Biochemical Engineering Journal 14 (2003) 217–225

Review
Acylated anthocyanins from edible sources and their
applications in food systems
M. Mónica Giusti a,∗ , Ronald E. Wrolstad b
a Department of Nutrition and Food Science, University of Maryland, 3304 Marie Mount Hall, College Park, MD 20742, USA
b Department of Food Science and Technology, Oregon State University, Corvallis, OR 97331, USA

Received 19 July 2002; accepted 2 November 2002

Abstract
There is considerable demand for food colorants from natural sources that can serve as alternatives to the use of synthetic dyes due
to both legislative action and consumer concerns over the use of synthetic additives. Interest in anthocyanin-rich foods and extracts has
intensified because of their possible health benefits. Anthocyanins are potent antioxidants and may be chemoprotective. Optimizing health
and performance through the diet is believed to be one of the largest and most lucrative markets in the US, and throughout the world.
Findings of acylated anthocyanins with increased stability have shown that these pigments may impart desirable color and stability for
commercial food products. Examples of suitable acylated anthocyanin sources may be radishes, red potatoes, red cabbage, black carrots,
and purple sweet potatoes. Among these, radishes and red potatoes stand out as potential alternatives for the use of FD&C Red No. 40
(allura red). Maraschino cherries with bright attractive and stable red color were obtained with radish extract. Radish and potato extracts
imparted color characteristics to model juices extremely close to those of allura red. Other potential applications for acylated anthocyanins
may include other challenging systems such as dairy products. The increased stability of these pigments together with their added value
due to potential beneficial effects opens a new window of opportunities for use of these extracts in a variety of food applications.
© 2003 Elsevier Science B.V. All rights reserved.
Keywords: Acylated anthocyanins; Natural food colorants; Color stability; Pigment stability; CIEL∗ ch; Chromatography; Treatment

1. Introduction vegetables, flowers, leaves, roots and other plant storage

Color plays a very important role in the acceptability of
foods. Consumers first judge the quality of a food product
by its color, and the food industry has used colorants for
centuries to enhance or restore original appearance of foods
or to ensure uniformity, as indicator of food quality. Color
is a vital constituent of foods because it is one of the first
characteristics perceived by the senses and is used by con-
sumers for the rapid identification and ultimate acceptance
of foods [1].
The safety of synthetic colorants has been questioned in
the past years, leading to a reduction in the number of per-
mitted colorants. Interest in natural colorants has signifi-
cantly increased as a consequence of both legislative action
and consumer awareness to the use of synthetic additives in
their foods.
Anthocyanins comprise a diverse group of intensely col-
ored pigments responsible for the appealing and often spec-
tacular orange, red purple and blue colors of many fruits,
∗ Corresponding author. Tel.: +1-301-405-5421; fax: +1-301-314-9327.
E-mail address: mg237@umail.umd.edu (M.M. Giusti).
organs. They are water soluble, which facilitates their in-
corporation into aqueous food systems [2], and have been
consumed for centuries without adverse effects.
Besides the color attributes, interest in anthocyanins
has intensified because of their possible health benefits.
Health benefits associated with anthocyanin extracts in-
clude enhancement of sight acuteness [3], antioxidant
capacity [4–8], treatment of various blood circulation dis-
orders resulting from capillary fragility [4], vaso-protective
and anti-inflammatory properties [9], inhibition of platelet
aggregation [10], maintenance of normal vascular perme-
ability [4], controlling diabetes, anti-neoplastic and chemo-
protective agents [11,12], radiation-protective agents [13],
and possibly others due to their diverse action on various
enzymes and metabolic processes [4]. These qualities make
anthocyanins attractive alternatives to synthetic dyes.
Some limitations that have restricted the use of natural
colorants in food systems are their relatively low stability
to several processing, formulation and storage conditions,
and that they may impart undesirable odor or flavor charac-
teristics. Successful applications of red cabbage and radish
extracts as coloring agents in foods have only been possible

1369-703X/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S1369-703X(02)00221-8
218 M.M. Giusti, R.E. Wrolstad / Biochemical Engineering Journal 14 (2003) 217–225
after the development of suitable procedures to remove or
drastically reduce the concentration of aroma and flavor
compounds that give a strong vegetative note to these ex-
tracts.
Research involving the development of anthocyanin-
containing food colorants has led to the discovery of antho-
cyanin molecules with complex patterns of glycosylation
and acylation that exhibit remarkable stability to pH
changes, heat treatment and light exposure [14–16]. The
improved stabilization has been attributed to intramolecular
and intermolecular copigmentation, self-association, metal
complexing and presence of inorganic salts [17–19].
The process of colorant selection usually is accomplished
by answering the following issues: the target shade, the phys-
ical/chemical attributes of the food matrix, stability to pro-
cessing and storage conditions, and regulatory issues. The
aim of this paper is to discuss the potential food applications
of a number of acylated anthocyanin extracts and to eval-
uate the relationship between chemical structure and color
characteristics and pigment stability.
2. Effect of anthocyanin chemical structure on
spectral characteristics and color attributes:
choosing the “right” chemistry
Under acidic conditions, the color of non- and mono-
acylated anthocyanins is determined largely by substitution
in the B-ring of the aglycon [20]. Mazza and Miniati [21]
reported that increased hydroxyl substitution on the B-ring
results in a shift of the visible absorption maximum (λmax )
to longer wavelengths (bathochromic shift) to yield a bluer
hue (Fig. 1).
The sugar residues may occur acylated with aromatic
acids including p-coumaric, caffeic, ferulic, sinapic, gallic
or p-hydroxybenzoic acids, and/or aliphatic acids such as
Fig. 1. Anthocyanidins more commonly found in nature.
malonic, acetic, malic, succinic or oxalic acids. These acyl
substituents are commonly bound to the C3 sugar, esterified
to the 6-OH or less frequently to the 4-OH group of the sug-
ars. However, anthocyanins containing rather complicated
acylation patterns attached on different sugar moieties have
been reported [18,22–24].
Substantial information can be obtained from the UV-Vis
spectral characteristics of anthocyanins. The presence of hy-
droxycinnamic acid acylation is revealed by the presence of
an absorption band in the 310–360 nm range. The ratio of
maximun absorbance in the 310–360 nm to the absorbance
at the visible λmax will give an estimation of the number
of aromatic acylating groups [25,26]. In contrast, acylation
with aliphatic acids has often been overlooked in the past,
due to their hydrolysis under typical extraction procedures
with acidified methanol and to the fact that they lack of a
typical UV-Vis absorption band.
Acylated and non-acylated pelargonidin derivatives iso-
lated from strawberry, radish, potato and from partially
hydrolyzed radish pigments were used to evaluate the effect
of glycosylation and acylation on spectral characteristics
and color attributes [27]. Individual pigments were isolated
using semi-preparative HPLC as follows: pelargonidin-
3-glucoside (pg-3-glu, from strawberry), pelargonidin-3-
[2-glucosyl-6-trans-p-coumaroyl-glucoside]-5-(6-malonyl-
glucoside) and pelargonidin-3-[2-glucosyl-6-trans-feruloyl-
glucoside]-5-(6-malonyl-glucoside), (pg-3-soph-5-glu acy-
lated with malonic and ferulic or p-coumaric acids from red
radish), pelargonidin-3-rutinoside-5-glucoside (pg-3-rut-5-
glu) acylated with p-coumaric acid (from red potato), pg-3-
soph-5-glu, pg-3-soph, pg-3-glu-5-glu, pg-3-glu, pg-5-glu,
pg-aglycon (from partially hydrolyzed radish pigments).
Spectral and color (CIEL∗ ch) attributes of purified pig-
ments were measured at room temperature in both acidi-
fied (pH 1) methanolic solutions and in aqueous buffer so-
lution, using chloride as the counter ion. We have found
[27] that differences in glycosylation and acylation patterns
of pelargonidin-based anthocyanins had a critical impact on
color and tinctorial strength of anthocyanin extracts, and that
small differences in chemical structure may have an impor-
tant impact on spectral characteristics and color attributes of
the pigments (Table 1, Fig. 2 shows color data in aqueous
solutions).
It has been reported as a general characteristic that an
increase in the number of sugar substitutions is accompa-
nied by an increase in the visible molar absorptivity of the
flavylium cation form when compared to their parent mono-
and di-glucosides [28]. When we compared the absorptiv-
ity of the different purified pelargonidin derivatives [27], we
found a slight drop in molar absorptivity when one sugar was
present, as compared to the aglycon. The addition of more
glucose units to the molecule seemed to have a hyperchromic
effect on the pigment, since pg-3-soph-5-glu showed a mo-
lar absorptivity substantially higher than the corresponding
mono-glucoside. Sugar substitution also showed an impor-
tant role on spectral and color characteristics of the pigment,
 M.M. Giusti, R.E. Wrolstad / Biochemical Engineering Journal 14 (2003) 217–225 219

Table 1
Color attributes of purified pelargonidin derivatives in pH 1 buffer containing 0.1 M KCl (modified from Ref. [27])a
Anthocyanin Concentration (×10−5 mol/l) Visible λmax Molar absorptivity L∗ Hue Chroma

pg-aglycon 1.5 505 18420 90 23 17

pg-3-glu 1.8 496 15600 90 44 18
pg-3-soph-5-glu (Raph) 1.9 497 25370 90 41 20
Raph + p-coumaric 1.7 506 28720 86 23 27
Raph + ferulic 2.8 506 24140 83 24 34
Raph + p-coumaric and malonic 2.5 508 33020 87 22 26
Raph + ferulic and malonic 2.7 508 31090 83 22 34
pg-3-rut-5-glu + p-coumaric 1.9 504 32080 87 23 25
a pg: pelargonidin; pg-3-glu: pelargonidin-3-glucoside; pg-3-soph-5-glu: pelargonidin-3-sophoroside-5-glucoside (also called raphanusin: Raph); Raph+

p-coumaric: pelargonidin-3-[2-glucosyl-6-trans-p-coumaroyl-glucoside]-5-glucoside; Raph+ferulic: pelargonidin-3-[2-glucosyl-6-trans-feruloyl-glucoside]-

5-glucoside; Raph + p-coumaric and malonic: pelargonidin-3-[2-glucosyl-6-trans-p-coumaroyl-glucoside]-5-(6-malonyl-glucoside); Raph + ferulic and mal-
onic: pelargonidin-3-[2-glucosyl-6-trans-feruloyl-glucoside]-5-(6-malonyl-glucoside); pg-3-rut-5-glu + p-coumaric: pelargonidin-3-rutinoside-5-glucoside
(pg-3-rut-5-glu) acylated with p-coumaric acid (position of acylation tentatively assigned to position C4 of the rhamnosyl moiety of rutinose).

with a hypsochromic shift (decrease in visible λmax ) caused
by the presence of glycosylation. pg-3-glu and pg-3,5-triglu
showed higher hue angle (>40◦ ) than the other pg-derivatives
evaluated (Table 1) when dissolved in buffer pH 1, corre-
sponding to the yellow-orange region of the solid of color.
The presence of cinnamic acid acylation showed great im-
pact on spectral and color characteristics. Our results showed
a bathochromic shift on visible λmax and a hyperchromic ef-
fect of acylation of pg-derivatives as compared to the parent
pg-3-soph-5-glu, in particular, when two acylating groups
were present. These results go in agreement with previous
studies [14,20,25,29] that reported that the presence of acy-
lation with cinnamic acids produces a bathochromic shift
in the visible λmax of the pigment coupled with a strongly
increased absorptivity (hyperchromic effect).
Under the conditions used (pH 1), acylation with malonic
acid showed little effect on λmax , the main spectral differ-
ence being the higher absorption of the major visible band
(Table 1, Fig. 2).
Fig. 2. Effect of acylation on spectral characteristics: spectra of non-
acylated pg-3-soph-5-glu, and the resulting spectra when acylation with
p-coumaric acid (pelargonidin-3-[2-glucosyl-6-trans-p-coumaroyl-gluco-
side]-5-glucoside) and malonic acids (pelargonidin-3-[2-glucosyl-6-trans-
p-coumaroyl-glucoside]-5-(6-malonyl-glucoside)) are present.
3. Looking at different edible sources of
acylated anthocyanins
3.1. Potential sources of anthocyanin-based
colorants
According to the USA regulations, color additives are de-
fined as any dye, pigment or substance capable of imparting
color when added or applied to a food, drug or cosmetic
(21CFR70.3). Anthocyanins are permitted as food colorants
in the USA under the category of fruit (21CFR73.250) or
vegetable (21CFR73.260) juice color. Under these categories
only the edible portion of plants are to be used. Extracts
from dried materials are permitted under this classification
but the extracting solvent is restricted to water.
A number of potential food plants have been suggested as
commercial sources of anthocyanin-based colorants, how-
ever their use has been limited by pigment stability, avail-
ability of raw material and economic considerations [20].
Highly pigmented fruits have been suggested as sources of
anthocyanin colorants, including extracts of red grapes and
its by-products [2], cranberry press cake [30–32], blueber-
ries [33], black chokeberries [34], elderberries [35], Hibiscus
calyces [36], black currents [37,38], purple corn [39] among
others. However, the anthocyanin composition of these con-
ventional sources involves mainly mono- and di-glucosides
which provides limited stability against hydration and pH
changes [40].
Acylation of the anthocyanin molecule improve their sta-
bility through intramolecular and/or intermolecular copig-
mentation, and self-association reactions. Therefore, sources
of acylated anthocyanins may provide the desirable stability
for food applications.
Examples of edible sources with acylated anthocyanins
that may impart desirable color and stability include
radishes, red potatoes, red cabbage, black carrots, and pur-
ple sweet potatoes. Among these, radishes and red potatoes
stand out as potential alternatives for the use of FD&C Red
No. 40 (allura red). We will discuss here some commercially
220 M.M. Giusti, R.E. Wrolstad / Biochemical Engineering Journal 14 (2003) 217–225

Table 2
Pigment composition of some edible sources of acylated anthocyaninsa
Source Type of pigments Composition

Radish (Raphanus sativus)
Potato (S. tuberosum)
Black carrot (Daucus carota L.)
Red cabbage (Brassica oleracea)
Grape (Vitis labrusca)
a Numbers in parenthesis match the molecular ion peaks which is shown in Fig. 5. Source from Ref. [55].
pg-derivative acylated with one cinnamic acid
and an aliphatic acid
pg-derivatives acylated with one cinnamic acid
cy-3-rut-glu-gal acylated with one cinnamic
acid
cy-3-diglu-5-glu acylated with one or two
cinnamic acids
A mixture of five different aglycones, acylated
and non-acylated with p-coumaric acid
pg-3-soph-5-glu acylated with: p-coumaric acid (1),
ferulic acid (2), p-coumaric acid and malonic acid
(3), ferulic acid and malonic acid (4)
pg-3-rut (1), pg-3-rut + p-coumaric acid (2),
pg-3-rut-5-glu (3), pg-3-rut-5-glu + p-coumaric acid
(4), pg-3-rut-5-glu + ferulic acid (5)
cy-3-gal-xyl (1), cy-3-gal-xyl-glu (2),
cy-3-gal-xyl-glu + p-coumaric (3) cy-3-gal-xyl-glu
+ ferulic (4), cy-3-gal-xyl-glu + sinapic (5)
Mono-acylated (1–5), cy-3-diglu-5-glu + sinapic
acid (3), di-acylated with hydroxycinnamic acids
(6–9), cy-3-diglu-5-glu + sinapic and ferulic acids
(8), cy-3-diglu-5-glu + two sinapic acids (9)
Anthocyanin glucosides (1–5), acylated with
p-coumaric acid (6–10), di-glucosides acylated with
p-coumaric acid (11–15)

available sources of acylated pigments (Table 2, Fig. 3) for
food purposes and some of their applications.
3.2. Anthocyanin-based colorants as alternatives to the
use of allura red (FD&C Red No. 40)
(mono-acylated cyanidin derivatives) had a more reddish
hue, while meganatural red grape (mono-acylated deriva-
tives of different anthocyanins) and red cabbage (mono- and
di-acylated cyanidin derivatives) extracts imparted a more
purplish color.

There is a particular demand for a red colorant that can
effectively substitute for FD&C Red No. 40 (allura red), the
certified dye with the highest per capita consumption in the
USA. The color attributes of different natural colorants were
compared to FD&C Red No. 40 [41]. Model juice solutions
containing 0.1 M citric acid, at pH 3 were colored with an-
thocyanin pigment to obtain the same final chroma of a solu-
tion of 150 ppm allura red. The two pelargonidin-based ex-
tracts obtained from red radish and red potato provided color
closest to the desired orange-red color of FD&C Red No.
40 (Fig. 3). The orange hue of pelargonidin aglycones shifts
into an intense orange-red color due to the presence of acy-
lation with cinnamic acids. Solutions of black carrot extract
Fig. 3. Hue angle of different anthocyanin extracts in solution (pH 3) as
compared to allura red at the same level of chroma. Pigment composition
were presented in Table 2. Modified from Ref. [41].
3.3. Stability to color changes with increasing pH
To test the effect of pH on different acylated pelargonidin-
based colorants, we evaluated the color characteristics of
radish extracts (radish cv. Fuego with pg-soph-5-glu acy-
lated with a cinnamic and a malonic acids, and a commer-
cial SN-radish extract, di-acylated with cinnamic acids) and
potato extracts (pg-3-rut-5-glu acylated with one cinnamic
acid) at pH values ranging from 1 to 7 at 15 mg monomeric
anthocyanin per 100 ml of a buffer solution. At pH 1–2
(0.1 M KCl buffer) the three extracts evaluated exhibited
very similar hue angles and chroma (Fig. 4). When the pH
was raised to 3 (0.1 M citrate buffer) the hue angles de-
creased from ∼40◦ to ∼20◦ . As the pH was increased fur-
ther, the hue and chroma of the solutions were dependant
on the chemical nature of the pigment extract. Potato an-
thocyanins showed a much larger decrease in chroma with
increase in pH, so that more pigment would be required
to achieve similar color intensities. At pH 4 (0.1 M citrate
buffer), the chroma had decreased by ∼85% and there were
only slight changes with further increases in pH. The hue
of the radish pigment di-acylated with cinnamic acids de-
creased continuously as the pH was increased, towards a
more purplish hue. However, the chroma of this solution de-
creased to about half of the initial chroma at pH 5–6 (0.1 M
phosphate buffer). The radish extract with malonic and cin-
namic acid acylation maintained a reddish hue angle at pH 4,
with further fading at higher pH. Both radish extracts showed
an increase in chroma when the pH was increased from 6
to 7 (0.1 M phosphate buffer). Di-acylation with cinnamic
 M.M. Giusti, R.E. Wrolstad / Biochemical Engineering Journal 14 (2003) 217–225
Fig. 4. Color changes of acylated pg-derivatives with changes in pH. Pigment concentration standardized to equal chroma, and measurements made in
the following buffers: 0.1 M KCl, pH 1 and 2 buffers; 0.1 M, pH 3 and 4 citrate buffers; 0.1 M, pH 5, 6 and 7 phosphate buffers.
acids (see SN-radish, Fig. 4) made the pigments more resis-
tant to color changes with pH, but consistent with the ex-
pected bathochromic shifts, also changes the hue angle to
a more purplish appearance. Fossen et al. [42] reported the
increase of color intensity of acylated petunidin derivatives
in the alkaline region.
Fig. 5. Anthocyanin profile of different edible plants as determined by
electrospray mass spectrometry. Modified from Ref. [55].
221
3.4. Radish anthocyanins as colorants for maraschino
cherries
Manufacturers of maraschino cherries have sought natu-
ral colorant which could serve as acceptable alternative to
FD&C Red No. 40, which gives the product its attractive
color. This is a challenging task since processors wanted to
match the hue of the allura red colored cherries and have
a reasonable shelf life for a product that can be packed in
glass and stored at ambient temperatures [43]. Also, the ma-
trix offers it own challenges since the pH is 3.5 (higher than
the typical pH ranges for anthocyanin applications) and the
residual SO2 after processing, which can lead to the degra-
dation of anthocyanins [43].
We evaluated the use of red radish anthocyanin ex-
tract for coloring maraschino cherries [43]. Primary and
secondary bleached cherries were sweetened to 40◦ Brix,
pH 3.5, and colored using two different concentrations of
radish anthocyanin extracts (60 and 120 mg monomeric an-
thocyanin per 100 ml syrup), and with FD&C Red No. 40
(200 ppm). The pigment composition of the extract was 70%
di-acylated pelargonidin derivatives (pelargonidin-3-[2-glu-
cosyl-6-trans-p-coumaroyl-glucoside]-5-(6-malonyl-gluco-
side) and pelargonidin-3-[2-glucosyl-6-trans-feruloyl-glu-
coside]-5-(6-malonyl-glucoside)), and 30% of mono-acy-
lated pelargonidin derivates (pg-3-soph-5-glu acylated with
ferulic or p-coumaric acids only). Color measurements
of cherries and syrup showed that acylated pelargonidin
derivatives extracted from red radishes imparted red color
to maraschino cherries extremely close to that of FD&C
Red No. 40 at pH 3.5. Color and pigment stability were
evaluated during a year of storage (Fig. 6) in the dark at
20 ◦ C (cherries and syrup), and exposed to light (syrup
only). Monomeric anthocyanin degradation, measured as
the changes in monomeric pigment by the pH-differential
method, followed first-order kinetics. The half lives of
syrups colored with 60 and 120 mg monomeric anthocyanin
222 M.M. Giusti, R.E. Wrolstad / Biochemical Engineering Journal 14 (2003) 217–225
Fig. 6. Pigment and color stability of mareschino cherries colored with
radish anthocyanin extract. (a) Comparison of light and dark storage
conditions; (b) comparison of the color stability when cherries were
colored with different concentrations of anthocyanins (RAE C1 and RAE
C2: starting concentration in syrup of 60 and 120 mg/100 ml syrup).
Modified from Ref. [44].
per 100 ml syrup were found to be 29 and 33 weeks, re-
spectively. Higher anthocyanin concentration exerted a pro-
tective effect on color stability. The color characteristics of
radish-colored brined cherries were extremely close to those
with FD&C Red No. 40 for ≈6 mo storage at room temper-
ature. The high color and pigment stability obtained were
attributed to the presence of hydroxycinnamic and malonic
acid acylating groups attached to the anthocyanin moiety.
Light exposure slightly accelerated pigment degradation
(Fig. 6).
These results suggested that radishes would be an ex-
cellent alternative to the use of FD&C Red No. 40 for
maraschino cherries, even with the challenging storage con-
dition of room temperature and light exposure.
3.5. Acylated pelargonidin derivatives in model juices
The pigment stability and chromatic properties (CIEL∗ ch)
of chemically related anthocyanins obtained from red radish
(cv. Fuego) and red-fleshed potato were evaluated in bev-
erage model systems [41]. Pelargonidin-based anthocyanins
from radishes and potatoes imparted color attributes similar
to those obtained with FD&C Red No. 40 in model juice
systems. However, differences in color and pigment stability
were found during storage. Radish extracts showed higher
stability during storage than potato extracts (Fig. 7).
Storage temperature had a clear effect on the pigment
degradation kinetics of colored model juices (Fig. 7). At
25 ◦ C, higher stability was obtained for juices colored with
radish extract (22 weeks half-life) than with potato extract
(11 weeks half-life). Refrigerated temperatures drastically
decreased the rate of anthocyanin degradation with esti-
mated half-life of over a year. The degradation kinetics of
acylated anthocyanins have been reported to follow linear
[45] or non-linear rates, probably due to the folding of the
acyl moiety protecting the aglycon [45,46]. In this study,
the anthocyanin degradation during storage at 25 ◦ C fitted a
quadratic model for the different treatments. Juices stored at
refrigerated temperature showed little degradation and the
regression analysis suggested simple linear models.
The initial color characteristics of the model juices de-
pended on the type of pigment used. Radish-colored juices
showed higher chroma and hue angle (more intense and
orange-red color) than potato-colored juices. These results
are in agreement with our previous finding that malonic acid
would not cause a bathochromic shift (which would result on
a shift toward a more blue-red color) on anthocyanins typi-
cal of cinnamic acid acylation [27]. During storage the light-
ness and hue angle of samples increased, while the chroma
decreased, with the juices becoming more orange/yellow.
The presence of di-acylation in the red radish antho-
cyanin as compared to mono-acylated anthocyanins in
red-fleshed potato might be responsible for its enhanced
stability (Fig. 8). Di-acylated anthocyanins can be stabilized
by a sandwich type stacking caused by hydrophobic inter-
actions between the planar aromatic residues of the acyl
groups and the positively charged pyrylium nucleus, and
thus diminishing the formation of the pseudobase [47,48].
In the case of mono-acylated anthocyanins, only one side of
the pyrylium ring can be protected against the nucleophillic
attack of water and therefore only a weak intermolecular
effect might occur [19].
Furthermore, differences in glycosidic substituent at C3
position of the aglycon (sophorose in red radish vs. ruti-
nose in red-fleshed potato) and position of acyl group in
the sugar moiety might account for the improved stability
showed by red radish anthocyanins. We reported [49] that
in red radish the acyl group was attached to the C6 of the
glucose moiety closest to the aglycon. Although there has
not been a conformational study on the anthocyanins of
these potato clones, Andersen et al. [50] reported that in
petanin (an acylated anthocyanin from Solanum tuberosum)
the acyl group (p-coumaric) was attached to the position
C4 of the rhamnosyl moiety of rutinose. Acylation through
position 6 of the sugar allows for free rotation of the acyl
group, allowing for folding of the molecule and intramolec-
ular stacking. The flexible saccharide chains can act as
linkers allowing the folding of the acyl aromatic rings over
the planar pyrylium ring. Formation of a sandwich type
complex has been proposed for anthocyanins with two cin-
namic acid acylating groups. This stacking phenomenon
excerpts a protective effect on anthocyanins but also con-
tributes to the color stabilization of the system (Fig. 8). It
has been proposed that cinnamic acid residues stack parallel
 M.M. Giusti, R.E. Wrolstad / Biochemical Engineering Journal 14 (2003) 217–225 223

Fig. 7. Pigment and color stability of model beverages (pH 3, 0.1 M citrate solution, 10◦ Brix) colored with radish or red potato anthocyanin extracts,
under refrigerated or ambient temperature storage. Modified from Ref. [41].

with the anthocyanidin nucleus protecting the chromophore
against water nucleophillic attack [51]. Two-dimensional
NMR spectroscopy analyses (NOESY) performed on radish
pigments [49] showed close spatial proximity between hy-
drogens from the flavylium cation and the acyl group. These
correlations are in agreement with the proposed fold-
ing of anthocyanins related to intramolecular copigmen-
tation.
3.6. Anthocyanins in dairy systems
Anthocyanins undergo reversible structural transforma-
tions with changes in pH, which is accompanied by dra-
matic changes in color. At pH of 3 or below, the color of
anthocyanins ranges from orange to bluish-red, depending
on the chemical structure, and exists predominantly as a
flavylium cation. As the pH is raised, hydration and proton

Fig. 8. Stabilization mechanisms of acylated anthocyanins—proposed spatial configuration and stacking of acylated anthocyanins. Modified from Ref. [20].
224 M.M. Giusti, R.E. Wrolstad / Biochemical Engineering Journal 14 (2003) 217–225
transfer reactions can occur with the generation of a number
of different chemical structures: the first reaction produces a
colorless carbinol pseudobase which can undergo ring open-
ing to a chalcone pseudobase, the latter reactions give rise
to quinonoidal bases, with formation of purplish quinonoid
anions after further deprotonation [52].
Due to these transformation with pH, anthocyanin appli-
cation for food systems were typically sought in acidic food
(pH < 3), to assure a predominance of the flavylium cation.
Reported by different researchers is the ability of acylated
anthocyanin (mostly di- or poly-acylated) to resist hydra-
tion, and hence posses higher color stability to pH changes,
exhibiting coloration on weakly acidic, neutral and slightly
alkaline solutions.
We tested the viability of using acylated anthocyanins
from edible sources to color dairy products such as yogurt
and sour cream, with pH levels ∼4.2–4.5. Extracts evaluated
included red radish, red cabbage, black carrot, and grape
skin extract. Preliminary results showed that radish and car-
rot alone or in combination could provide desirable red hue
to these systems at concentrations as low as 5 mg monomeric
anthocyanin per 100 g sample. Color measurements and vi-
sual appearance showed higher color intensity in samples
colored with black carrot, as compared to the radish-colored
samples. Red cabbage gave an attractive purple color, resem-
bling the color of blueberries. We also found that fat content
had an effect on color characteristics (data not shown), sug-
gesting that fat content may affect the copigmentation and
stacking of the acylated anthocyanin pigments. The shelf life
of these dairy products is only a few weeks under refrigera-
tion, and these conditions would favor the stability of these
natural pigments, making them viable alternatives.
In the pH region of 5–6, anthocyanins are unstable and
quickly decolorize by hydration at the two-position of the
anthocyanidin skeleton [52]. We found evidence that the
chroma of some acylated pelargonidin derivatives increased
when the pH was further increased to neutral conditions
(pH 7). Fossen et al. [42] reported that acylated petunidin
derivatives exhibited color intensity in the neutral to slightly
alkaline region even higher than at low pH values. These
results suggest that other potential applications for certain
acylated anthocyanins may be in neutral or slightly alkaline
products.
3.7. Anthocyanins: value-added natural colorants
Acylated anthocyanins have shown to be promising al-
ternatives to the use of synthetic dyes in food systems. De-
pending on anthocyanin chemical structure and pH of food
matrix, a wide variety of hues can be achieved. Anthocyanin
acylation improves color and pigment stability. Acylated
pelargonidin derivatives from radish and potato can closely
match the color characteristics of allura red under acidic
conditions. Other acylated anthocyanidin derivatives, such
as cyanidin from black carrots or cabbage, can provide hues
ranging from deep red to purple, depending on the pH. Al-
though anthocyanins undergo transformations with pH, and
typically are more colored at pH of 3 or below, a number of
applications in foods with higher pH levels are also viable
when the “right” extract is chosen.
A number of studies point out a relationship between con-
sumption of anthocyanin-rich foods and improved health.
Optimizing health and performance through the diet is be-
lieved to be one of the largest and most lucrative markets in
the US. The Food Marketing Institute has estimated the “do
it yourself health” food market as a US$ 42 billion retail op-
portunity [53]. There is an increased market for nutraceutical
or medicinal foods and consumers are interested in foods that
may help to prevent or reduce the incidence of illness. The
potential impact of health benefits on the demand for spe-
cific foods has been well illustrated by the dramatic increase
in US consumer consumption of soy foods and soy-based
food ingredients. The near doubling from 1998 to 2000, and
projected future tripling of soy food consumption, has been
attributed to reported health benefits of soy including pre-
vention of cancer, osteoporosis and coronary heart disease
[54]. Therefore, identification of health-promoting compo-
nents in anthocyanin extracts would increase the demand of
these commodities by consumers and the food industry as
added value natural pigments, and would open a new win-
dow of opportunities for use of these extracts in a variety of
food applications.
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