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J. of Supercritical Fluids 45 (2008) 32–36

Extraction and formulation of anthocyanin-concentrates

from grape residues
Tünde Vatai a,∗ , Mojca Škerget a , Željko Knez a , Sabine Kareth b,1 ,
Manuel Wehowski b,1 , Eckhard Weidner b,1
a Laboratory for Separation Processes and Product Design, Faculty of Chemistry and Chemical Engineering,
University of Maribor, Smetanova 17, SI-2000 Maribor, Slovenia
b Chair for Process Technology, Faculty of Mechanical Engineering, Ruhr-University Bochum,

Universitätstr. 150, IB 6/126, DE-44780 Bochum, Germany

Received 15 October 2007; received in revised form 12 December 2007; accepted 22 December 2007

Abstract
The interest in natural food colorants shows an increasing tendency due to the strictening rules and consumers’ concerns over synthetic additives.
One of natural pigments with high potential are anthocyanins, which are providing colours from yellow to blue in berries, flowers and vegetables.
However the use of anthocyanins as food colorants is still limited because of their relatively low stability during processing, formulation and
storage. In this work extraction of anthocyanins from grape marc was investigated with different concentrations of acetone (50 wt%, 70 wt% and
100 wt%) at 20 ◦ C and at 60 ◦ C temperatures. Pilot-scale extraction of grape marc was performed upon the results obtained from laboratory scale
experiments. High-pressure concentrated powder form (CPF) technique was applied in order to formulate the obtained extracts in powderous form.
Carrier materials, such as starch and silica were tested. Colorimetric properties (lightness, chroma and hue angle) of powderous products were
measured and compared with synthetic colorants. Colour stability was monitored during storage at different conditions (light/dark and ambient
temperature/refrigerator). The results were compared with the non-formulated extracts, which were stored at same conditions. Product formulation
with CPF improved the colour stability of anthocyanin extracts, while for the non-formulated extracts degradation of colour rapidly occurred. The
results show that formulated anthocyanin extracts have potential use as natural colorants. However, the major difference compared to synthetic
colorants is still in the saturation of the colour.
© 2008 Elsevier B.V. All rights reserved.

Keywords: Anthocyanins; Grape marc; Extraction; Stabilization; Formulation; Concentrated powder form

1. Introduction and on new methods for stabilization and formulation are

Anthocyanins belong to the flavonoid phenolic compounds
and they are responsible for the orange, red, pink and blue
colours in most of the fruits, flowers and vegetables. Beside the
colour properties, interest in anthocyanins has increased because
of their possible health benefits [1].
The stability of the anthocyanin pigments is influenced by
environmental and processing factors such as pH value, tem-
perature and presence of oxygen, enzymes and condensation
reactions [2,3]. Research on new viable sources of anthocyanins
∗
Corresponding author. Tel.: +386 2 229 44 68; fax: +386 2 251 67 50.
E-mail addresses: tvatai@gmail.com (T. Vatai), zeljko.knez@uni-mb.si
(Ž. Knez), kareth@vtp.ruhr-uni-bochum.de (S. Kareth).
1 Tel.: +49 234 32 26680, fax: +49 234 32 14277.
required to develop the products of natural colorants in a proper
form with desirable stability, low cost and high tinctorial strength
[4–7]. The possible stabilizations are complex formation (e.g.
surface adsorption), intra- and intermolecular co-pigmentation,
self-association, etc. Product formulation improves the stability
of the anthocyanins: spray- and freeze-dried extracts show better
stability, probably due to low moisture environment [8].
In this work applicability of a high-pressure technique using
dense carbon dioxide (CO2 ) for formulation and stabilization of
anthocyanin extracts was investigated. The applied method was
concentrated powder form (CPF) technology [9]. CPF is already
implemented in the spice industry, spraying extracts of celery,
oregano, laurel, paprika, etc., but also low and high viscous oils,
fats and aromas were sprayed and pulverized [10]. In this tech-
nique the liquid extract and a supercritical gas, mostly CO2 , are
mixed. The formed solution is then expanded rapidly and during

0896-8446/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.supflu.2007.12.008
 T. Vatai et al. / J. of Supercritical Fluids 45 (2008) 32–36
the expansion, the liquid dissipates in very fine droplets, which
are mixed with the powder, which acts as a carrier [11]. Due to
the rapid release of CO2 , the temperature in the spray tower is
very low, usually between 0 ◦ C and −50 ◦ C.
Prior the CPF formulation, extraction of anthocyanins from
grape marc was performed with different concentrations of ace-
tone (50 wt%, 70 wt% and 100 wt%) at 20 ◦ C and at 60 ◦ C
temperatures, and the total phenolic and total anthocyanin con-
tent was measured. Pilot-scale extraction of grape marc was
performed upon the results obtained from laboratory scale exper-
iments. Extract of grape marc was sprayed on different carrier
materials in order to investigate the possible stabilization with
CPF. As a liquid, solutions of the extract were used with dif-
ferent dilutions (1 wt%, 10 wt% and 30 wt%), comparing water
and 50 wt% ethanol (in water) as solvents. Shifting the colour
towards ‘redness’ was investigated by acidifying the solutions
with citric acid. As carriers silica and starch were tested. The
colour was measured using the method of CIEL (International
Commission on Illumination, Vienna), based on uniform colour
spaces (CIELAB) which enables objective measurement and
characterization of colour properties. One part of the obtained
formulated product was stored in dark in refrigerator and the
other part at light at room temperature. The stability of the CPF
powder was monitored during storage. The results were com-
pared with the pure extract (not CPF formulated) stored at same
conditions.
2. Materials and methods
2.1. Materials and reagents
The red grape marc (variety of Refošk) used in the present
study was kindly donated by Vinakoper (Slovenia). The grape
was harvested and processed for wine in 2005. The marc was
dried in vacuum dryer at 50 ◦ C, milled and stored in a cool
and dry place. The moisture of the sample determined by Karl-
Fischer method was 2.6%. The carrier materials were purchased
from Degussa GmbH (Düsseldorf, Germany). All the reagents
and solvents were obtained from Merck (Darmstadt, Germany).
2.2. Extraction and analyses of extracts
Extraction experiments of Refošk grape marc (RF) in labo-
ratory scale were performed in batch mode using acetone–water
mixtures of different concentrations (50 wt%, 70 wt% and
100 wt% acetone) at temperatures 20 ◦ C and 60 ◦ C. The ratio of
solvent to plant material (v/w) was 20 L/kg. After 2 h of extrac-
tion the mixture was filtered and the solvent was removed by
vacuum evaporation. Upon the obtained results from labora-
tory scale investigations the parameters for pilot-scale extraction
were chosen. Extraction in a 3 m3 vessel was performed with
50 wt% acetone and a solvent-to-solid ratio of 20 L/kg at
60 ◦ C for 2 h. Afterwards, the mixture was filtered, the sol-
vent evaporated and the extract was dried in a vacuum dryer at
50 ◦ C.
The content of total phenols was measured by the Folin-
Ciocalteu method using a spectrophotometer (Varian Cary 50),
33
based on a colorimetric oxidation/reduction reaction of phenols
[12]. To 0.5 mL of diluted extract (20 mg in 10 mL distilled
water) 2.5 mL of Folin-Ciocalteu reagent (diluted 1:10 with
water) and 2 mL of Na2 CO3 (75 g/L) was added. The sample
was incubated for 5 min at 50 ◦ C and then cooled. For control
sample 0.5 mL distilled water was taken. The absorbance was
measured at 760 nm. The results were calculated on the basis
of the standard curve of the gallic acid (GA) and expressed as
mg GA/g dry material.
The content of monomeric anthocyanins was measured by
the pH-differential method, which relies on the structural trans-
formation of the anthocyanin chromophore as a function of pH
value, which can be measured using optical spectroscopy [13].
Twenty milligrams of extract was dissolved in 10 mL distilled
water. Two dilutions of the sample were prepared, one with
potassium chloride buffer pH 1 (1.86 g of KCl in 1 L of dis-
tilled water) and the other with sodium acetate buffer pH 4.5
(32.8 g of CH3 COONa in 1 L of distilled water). The pH value
of the buffers was adjusted with cc. HCl. The dilutions were left
to equilibrate for 15 min. The absorbances of each dilution were
measured at 520 and 700 nm. The anthocyanin content was cal-
culated on the basis of cyanidin-3-glycoside (MW = 449.2 and
ε = 26,900) and the results were expressed as mg/g dry material.
2.3. CPF experiments
For the CPF experiments the extract solution was prepared
as follows: extract of grape marc (from pilot-scale extraction)
was dissolved in solvent (water or 50 wt% ethanol–water mix-
ture). The concentrations of RF extract were 1 wt%, 10 wt%
and 30 wt% in solvent. In the case of 50 wt% ethanol solution,
the effect of the change of the pH value on colour was investi-
gated by setting the pH value to 1 using citric acid. As carrier
material modified silica and starch were used. Fig. 1 shows the
flow scheme of the CPF plant. The solution was filled into the
autoclave. CO2 was dissolved in the extract solution at a pres-
sure ranging from 10 to 18 MPa at ambient temperature. The
carrier was filled into a vessel. The carrier was dozed to the
spraying zone by an additional CO2 cylinder with a pressure of
0.1–0.2 MPa. An inert atmosphere was provided by leading N2
to the system. The solution formed in the autoclave was depres-
surized over a nozzle into the spray tower. Due to the rapid
expansion of the gas the liquid was disintegrated into very fine
droplets. Concurrently the solid, powderous carrier was added
to the spraying zone. The turbulence in the tower, caused by the
expanding gas led to an intensive mixing of the droplets and the
solid carrier. The coarse fraction was collected at the bottom
of the spray tower while the fine fraction was obtained in the
cyclone.
2.4. Colour evaluation
The CIEL*a*b* system is a method for measuring colour
differences and tracking colour changes during processing and
storage [14]. The colorimeter measures L* which is a measure
of ‘lightness’ and two coordinates a* and b*. Positive values
of a* are in the direction of ‘redness’ and negative values in
34 T. Vatai et al. / J. of Supercritical Fluids 45 (2008) 32–36

Fig. 1. Flow scheme of the CPF process.

the direction of the complement ‘green’. Positive values of b*
are in the direction of the vector for ‘yellowness’, and nega-
tive for ‘blueness’. For better descriptions of colour changes
L*a*b* are converted to the L·C·h system where the L = lightness
(L) with 100 = absolute white and 0 = absolute black. Chroma
(C) is a measure of intensity (or saturation) and calculated
as (a* + b*)1/2 . Hue angle (h) is derived from the two coor-
dinates a* and b* and is determined as arc tan (b*/a*). Hue
angle is expressed on a 360◦ grid where 0◦ = red, 90◦ = yellow,
180◦ = green and 270◦ = blue. This system avoids the use of neg-
ative numbers and differences in hue angle of 1◦ are readily
discernible by the human eye.
The obtained CPF samples were stored at different condi-
tions. They were divided into two parts. One part was put to
dark into refrigerator and the other part was stored at ambient
temperature and light. The colour stability was compared to non-
formulated extracts, which were stored at the same conditions
as the CPF formulated samples. For colorimetric measurements
the non-formulated extracts were prepared as solutions: 100 mg
dissolved in 100 mL of 50 wt% ethanol.
3. Results and discussion
In laboratory scale the highest amounts of total phenols and
total anthocyanins were generally extracted with 50 wt% acetone
(Fig. 2). By using 50 wt% acetone as solvent, the highest amount
of total phenols was obtained at 60 ◦ C (16.7 ± 0.4 mg GA/g
dry material), while the amounts of total anthocyanins at 20
and 60 ◦ C were 0.74 ± 0.05 and 0.54 ± 0.06 mg/g dry material,
respectively. Amounts of total phenols and total anthocyanins
obtained by the extraction of grape marc in pilot-scale (at con-
ditions described in Section 2.2) were 15.4 mg GA/g dry material
and 0.68 mg/g dry material, respectively.
In the CPF experiments starch as a carrier was not successful,
because the obtained powder was sticky and wet; while silica
resulted in a homogenous coloured powder. In the case of 50 wt%
ethanol – as the solvent for the extract – more intensive colour
was observed than in the case of pure water. Anthocyanins at an
acidic pH value are present in the most coloured form (flavylium
cation), thus by adding acid to the extract solution the colour was
shifted towards ‘redness’. By increasing the concentration of the
extract in the liquid solution (to 30 wt%) powder obtained was
of more intense colour.
The “visually” observed differences by the obtained CPF
powders were confirmed by the colorimetric measurements. The
initial values of L, C and h of some of the products are shown
in Table 1. The 10 wt% extract solutions showed similar light-
ness values (L ≈ 60), while the 30 wt% solutions where darker
(L ≈ 50). Commonly used synthetic colorants in FD&C (Food,
Drug and Cosmetics) industry, such as Red 3 and 40, have L

Fig. 2. Extraction of Refošk grape marc with different concentrations of acetone in mixture with water (50 wt%, 70 wt% and 100 wt%) at 20 ◦ C and at 60 ◦ C. (a)
total phenols, (b) total anthocyanins extracted. Abb.: GA, gallic acid.
 T. Vatai et al. / J. of Supercritical Fluids 45 (2008) 32–36 35

Table 1
Comparison of the initial colorimetric values of three CPF products and two commercial colorants
Samples Liquid Liquid/carrier ratio (wt) Lightness Hue angle Saturation

CPF product
Carrier: silica 10 wt% RF extract in 50% ethanol 60/40 62 35 11
Liquid: RF extract in solvent
CPF product
10 wt% RF extract in 50%
Carrier: silica 56/44 57 6 17
ethanol (acidified with citric acid)
Liquid: RF extract in solvent
CPF product
30 wt% RF extract in 50%
Carrier: silica 44/56 53 7 18
ethanol (acidified with citric acid)
Liquid: RF extract in solvent
Commercial colorant: FD&C Red 40 (erythrosine) 72 39 70
Commercial colorant: FD&C Red 3 (allura red) 71 25 73

Abbreviations: RF Refošk, FD&C Food, Drug and Cosmetics.

values around 70 [5]. The hue angle of the non-acidified solu-
tion was higher (35◦ ) compared to the acidified samples (1–2◦ ),
which implies that by acidification the colour was shifted to the
red angle, towards 0◦ . Further, for the acidified samples, also the
chroma values showed higher saturation. The significant differ-
ences compared to the synthetic colorants were observed by the
values of chroma: for synthetic colorants around 70 and for the
CPF product ranging from 10 to 22.
During 21 weeks of storage no significant changes were
observed in the colorimetric values of all CPF samples stored at
dark place and at ambient light. In general, the CPF formulated
products showed good colour stability. The values for the hue
angle, chroma and lightness monitored for 21 weeks are shown
in Fig. 3.
The non-formulated extracts stored at the same conditions as
formulated ones showed lower stability during 15 weeks of mon-
itoring. For the extracts which were extracted with non-acidified
solvent, the lightness increased from around 40 to 50, while the
increase was even higher for the extracts obtained with acidified
solvent (to 60 and above) (Fig. 4c). For the samples which were
stored in dark the increase was slightly lower compared to the
extracts which were stored at ambient light. The chroma values –
which represent the saturation of the colour – decreased for both
extracts (Fig. 4b). The initial hue values for the extracts were 2
(non-acidified extraction solvent) and 15 (acidified extraction
solvent). For both samples high increase in hue angle values
was observed, especially for the extracts which were stored at
ambient temperature and light (Fig. 4a).

Fig. 3. (a) Hue angle, (b) chroma and (c) lightness values of the CPF products stored at dark and at light for 21 weeks. Concentration of Refošk (RF) extract in liquid:
10 wt% and 30 wt%. Comparison of acidified (with citric acid to pH 1) and non-acidified extracts.
36 T. Vatai et al. / J. of Supercritical Fluids 45 (2008) 32–36
Fig. 4. (a) Hue angle, (b) chroma and (c) lightness values of the Refošk (RF) extracts obtained with acidified and non-acidified solvents stored at dark and at light
for 15 weeks.
4. Conclusions
The extract from Refošk grape marc was formulated in a pow-
derous form with CPF technique by using silica gel as carrier.
The powder obtained was homogenously coloured and showed
better stability during prolonged storage compared to the non-
formulated extract. The results suggest that application of CPF
high-pressure technique, which enables the attachment of com-
pounds on solid carrier, is a suitable method for stabilizing the
colour of the anthocyanin extracts. However, the difference to
synthetic colorants was observed in the saturation of the colour,
which should be improved for further applications in the indus-
try.
Acknowledgements
This research was financially supported through a European
Community Marie Curie Fellowship (Project MEST-CT-2004-
007767). The European Commission DG Research—Improving
The Human Research Potential is gratefully acknowledged. For
further information: http://www.cordis.lu/improving.
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