therefore, acylated anthocyanins with

a high degree of glycosylation being a source of colour and bioac-

tivity may maintain the desired stability during food processing.

Anthocyanins are absorbed by humans in the aglycone, gluco-

sidic and acylated forms (Charron, Clevidence, Britz, & Novotny,

2007). It has been indicated that anthocyanins consumed do not

have any toxic, teratogenic and mutagenic properties even at high

doses of these compounds (Clifford, 2000). The intake of anthocy-

anins has been considered to exert a beneficial effect on human

health (Zafra-Stone et al., 2007), however mechanisms of this

action have not been entirely explained. These natural red colou-

rants have been demonstrated to have anticancer, cardioprotec-

tive, antineurodegenerative, vision improving and diabetes

preventing activities (De Pascual-Teresa & Sanchez-Ballesta, 2008).

Red cabbage is gaining popularity all over the world and is

eaten raw and after both technological and home treatment. Red

cabbage is an attractive for consumers not only because of its cru-

cial dietetic and taste values, but also its intense purple/red colour.

It has been indicated that anthocyanins are responsible for forma-

tion of this colour. As was presented in the previous study

(Podsedek, 2007), anthocyanins are one of the major groups of

phytochemicals in red cabbage. The concentration of anthocyanins

in red cabbage is relatively large and varies significantly in plants

grown in different years. From nine to thirty-six different anthocy-

anin derivatives have been detected in various red cabbages.

Among them, a large number occurs in acylated forms. Red cab-

bage varieties are characterised by a specific and individual profile

http://dx.doi.org/10.1016/j.foodchem.2014.06.087

0308-8146/2014 Elsevier Ltd. All rights reserved.

Corresponding author. Tel.: +48 89 5234604; fax: +48 89 5240124.

E-mail address:[email protected](W. Wiczkowski).

Food Chemistry 167 (2015) 115123

Contents lists available at ScienceDirect

Food Chemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m
http://dx.doi.org/10.1016/j.foodchem.2014.06.087mailto:[email protected]://dx.doi.org/10.1016/j.foodchem.2014.06.087http://www.sciencedirect.com/science/journal/03088146http://www.elsevier.com/locate/foodchemhttp://www.elsevier.com/locate/foodchemhttp://www.sciencedirect.com/science/journal/03088146http://dx.doi.org/10.1016/j.foodchem.2014.06.087mailto:[email protected]://dx.doi.org/10.1016/j.foodchem.2014.06.087http://-/?-http://-/?-http://-/?-http://-/?-http://crossmark.crossref.org/dialog/?doi=10.1016/j.foodchem.2014.06.087&domain=pdfhttp://-/?-

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of anthocyanins (Charron et al., 2007; Pliszka, Huszcza-Ciolkowska,

Mieleszko, & Czaplicki, 2009; Wu & Prior, 2005). In addition, in the

previous work, it has been found that red cabbage has its own

characteristic antioxidant capacity where the kind of acylation

affects the antioxidant activity of acylated anthocyanins

(Wiczkowski, Szawara-Nowak, & Topolska, 2013). Published

reports prove that red cabbage is considered to be a vegetable of

a considerably high antioxidant activity (Hassimotto, Genovese, &Lajolo, 2005; Wu et al., 2004).

Taking the above into account, measurement of anthocyanins

content and determination of their profile appearing in products

after treatment are essential requirements for exploring the fate

of anthocyanins during processing, as well as for development of

suitable procedures to reduce the degradation of these red natural

compounds. Examination of the manner in which red cabbage is

processed and consumed has to be accompanied by the consider-

ation of its role in preventing diseases and obtaining maximum

health effects. We have therefore investigated the effects of fer-

mentation, storage and stewing processes on the red cabbage

anthocyanin concentration and antioxidant activity. The composi-

tion of individual red cabbage anthocyanins in fresh, fermented

and stewed products by means of HPLC-DAD and HPLC-MS/MS

methods was also determined. Five different assays were used

for determination of antioxidant capacity in red cabbage products.

2. Materials and methods

2.1. Reagents

2,20-Azobis(2-amidopropane) hydrochloride (AAPH), 2,20-azin-

obis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt

(ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 6-hydroxy-

2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were pur-

chased from Sigma Chemical Co. (Sigma Chemical Co., St. Louis,

MO). Sodium fluorescein was obtained from Fluka (Buchs, Switzer-

land). ACW (hydrophilic condition) and ACL (lipophilic condition)kits (model no. 400.801) for the photochemiluminescence (PCL)

assay were received from Analytik Jena AG (Jena, Germany). Cyani-

din aglycone was obtained from Extrasynthese (Genay, France). All

other reagents of gradient-grade including acetonitrile, methanol,

trifluoroacetic and formic acid were purchased from Merck KGaA

(Dramstad, Germany). Water was purified with a Mili-Q system

(Millipore, Bedford, MA).

2.2. Plant material and processes

Red cabbage (Brassica oleraceaL. var. capitata L. f. rubra) plants

of the cultivar Langedijker Polona were grown in the experimental

fields of the Research Centre for Cultivar Testing (COBORU, Szcze-

cin-Dabie, Poland). The plants harvested were planted in 2009.Before cutting the bulbs (7 bulbs) in a shredder into 2 mm thick

strips their dried outer leaves were removed. After mixing red cab-

bage strips, three samples of 200 g were taken to determine the

initial composition and content of anthocyanins and antioxidant

properties. Subsequently, samples were immediately frozen

together with liquid nitrogen. After lyophilisation, the samples

were pulverised and stored at 80C until the analysis. The

remaining shredded red cabbage was divided into two parts: 10%

process of stewing and 90% process of fermentation.

2.2.1. Fermentation and storage processes

The shredded red cabbage was thoroughly mixed with grated

carrots (1%) and NaCl (3%), and after mixing, the whole was

transferred to three traditional stoneware pots to run three inde-pendent fermentations. For the first 3 days cabbage pots were kept

at a temperature of 24C and for further 11 days at a temperature

of about 18 C. For the proper conduct of fermentation process, the

cabbage was pricked in order to remove releasing fermentation

gases. During the fermentation process changes of pH (Radiometer

PHM85, Denmark) were measured. The results obtained clearly

showed that the process was run properly (Table 1). After 14 days,

the process of fermentation was ended and the sauerkraut juice

was collected. Next, the samples from each stoneware pot(200 g) mixed with the proportional volume of the sauerkraut juice

collected were taken to determine the composition and content of

anthocyanins as well as antioxidant properties of fermented red

cabbage. Next, the samples were immediately frozen together with

liquid nitrogen and then lyophilisated. The samples obtained were

pulverised and stored at 80C until the analysis. The remaining

red cabbage from each stoneware pot mixed with the proportional

volume of the sauerkraut juice collected was transferred into five

Weck jars (volume of 900 mL). The jars were filled, by strongly

pressing down, to 1 cm from their upper edges and stored at4 C in a refrigerator. The composition and content of anthocya-

nins and antioxidant properties of fermented red cabbage were

analysed after 7, 30, 60, 90, and 180 days of storage. At the due

time the samples from jars were immediately frozen together with

liquid nitrogen and then lyophilisated. The samples obtained were

pulverised and stored at 80C until the analysis.

2.2.2. Stewing process

The shredded red cabbage samples (200 g) were placed into a

stainless steel pot with a small volume of boiling distilled water

(50 mL) and covered with lid. After bringing the water to boil,

red cabbage was stewed for 30 min or 60 min, and mixed from

time to time. Following this procedure, the stewed red cabbage

was immediately frozen together with liquid nitrogen and then

lyophilisated. The samples obtained were pulverised and stored

at 80C until the analysis.

2.3. Extraction and chromatographic analysis

Extraction and analysis of anthocyanins in red cabbage products

were carried out as described previously by Wiczkowski et al.

(2013). About 0.05 g of dried and pulverised red cabbage tissues

were extracted by 30 s sonication (VC 750, Sonics & Materials,

USA) with 1 mL of mixture consisting of methanol/water/trifluoro-

acetic acid (0.58/0.38/0.04, v/v/v). Subsequently, the mixture was

Table 1

The changes of environmental pH value during process of fermentation and storage.

Time of fermentation and storage pH value SD

Shredded red cabbage 6.21 0.02

1 Day of fermentation 5.81 0.022 Day of fermentation 4.80 0.01

3 Day of fermentation 4.20 0.03












7 Days of storage 3.98 0.03





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vortexed for 30 s, again sonicated and vortexed, and centrifuged

(Centrifuge 5415R, Eppendorf, Niemcy) for 10 min (13,200g at

4C). Supernatant was collected in 5 mL flask. This step was

repeated 5 times. Finally, before the analysis, the extract was cen-

trifuged (20 min, 13,000g). Extracts of red cabbage products were

analysed by HPLC-DAD method. Aliquots (5 lL) of sample solutions

were injected into the HPLC system (Shimadzu, Kyoto, Japan)

equipped with a 150

2.1 mm i.d. XBridge C18 3.5 lm column(Waters, USA). The HPLC system consisted of two pumps (LC-10

ADVP), DAD detector (SPD-M10 AVP) set at 520 nm, autosampler

(SIL-10 ADVP), column oven (CTO-10 ASVP) and system controller

(SCL-10 AVP). All chromatographic determinations were performed

at 45C with the flow rate of 0.2 ml/min. The elution was con-

ducted using a solvent gradient system consisting of solvent A

(6% formic acid aqueous solution) and solvent B (6% formic acid

acetonitrile solution). Gradient was as follows: 317% B (0

77 min), 1780% B (7780 min), 803% B (8084 min), and 3% B

(84105 min). Anthocyanins were identified basing on the com-

parison of their retention time, UVvisible spectrum and MS/MS

fragmentation spectrum (m/z values) with the previously

published data (Charron et al., 2007; Wiczkowski et al., 2013).

Anthocyanins quantity was calculated from HPLC-DAD peak area

at 520 nm against cyanidin as the external standard. The

calibration curve (the range of 0.340 lM) was linear with a

correlation coefficient of 0.998.

Identification of anthocyanins was carried out using mass spec-

trometer QTRAP 5500 (AB SCIEX, USA) equipped with a triple

quadrupole, ion trap, and ion source of electrospray ionisation.

Qualitative analysis was performed, among other, basing on

scanning in positive ion mode in the quadrupoles and in the ion

trap. Scanning of fragmentation ions derived from the selected

parent ion for observation of all the ions formed by the

disintegration of the parent ion as well as scanning precursor ion

and neutral particles was also conducted. Optimal identification of

anthocyanins was achieved under the following conditions: curtain

gas 20 L/min, collision gaz 9 L/min, ionspray voltage 5300 V,

temperature 550 C, 1 ion source gas 55 L/min, 2 ion sourcegas 70 L/min, declustering potential 50120 V, entrance

potential 10 V, collision energy 3070 eV, collision cell exit

potential 1045 V.

2.4. Antioxidant capacity

2.4.1. ABTS assay

The method presented byRe et al. (1999)with a minor modifi-

cation was used to determine the antioxidant activity of red cab-

bage products extract. The analysis was conducted using

spectrophotometer UV-160 1PC (Shimadzu, Japan). Briefly, the

ABTS+ solution was diluted with 80% methanol to an absorbance

of 0.70 0.02 at 734 nm. Next, 1.48 mL of the ABTS + solution and

0.02 mL of the isolated red cabbage standard solution or red cab-bage extract or Trolox solution were mixed. Then, the absorbance

was measured immediately after 6 min at 734 nm at 30 C. Appro-

priate solvent blanks were analysed in each assay. The antioxidant

assay was carried out in triplicate for each sample. The antioxidant

activity of 80% methanol solution of the red cabbage products

extract was calculated, using Trolox standard curve, on the basis

of percent inhibition of the absorbance of the ABTS + solution at

734 nm. The 80% methanol solution of Trolox within the concen-

tration range of 0.12.5 mM was used for building the calibration

curve.

2.4.2. PCL assay

The PCL method was used to measure the antioxidant activity of

red cabbage products extract with a Photochem apparatus(Analytik Jena, Leipzig, Germany) against superoxide anion radicals

generated from luminol (a photosensitiser) when exposed to UV

light. The antioxidant activity of red cabbage products extract

was analysed using both ACW (hydrophilic condition) and ACL

(lipophilic condition) kits as well as the protocol of measurement

provided by the manufacturer. The assay was carried out as previ-

ously described byZielinska, Wiczkowski, and Piskula (2008). The

80% methanol solution of extracts were centrifuged by 10 min at

16,000g, and at 4

C prior to the analysis. The antioxidant assaywas carried out in triplicate for each sample. The antioxidant

capacity was calculated by means of the comparison with a Trolox

standard curve (0.253.00 nM).

2.4.3. ORACFL assay

The oxygen radical absorbance capacity (ORACFL) method was

used as described by Del Castillo, Gordon, and Ames (2005). The

reaction mixtures for ORACFLvalue determination were prepared

at 37C by mixing 2.25 mL of 42 nM fluorescein solution (sub-

strate), 375 lL of 153 mM AAPH (peroxyl radical generator), and

diluted extracts or blank or Trolox standard. The intensity of

fluorescence was measured at an excitation wavelength of

Ex = 493 nm and an emission wavelength of Em = 515 nm. The

ORACFLvalue was calculated as Trolox equivalent. A standard curvewas prepared by Trolox concentration range of 10100 lM and

used for calculation of the peroxyl radical scavenging properties

of the samples. The results were expressed as lmol Trolox/g of

dry matter (dm). The test was carried out in triplicate for each

variety.

2.4.4. DPPH assay

The DPPH scavenging activity was determined using 80%

methanol extract of red cabbages products as described previously

by Zielinska et al. (2008). The Trolox standard (concentration

0.12.0 mM) in 80% methanol were assayed under the same

conditions, and then the DPPH scavenging activity of the samples

was expressed in terms of Trolox equivalents on the basis of

reduction in the absorbance of the DPPH

solution by standardsat 515 nm. The measurements were carried out using a

spectrophotometer UV-160 (Shimadzu, Japan).

2.5. Statistical analysis

The data are presented as mean SD for triplicate analysis. The

results were subjected to one-way analysis of variation ANOVA

with Fishers Least Significant Difference test. P< 0.05 was consid-

ered significant. The Pearson Correlation test for correlation analy-

sis was used. The statistical analysis was performed using Statistica

(Stat Soft, USA).

3. Results and discussion

3.1. The profile of red cabbage anthocyanin

Anthocyanins in red cabbage products were analysed using

HPLC-DAD-MS/MS method. The identification of anthocyanins was

carried out by means of the comparison of their retention time,

UVVis and MS/MS spectra, and the previous data ( Charron et al.,

2007; Wiczkowski et al., 2013). As presented inFig. 1andTable 2,

anthocyanin profiles of red cabbage products obtained in this study

is characterised by twenty derivatives of cyanidin with the main

structureof cyanidin-3-diglucoside-5-glucosides.Two of themwere

nonacylated while eighteen acylated. Among acylated compounds,

eleven were monoacylated and seven were diacylated. Sinapic,

ferulic, caffeic and p-coumaric acids were found as acyl residues

in the acylated structure. Among twenty anthocyanins identifiedand quantified seven of them predominated: one nonacylated

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(cyanidin-3-diglucoside-5-glucoside), three monoacylated (cyanidin-3-

(p-coumaroyl)-diglucoside-5-glucoside, cyanidin-3-(feruloyl)-digluco-

side-5-glucoside, cyanidin-3-(sinapoyl)-diglucoside-5-glucoside),and three diacylated (cyanidin-3-(feruloyl)(feruloyl)-diglucoside-

5-glucoside and cyanidin-3-(feruloyl)(sinapoyl)-diglucoside-5-gluco-

side, and cyanidin-3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside).

These seven main compounds covered almost 68% of the total

anthocyanins content in fresh red cabbage. The profiles of anthocy-

anins containing twenty cyanidin derivatives were observed for all

samples processed, however, it has to be noted that the content of

individual constituents differed depending on treatments applied

(Table 3). Similar profile of anthocyanins in fresh red cabbage was

observed in authors previous study(Wiczkowski et al., 2013). Other

previous studies of red cabbage anthocyanins exhibited differences

when compared with authors study in the following respects: (1)

number of anthocyanin in the profile (from nine to thirty-six

anthocyanins derivatives); (2) type of aglycone appearing in antho-cyanins structure(pelargonidin, peonidin,and malvidinexceptfrom

cyanidin); (3) kind of sugars bonded to anthocyanins aglycone

(xylose besides glucose); (4) type of acyl group (p-hydroxybenzoic

and malonic acids except from cinnamic acid derivatives); and (5)acylation pattern (triacylation) (Charron et al., 2007; Lin, Li, &

Hwang, 2008; McDougall, Fyffe, Dobson, & Stewart, 2007; Pliszka

et al., 2009; Wu & Prior, 2005; Wu et al., 2004). These disparity

may result from varietal differences and the impact of biotic

(diseases and plant pests) and abiotic factors (temperature,

precipitation, insolation) (Podsedek, 2007).

3.2. Changes in the content of anthocyanins in red cabbage during

processing

Red cabbage constitute a rich source of nonacylated, monoacy-

lated, and diacylated forms of cyanidin and, therefore, provides an

unique ground for investigating the relationship between the con-

tent and profile of anthocyanins versus the type of food processing.In this study the impact of processing was evaluated by comparing

Fig. 1. HPLC chromatogram of anthocyanins profile of red cabbage detected at 520 nm. Names of anthocyanins identified correspond to a number referred to in Table 1.

Table 2

The UVVis and MS data of anthocyanins from red cabbage products.

Peak Compounds kvis (nm) kacyl(nm) [M]+ (m/z) MS/MS (m/z)

1 Cyanidin-3-diglucoside-5-glucoside 513 x 773 611/449/287

2 Cyanidin-3-glucoside-5-glucoside 512 x 611 449/287

3 Cyanidin-3-(sinapoyl)-diglucoside-5-glucoside 527 330 979 817/449/287

4 Cyanidin-3-(sinapoyl)-triglucoside-5-glucoside 525 321 1141 979/449/287

5 Cyanidin-3-(caffeoyl)(p-coumaroyl)-diglucosides-5-glucoside 521 314 1081 919/449/287

6 Cyanidin-3-(feruloyl)-triglucosides-5-glucoside 522 320 1111 949/449/287

7 Cyanidin-3-(sinapoyl)-triglucoside-5-glucoside 525 321 1141 979/449/287

8 Cyanidin-3-(feruloyl)(feruloyl)-triglucoside-5-glucoside 536 321 1287 1125/449/287

9 Cyanidin-3-(feruloyl)-diglucoside-5-glucoside 522 328 949 787/449/28710 Cyanidin-3-(feruloyl)(sinapoyl)-triglucoside-5-glucoside 536 324 1317 1155/449/287

11 Cyanidin-3-(caffeoyl)-diglucoside-5-glucoside 522 315 935 773/449/287

12 Cyanidin-3-(p-coumaroyl)-diglucoside-5-glucoside 521 312 919 757/449/287

13 Cyanidin- 3-(caffeoyl)(p-coumaroyl)-diglucoside-5-glucoside 521 314 1081 919/449/287

14 Cyanidin-3-(feruloyl)-diglucoside-5-glucoside 523 329 949 787/449/287

15 Cyanidin-3-(sinapoyl)-diglucoside-5-glucoside 526 328 979 817/449/287

16 Cyanidin-3-(feruloyl)-glucoside-5-glucoside 522 328 787 449/287

17 Cyanidin-3-(sinapoyl)-glucoside-5-glucoside 527 330 817 449/287

18 Cyanidin-3-(feruloyl)(feruloyl)-diglucoside-5-glucoside 535 328 1125 963/449/287

19 Cyanidin-3-(feruloyl)(sinapoyl)-diglucoside-5-glucoside 535 330 1155 993/449/287

20 Cyanidin-3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside 535 332 1185 1023/449/287


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fresh red cabbage with fermented, stored and stewed red cabbage.

The analysis conducted shown, for the first time, that processes of

fermentation, storage and stewing affect the concentration and

profile of anthocyanins in red cabbage. Generally, results indicated

that processes applied contributed to decrease in the total contentof anthocyanins in red cabbage products (Table 3). This observa-

tion is in agreement with previous results which showed that both

technological and home treatment of plant materials led to a

decrease in the content of anthocyanins (Bakowska-Barczak,

2005). In our study, the concentration of anthocyanins in fresh

red cabbage var. Langedijker Polona determined by HPLC-DAD

was 6.30 0.09 mg of cyanidin/g dm. The loss of total anthocya-

nins during fermentation process was at the level of 24%. A similar

reduction in anthocyanins content (25%) was observed during

30 min of the stewing process. When the process of stewing was

set at 60 min the degradation of anthocyanins increased respec-

tively (34%). In case of storage of fermented red cabbage at 4 C,

the intensity of anthocyanins degradation depended on the storage

length. In red cabbage stored for 7 days the intensity of this processwas found at a significantly low level. On the other hand, after

180 days of storage under the same conditions, a very intense deg-

radation of anthocyanins was observed. The order of anthocyanins

degradation intensity was as follows: 7 days (2%)

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The negative influence of room temperature (2022C) on the con-

centration of phytochemicals during food processing has been pre-

viously reported (Aaby, Wrolstad, Ekeberg & Kkrede, 2007). Also,

the impact of fermentation microbiota may determine anthocya-

nins content and composition of red cabbage. The previous report

ofBisakowski, Atwal, Gardner, and Champagne (2007) on the onion

flavonoids indicated that lactic acid fermentation influences the

composition of these compounds. Moreover, during the preparationof red cabbage for processes, thebulbs were cutinto stripsand, con-

sequently, cell structure was disrupted and the anthocyanins

became more prone to enzymatic and nonenzymatic oxidation

(Pourcel, Routaboul, Cheynier, Lepiniec, & Debeaujon, 2007). In this

respect, it can be stated that plant enzymes may affect the content

and composition of anthocyanins until they are deactivated by

environmental factors of the fermentation process.

The impact of all above factors on anthocyanins stability during

fermentation could depend on the pH of the processes environ-

ment (Castaneda-Ovando, Pacheco-Hernandez, Paez-Hernandez,

Rodriguez & Galan-Vidal, 2009; Clifford, 2000). A pH value within

the first several days of fermentation could constitute another neg-

ative factor for the stability of anthocyanins (Table 1). At the begin-

ning, the pH of fermentation juice was under 6.0, to gradually

decline to a level of 3.8 in the 8 day of the process, and remained

at this level over the next 7 days. This high pH value during the

first days of fermentation and its rate of change could result in

the degradation of anthocyanins. Previous studies suggest that

pH below 4.0 is favourable for anthocyanins stability, while pH

around neutral is negative (Markakis, 1982). On the other hand,

the reports ofGiusti and Wrolstad (2003) and McDougall et al.

(2007) showed that acylated anthocyanins were more stable at

pH near neutral than nonacylated anthocyanins, since acylation

increases anthocyanins stability. Taking this information into

account, it can be stated that a very high percentage of acylated

anthocyanins (approximately 80%) in the studied red cabbage

had limited the rate of degradation of anthocyanins (only 24%

decrease) during 14 days of fermentation (Table 3).

Our findings revealed that storage time significantly affectedthe level of anthocyanins (Table 3). Also the previous report of

Giusti and Wrolstad (2003) indicated that the length of storage

lead to anthocyanins degradation. In addition, these authors found

that the temperature of storage exerted an effect on the anthocya-

nins degradation kinetics at 25 C it was higher than at 2 C. In

our study, under the storage at 4 C the rate of anthocyanins

decline during 180 day was 0.32% of total anthocyanins per day.

The highest decrease in the anthocyanins content was noted in

the period between 90 and 180 days of storage, while the highest

degradation rate of anthocyanins (0.59% of total anthocyanins/

day) in the period between 7 and 30 days. A similar rate of total

anthocyanins decrease (0.34% of total anthocyanins/day) in a

strawberry juice stored for 77 days at 8C by Hartmann et al.

(2008) was indicated. Also storage of 112 days at 6 C showed aconsiderable loss of total anthocyanins (0.38% of total anthocya-

nins/day) in strawberry puree (Aaby, Wrolstad, Ekeberg &

Kkrede, 2007). Regarding the pH value during storage, it increased

slightly during this process but remained favourable for anthocya-

nins stability (Markakis, 1982), close to 4.0 (Table 1). Therefore,

apart from the storage time and mentioned temperature, other fac-

tors such as uncontrolled growth of microorganisms may influence

the level of anthocyanins during storage.

A 30 and 60-min process of stewing in low amounts of water

lead to a significant decrease in the content of red cabbage antho-

cyanins, whereas in case of shorter time of treatment the decline in

anthocyanins concentration was lower (Table 3). In addition, in our

experiment stewing at high temperature (around 100C) for

60 min was one of the processes applied causing the highest lossesin anthocyanins. This may result from high temperature and dura-

tion of the process. Also in case of different food products heating

lead to a significant decrease in the total content of anthocyanins.

The heat treatment of strawberry juice at high temperature (85C)

for a very short time (5 s) indicated a loss of 12% in anthocyanins

content, while prolongation of the process time up to 15 min

resulted in higher losses of anthocyanins (21% diminution)

(Hartmann et al., 2008). In case of other heat treatments, such as

boiling, blanching and steaming, the reduction of monomericanthocyanins measured by pH-differential method in red cabbage

was noted in the range of 1222% (Volden et al., 2008). However, in

the report cited a part of recovered anthocyanins (638%) was

found in the processing water. AlsoScalzo et al. (2008)indicated

that blanching and cooking had reduced the total cauliflower

anthocyanins content. On the other hand, the same authors exhib-

ited that in case of microwave treatment the content of anthocya-

nins had remained almost unchanged. At the stewing process of

red cabbage the plant enzyme system could also operate but to a

very small extent, since upon cutting of cabbage, it was subjected

to high temperatures inactivating enzymes (Jang, Ma, Shin, & Song,

2005).

3.3. Changes in the composition of anthocyanins in red cabbage during

processing

The effect of fermentation, storage and stewing on the

individual cyanidin derivatives of red cabbage was studied in

detail. From the results obtained, shown inTable 3, it was observed

that in fresh red cabbage, cyanidin-3-diglucoside-5-glucoside

was the major compound followed by cyanidin-3-(sinapoyl)

(sinapoyl)-diglucoside-5-glucoside and cyanidin-3-(p-coumaroyl)-

diglucoside-5-glucoside. Similarly, after fermentation and stewing

as well as during storage, the mentioned nonacylated derivative

was the main form, but followed by different acylated derivatives

of cyanidin. Regarding the fermented cabbage, cyanidin-3-

(p-coumaroyl)-diglucoside-5-glucoside and cyanidin-3-(sinapoyl)

(sinapoyl)-diglucoside-5-glucoside were the second and the third

major constituents, respectively. In terms of stored and stewedproducts, besides cyanidin-3-diglucoside-5-glucoside and cyani-

din-3-(p-coumaroyl)-diglucoside-5-glucoside, cyanidin-3-(feruloyl)-

diglucoside-5-glucoside was noted as one of the three main

anthocyanin compounds. Both processes, fermentation and stewing,

lead to a decrease in the content of all analysed anthocyanins. How-

ever, the intensity of decrease depended on the chemical structure

of these compounds. Generally, among seven main individual red

compounds found in fermented and stewed red cabbage products,

the lowest rate of decrease was noted in the case of nonacylated

anthocyanins. Among main monoacylated anthocyanins, derivatives

acylated with sinapic acid were characterised by the highest losses

(Table 3). The order of stability of monoacylated anthocyanins for

fermented and stewed red cabbage was similar: cyanidin-3-

(p-coumaroyl)-diglucoside-5-glucoside = cyanidin-3-(feruloyl)-dig-lucoside-5-glucoside > cyanidin-3-(sinapoyl)-diglucoside-5-glucoside

and cyanidin-3-(feruloyl)-diglucoside-5-glucoside > cyan idin-3-

(p-coumaroyl)-diglucoside-5-glucoside > cyanidin-3-(sinapoyl)-dig-

lucoside-5-glucoside, respectively. These results are in accordance

with study ofSadilova et al. (2007)which showed that anthocya-

nins acylated with sinapic acid have the lowest half-life when

compared to the compounds acylated with ferulic and coumaric

acids. Also, during simulation of neutral pancreatic digestion red

cabbage anthocyanins acylated with sinapic acid were less stable

than those acylated with p-coumaric and ferulic acids

(McDougall et al., 2007). In our study, within the diacylated forms

of cyanidin-3-diglucoside-5-glucoside, anthocyanins conjugated

with two sinapic acids were less stable than forms bond with

two ferulic acids. These findings stay in agreement with theobservations made for the fate of diacylated anthocyanins during


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treatment under small intestine conditions (McDougall et al.,

2007). In contrast to the results obtained for fermentation

and stewing processes, during 7 and 30 days of storage, an

increase in the content of four derivatives (cyanidin-3-digluco-

side-5-glucoside, cyanidin-3-(p-coumaroyl)-diglucoside-5-glucoside,

cyanidin-3-(feruloyl)-diglucoside-5-glucoside, cyanidin-3-(sinapoyl)-

diglucoside-5-glucoside) was found. In the other point of

observation (60, 90, and 180 days of storage) the concentrationof anthocyanins analysed was lower when compared to the

non-stored fermented red cabbage. Moreover, in case of storage,

an entirely different profile of stability of acylated anthocyanins

was found. The derivatives acylated with sinapic acid were

characterised by lower losses when compared to forms conjugated

with ferulic acid.

Since the anthocyanins identified had the same main core

cyanidin glucosides all applied processes could lead to a

conversion of one compound to another. Diacylated derivatives

of cyanidin-3-diglucoside-5-glucoside could degrade to monoacy-

lated forms, and next to nonacylated constituents. Also, a direct

transformation from diacylated to nonacylated derivatives was

possible. In addition, the hydrolysis of glucosidic bond in chemical

structure of anthocyanins present in red cabbage could result in

the formation of anthocyanins with a lower degree of glucosyla-

tion. Previously, the deacylation of vegetable flavonoid was found

byDuPont, Mondin, Williamson, and Price (2000). Furthermore, a

cleaving of acylated anthocyanins into the acyl-glycosides and

the aglycone was observed (Sadilova et al., 2007). These phenom-

ena may constitute the basis for explaining the increase of nonacy-

lated anthocyanins content and their contribution to the total

anthocyanins concentration during treatments used when com-

pared to the concentration of acylated forms (Table 3).

3.4. The contribution of nonacylated, monoacylated and diacylated

anthocyanins in the total anthocyanin content of red cabbage products

The percentages of nonacylated, monoacylated and diacylated

cyanidin derivatives in the total anthocyanin content of red cab-

bage products were studied. After the fermentation process, the

contribution of nonacylated compounds to the total content of

anthocyanins increased when compared to the fresh red cabbage

anthocyanins profile. The percentages of both nonacylated antho-

cyanins identified were higher than in fresh red cabbage ( Table 3).

In contrast to nonacylated compounds, the presence of acylated

forms in the total anthocyanins concentration decreased during

fermentation. During 180 days of storage the participation of non-

acylated, monoacylated, and diacylated forms in the total anthocy-

anins concentration was variable. In case of nonacylated

compounds, during the first 7 days of storage an increase in the

presence of these derivatives in the total content of anthocyanins

was observed (Table 3). After 30, 60 and 90 days of storage the

contribution of nonacylated compounds to the total content of

anthocyanins was very similar to this noted after 7 days of storage.

However, after 180 day of storage the value of these parameters

decreased to the level of 23.4%. In terms of monoacylated deriva-

tives of cyanidin, after 7 days of storage, higher contribution of

these compounds to the total content of anthocyanins was

observed when compared to non-stored fermented product. In

the other point of observation (30, 60, 90, and 180 days of storage)

the presence of cyanidin monoacylated derivatives in the total con-tent of anthocyanins decreased gradually. An entirely opposite

phenomenon was found in case of diacylated anthocyanins. During

the first 7 days of storage the contribution of these compounds to

the total content of red pigment decreased when compared to non-

stored fermented cabbage. Subsequently, the value of this param-

eter in other points of measurements increased gradually. After

stewing, the contribution of nonacylated derivatives of anthocya-

nins to the total anthocyanins content was higher when compared

to the results obtained for the fresh product. In addition, it was

observed that the presence of monoacylated compounds in the

total concentration of analysed substances increased. An opposite

phenomenon was noted in the case of diacylated derivatives of

cyanidin. During 30 min of stewing the contribution of diacylated

compounds decreased from 34.3% to 30.6%. Subsequent 30 min of

high temperature treatment caused further degradation of diacy-

lated anthocyanins up to level of 29.2% of the total anthocyanins

content. As has been mentioned in previous subpart, anthocyanins

found in red cabbage had identical main core therefore the changes

in the contribution of nonacylated, monoacylated and diacylated

anthocyanins in the total anthocyanin content of red cabbage

during processing, may result from a conversion of one anthocyanin

to another.

3.5. Changes in red cabbage antioxidant capacity during processing

In this study, five methods (ACW PCL, ACL PCL, ORAC, ABTS and

DPPH) to determine the antioxidant capacity of red cabbage prod-

ucts were used. For a comparison, previous studies ( Prior, Wu, &

Schaich, 2005) suggested the use of the three methods (ORAC,

ABTS, and FolinCiocalteu assay) to be applied for standardisation

of antioxidant capacity measurements of food samples. In conse-

quence, the application of these five methods and the use of nine

different products of red cabbage (fresh, fermented, stored by 7,

30, 60, 90 and 180 days, stewed 30 min and 60 min) for the tests

allowed to obtain a full range of antioxidant capacities of this veg-

etable during food processing. Ultimately, a specific antioxidant

capacity of different red cabbage products was determined

(Table 4). The values of antioxidant capacity of red cabbage

products provided by ABTS assay ranged from 133.1 to 166.5 lmol

Trolox/g dm. In case of ORAC assay, these values ranged from 318.3

to 411.3 lmol Trolox/g dm. The antioxidant capacity estimated by

ACW PCL assay oscillated between 71.6 and 103.5 lmol Trolox/g dm,

while by ACL PCL assay from 413.7 to 632.8 lmol Trolox/g dm.

Table 4

The antioxidant capacity of red cabbage products (fresh, fermented, stored, stewed) determined by five different assays.

Antioxidant activity assays lmol Trolox/g dma

Red cabbage products

Fresh Fermented Stored after fermentation Stewed red cabbage

7 days 30 days 60 days 90 days 180 days 30 min 60 min

TEAC 166.5 2.9A 137.9 2.5BC 136.6 2.4BC 133.1 2.4C 139.9 1.6B 136.5 1.9BC 135.5 1.7C 135.7 1.9C 133.8 2.8C

ORACFL 411.3 8.6A 380.6 5.6C 397.0 7.1B 364.2 5.0D 347.8 7.3E 357.7 7.1DE 318.3 2.2F 397.9 1.5B 368.4 1.7D

ACW PCL 103.5 1.2A 94.0 1.2B 83.5 1.7C 80.5 1.7CD 79.1 1.2D 80.2 1.7CD 71.6 0.9E 100.7 1.3A 92.0 1.4B

ACL PCL 632.8 5.4A 499.6 3.4D 496.7 1.2D 478.2 3.4E 474.5 2.0E 452.3 1.0F 413.7 1.1G 537.4 7.7B 512.2 5.8C

DPPH 47.0 1.0A 45.7 1.3A 43.6 1.2B 41.9 1.0C 38.3 2.8CD 38.7 0.7D 38.1 1.2D 45.7 1.2A 42.6 0.6BC

a

Data are expressed as means SD (n= 3). All values were expressed as micromoles of Trolox per gram dry matter of red cabbage. Means in line related to a respective testfollowed by the different letters are significantly different ( P< 0.05).

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In terms of DPPH method the values of antioxidant capacity ranged

from 38.1 to 47.0 lmol Trolox/g dm. Generally, the highest antiox-

idant capacity of red cabbage varieties was noted for in the case of

evaluation conducted by ACL PCL method, while the lowest by

DPPH. The order of antioxidant capacity of red cabbage was as fol-

lows: ACL PCL > ORACFL> ABTS > ACW PCL < DPPH. The antioxidant

capacity of red cabbage products analysed by ACL PCL was highly

correlated with that provided by ACW PCL (r= 0.865), ORACFL(r= 0.859) and DPPH (r= 0.832). Moreover, high correlation

between ACW PCL: ORACFL (r= 0.846), ACW PCL: DPPH

(r= 0.904) and ORACFL: DPPH (r= 0.900) was found.

All samples of red cabbage products scavenged radicals and

their antiradical potential differed significantly across the products

studied (P< 0.05). Fresh red cabbage was characterised by a stron-

ger ability to radical scavenge than fermented, stored and stewed

red cabbage. The highest reduction of this parameter occurred for

red cabbage stored for 180 days (average 28.1%). The lowest

decrease in the antioxidant capacity was determined for red cab-

bage stewed for 30 min (average 10.4%). The order of scavenging

capacity was as follows: fresh > stewed 30 min > fermented =

stored 7 days > stewed 60 min > stored 30 days > stored 60 day-

s > stored 90 days > stored 180 days (Table 4). The results obtained

indicate, for the first time, that differences in the antioxidant

capacity of red cabbage products occurred depending on the type

treatment applied. Other reports also demonstrated that the anti-

oxidant activity of food products can depend on the kind of food

processing used (Aaby et al., 2007). After heating, a decline of TEAC

in black carrot, elderberry and strawberry samples was found by

Sadilova et al. (2007), what the authors assigned among others to

anthocyanins degradation following thermal exposure. Moreover,

authors suggested that the loss of antioxidant capacity supplied

by anthocyanins could not be compensated by the activity of newly

formed phenolics upon heating. Other study indicated that cooking

increased free radical scavenging activity for red cabbage (Wu

et al., 2004).

In our analysis, the antioxidant capacity of red cabbage products

is positively and significantly correlated (P< 0.05) with the totalanthocyanins concentration, determined by four assays used:

ORACFL(r= 0.949), ACL PCL (r= 0.938), DPPH (r= 0.926) and ACW

PCL(r = 0.861). The results clearly showed that anthocyanins occur-

ring in fermented, stored and stewed red cabbage were responsible

for their antioxidant capacity. In addition, the contribution of seven

main derivatives of cyanidin (cyanidin-3-diglucoside-5-glucoside,

cyanidin-3-(p-coumaroyl)-diglucoside-5-glucoside, cyanidin-3-(feru-

loyl)-diglucoside-5-glucoside, cyanidin-3-(sinapoyl)-diglucoside-

5-glucoside, cyanidin-3-(feruloyl)(feruloyl)-diglucoside-5-glucoside,

cyanidin-3-(feruloyl)(sinapoyl)-diglucoside-5-glucoside, and cyani-

din-3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside) to the antioxi-

dant capacity of red cabbage products was determined. Upon

Pearson correlation analysis between the concentration of these

major red cabbage anthocyanins and antioxidant capacity of redcabbage products extracts it was found that the content of cyani-

din-3-diglucoside-5-glucoside indicated positive and significant

correlation (P< 0.05) with antioxidant capacity provided by the

two methods used (for ORACFL r= 0.934, for ACL PCL r= 0.918).

The level of both cyanidin-3-(p-coumaroyl)-diglucoside-5-gluco-

side and cyanidin-3-(feruloyl)-diglucoside-5-glucoside was corre-

lated (P< 0.05) with the antioxidant activity measured with only

one assay (for ORACFL r= 0.913 and r = 0.884, respectively). The

concentration of cyanidin-3-(feruloyl)(feruloyl)-diglucoside-5-glu-

coside was associated (P< 0.05) with the value of antioxidant

capacity determined by the three following tests: ACW PCL

(r= 0.924), ACL PCL (r= 0.916) and DPPH (r = 0.904). In case of

cyanidin-3-(feruloyl)(sinapoyl)-diglucoside-5-glucoside, its indi-

vidual content was positively and significantly correlated(P< 0.05) with the antioxidant capacity provided by until four

methods used (ACL PCL r= 0.926, DPPH r= 0.882, ACW PCL

r= 0.29, ORACFL r= 0.828). A similar observation was noted for

cyanidin-3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside (ACL PCL

r= 0.944, ABTS r= 0.861, ORACFL r= 0.836, DPPH r= 0.825). Our

data supported previous observations which demonstrated that

anthocyanins contribute to the antioxidant capacity (Sadilova

et al., 2007). Moreover, it has been recently exhibited that fermen-

tationand heat treatmentof white cabbage increasedthe initial val-ues of the antioxidant activity (Kusznierewicz, Smiechowska,

Bartoszek, & Namiesnik, 2008). Conversely, in our study the antiox-

idant capacity of red cabbage products after fermentation, storage

and stewing was lower when compared to the fresh product. This

probably stems from the fact that since anthocyanins are strong

in vitro antioxidants and the antioxidant capacity of red cabbage

products studied in our experiments is positively and significantly

correlated with anthocyanins concentration, the degradation of

anthocyanins during these processes resulted in a decrease of

antioxidant capacity of the products analysed. In addition, taking

into account the findings indicating that anthocyanins acylated

with sinapic acid have the highest antioxidant capacity among

anthocyanins conjugated with others hydroxylcinnamic acids

(Wiczkowski et al., 2013) together with the assumptions derived

from this study that cyanidin-3-diglucoside-5-glucoside acylated

with sinapic acid show the highest rate of degradation, the

reduction of antioxidant capacity of red cabbage products is

observed justified.

4. Conclusion

The results indicated that all red cabbage processing applied

reduced the content of anthocyanins in the products obtained.

However, it is necessary to emphasise that the extent of respective

losses depended on the kind of the processing introduced. Among

individual red compounds found in red cabbage products, acylated

with sinapic acid derivatives of cyanidin-3-diglucoside-5-gluco-

side were characterised by the highest losses. Contrary, the lowestrate of decrease was noted for nonacylated anthocyanins, however

these results might stem from the decomposition of acylated

anthocyanins. In case of antioxidant activity, fresh red cabbage

was characterised by a stronger ability to radical scavenge than

fermented, stored and stewed red cabbage where the diminution

of anthocyanins content entailed the reduction of antioxidant

capacity.

When it comes to the pro-health food, maximal retention of

bioactive compounds during processing is strongly desired since

beneficial biological properties of food are to some extend attrib-

uted to their active constituents. On the basis of all the facts pre-

sented, red cabbage products in the fresh, fermented or short

time stewed form constitute valuable vegetables for daily con-

sumption as they remain rich in anthocyanins. What should also

be noted, fermented red cabbage stored up to 30 days is a signifi-

cant source of anthocyanins, acting as strong antioxidants. Quanti-

fication of the losses and estimation of anthocyanin profile changes

allows for the consideration of optimisation possibilities as well as

the quest of processing techniques, manner of storage, and the raw

material in order to reduce losses and changes, as well as to

improve the pro-health potential of red cabbage products. Cur-

rently, further studies are needed to determine how anthocyanins

from different forms of red cabbage products behave after

consumption.

Acknowledgement

The research was supported by the National Science Centre(Poland, project 1902/B/P01/2008/35).


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References

Aaby, K., Wrolstad, R. E., Ekeberg, D., & Kkrede, G. (2007). Polyphenol compositionand antioxidant activity in strawberry puree; impact of achene level andstorage. Journal of Agricultural and Food Chemistry, 55, 51565166.

Bakowska-Barczak, A. (2005). Acylated anthocyanins as stable, natural foodcolorants a review. Polish Journal of Food and Nutrition Sciences, 14(55),107116.

Bisakowski, B., Atwal, A. S., Gardner, N., & Champagne, C. P. (2007). Effect of lactic

acid fermentation of onions (Allium cepa) on the composition of flavonolglucosides.International Journal of Food Science and Technology, 42, 783789.Castaneda-Ovando, A., Pacheco-Hernandez, Ma. L., Paez-Hernandez, Ma., Rodriguez,

J. A., & Galan-Vidal, C. A. (2009). Chemical studies of anthocyanins: a review.Food Chemistry, 113, 859871.

Charron, C. S., Clevidence, B. A., Britz, S. J., & Novotny, J. A. (2007). Effect of dose sizeon bioavailability of acylated and nonacylated anthocyanins from red cabbage(Barssica oleraceaL. var.capitata). Journal of Agricultural and Food Chemistry, 55,53545362.

Clifford, M. N. (2000). Anthocyanins nature, occurrence and dietary burden.Journal of the Science of Food and Agriculture, 80, 10631072.

Davey, M. W., Van Montagu, M., Inze, D., Sanmartin, M., Kanellis, A., Smirnoff, N.,et al. (2000). Plant L-ascorbic acid: chemistry, function, metabolism,bioavailability and effects of processing. Journal of the Science of food and

Agriculture, 80, 825860.Del Castillo, M. D., Gordon, M. H., & Ames, J. M. (2005). Peroxyl radical-scavenging

activity of coffee brews. European Food Research and Technology, 221 , 471477.De Pascual-Teresa, S., & Sanchez-Ballesta, M. T. (2008). Anthocyanins: from plant to

health.Phytochemistry Reviews, 7, 281299.

DuPont, M. S., Mondin, Z., Williamson, G., & Price, K. R. (2000). Effect of variety,processing, and storage on the flavonoid glycoside content and composition oflettuce and endive. Journal of Agricultural and Food Chemistry, 48, 39573964.

Giusti, M. M., & Wrolstad, R. E. (2003). Acylated anthocyanins from edible sourcesand their applications in food systems. Biochemical Engineering Journal, 14,217225.

Hartmann, A., Patz, C.-D., Andlauer, W., Dietrich, H., & Ludwig, M. (2008). Influenceof processing on quality parameters of strawberries. Journal of Agricultural andFood Chemistry, 56, 94849489.

Hassimotto, N. M. A., Genovese, M. I., & Lajolo, F. M. (2005). Antioxidant activity ofdietary fruits, vegetables, and commercial frozen fruit pulps. Journal of

Agricultural and Food Chemistry, 53, 29282935.Jang, J., Ma, Y., Shin, J., & Song, K. (2005). Characterization of polyphenoloxidase

extracted from Solanum tuberosum Jasim. Food Science and Biotechnology, 14,117122.

Kusznierewicz, B., Smiechowska, A., Bartoszek, A., & Namiesnik, J. (2008). The effectof heating and fermenting on antioxidant properties of white cabbage. FoodChemistry, 108, 853861.

Lin, J.-Y., Li, Ch.-Y., & Hwang, I.-F. (2008). Characterization of the pigmentcomponents in red cabbage (Brassica oleracea L. var.) juice and their anti-inflammatory effects on LPS-stimulated murine splenocytes. Food Chemistry,109, 771781.

Markakis, P. (1982). Stability of Anthocyanins in Foods. In P. Markakis (Ed.),Anthocyanins as Food Colors (pp. 163180). New York: Academic Press.

McDougall, G. J., Fyffe, S., Dobson, P., & Stewart, D. (2007). Anthocyanins from redcabbage stability to simulated gastrointestinal digestion. Phytochemistry, 68,12851294.

Pliszka, B., Huszcza-Ciolkowska, G., Mieleszko, E., & Czaplicki, S. (2009). Stabilityand antioxidative properties of acylated anthocyanins in three cultivars of redcabbage (Brassica oleracea L. var. capitata L. f. rubra). Journal of the Science ofFood and Agriculture, 89, 11541158.

Podsedek, A. (2007). Natural antioxidants and antioxidant capacity of Brassicavegetables: A review. LWT Food Science and Technology, 40, 111.

Pourcel, L., Routaboul, J.-M., Cheynier, V., Lepiniec, L., & Debeaujon, I. (2007).

Flavonoid oxidation in plants: from biochemical properties to physiologicalfunctions.Trends in Plant Science, 12, 2936.

Prior, R. L., Wu, X., & Schaich, K. (2005). Standardized methods for thedetermination of antioxidant capacity and phenolics in foods, and dietarysupplements.Journal of Agricultural and Food Chemistry, 53, 42904302.

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. A. (1999).Antioxidant activity applying an improved ABTS radical cation decolorizationassay. Free Radical Biology and Medicine, 26, 12311237.

Sadilova, E., Carle, R., & Stintzing, F. C. (2007). Thermal degradation of anthocyaninsand its impact on color and in vitro antioxidant capacity.Molecular Nutrition &Food Research, 51, 14611471.

Scalzo, R. L., Genna, A., Branca, F., Chedin, M., & Chassaigne, H. (2008). Anthocyanincomposition of cauliflower (Brassica oleracea L. var. botrytis) and cabbage (B.oleraceaL. var. capitata) and its stability in relation to thermal treatments. FoodChemistry, 107, 136144.

Truong, V.-D., Deighton, N., Thompson, R. T., McFeeters, R. F., Dean, L. O. D., Pecota,K. V., et al. (2010). Characterization of anthocyanins and anthocyanidins inpurple-fleshed sweetpotatoes by HPLC-DAD/ESI-MS/MS. Journal of Agriculturaland Food Chemistry, 58, 404410.

Volden, J., Borge, G. I. A., Bengtsson, G. B., Hansen, M., Thygesen, I. E., & Wicklund, T.(2008). Effect of thermal treatment on glucosinolates and antioxidant-relatedparameters in red cabbage (Brassica oleracea L. ssp. capitata f. rubra). FoodChemistry, 109, 595605.

Wiczkowski, W., Szawara-Nowak, D., & Topolska, J. (2013). Red cabbageanthocyanins: profile, isolation, identification, and antioxidant activity. FoodResearch International, 51, 303309.

Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D., Gebhardt, S. E., & Prior, R. L.(2004). Lipophilic and hydrophilic antioxidant capacities of common foods inthe United States. Journal of Agricultural and Food Chemistry, 52, 40264037.

Wu, X., & Prior, R. L. (2005). Identification and characterization of anthocyanins byhigh-performance liquid chromatographyelectrospray ionizationtandemmass spectrometry in common foods in the United States: vegetables, nuts,and grains. Journal of Agricultural and Food Chemistry, 53, 31013113.

Zafra-Stone, S., Yasmin, T., Bagchi, M., Chatterjee, A., Vinson, J. A., & Bagchi, D.(2007). Berry anthocyanins as novel antioxidants in human health and diseaseprevention.Molecular Nutrition & Food Research, 51, 675683.

Zielinska, D., Wiczkowski, W., & Piskula, M. K. (2008). Determination of the relativecontribution of quercetin and its glucosides to the antioxidant capacity of onionby cyclic voltammetry and spectrophotometric methods. Journal of Agriculturaland Food Chemistry, 56, 35243531.

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