Eur Food Res Technol (2012) 234:207–222
DOI 10.1007/s00217-011-1601-y
ORIGINAL PAPER
Impact of enzymatic mash maceration and storage on anthocyanin
and color retention of pasteurized strawberry purées
Melanie Holzwarth • Sabine Korhummel •
Reinhold Carle • Dietmar R. Kammerer
Received: 1 August 2011 / Revised: 22 September 2011 / Accepted: 30 September 2011 / Published online: 26 November 2011
Ó Springer-Verlag 2011
Abstract Strawberry purées were prepared using a
commercial polygalacturonase (PG) and a highly purified
pectinesterase (PE) preparation, respectively. To elucidate
the effect of pectin on color stability following enzymatic
pulp maceration, pectin composition was studied by iso-
lating and fractionating the alcohol-insoluble residue from
the strawberry purées. The purées were stored at ?20 and
?4 °C in the dark over a period of 24 weeks monitoring
the amounts of monomeric and polymeric anthocyanins as
well as antioxidant activities (FRAP, TEAC). Individual
anthocyanins were analyzed by HPLC–DAD–MSn, and
color measurements were obtained in the CIE L*a*b*
system. Pectin composition was significantly modified
following enzymatic maceration of the purées. While PG
treatment generally resulted in pectin losses, oxalate-solu-
ble pectins were increased in PE-treated purées. After
24 weeks of storage, the best anthocyanin retention was
observed in PE-treated purées. Such products also revealed
greatest anthocyanin half-life values and lowest proportion
of polymeric pigments. Compared to an untreated control,
enzymatic purée maceration using the PG was also bene-
ficial for pigment retention, but less effective than PE. In
contrast, color and antioxidant activity were independent of
both enzymatic treatments. An initial heating step (90 °C,
10 s) for immediate inactivation of native enzymes such as
polyphenoloxidases slightly improved pigment stability,
while lowered temperature during mash maceration was
less effective. However, by far best color and pigment
M. Holzwarth  S. Korhummel  R. Carle 
D. R. Kammerer (&)
Institute of Food Science and Biotechnology,
Chair Plant Foodstuff Technology, Hohenheim University,
Garbenstrasse 25, 70599 Stuttgart, Germany
e-mail: Dietmar.Kammerer@uni-hohenheim.de
retention were achieved when the purées were stored at
4 °C in the dark.
Keywords Strawberry anthocyanins  Purée processing 
Color stability  Storage  Antioxidant activity
Introduction
Strawberries (Fragaria 9 ananassa Duch.) belong to the
most important berry fruits used for industrial food pro-
cessing, therefore being of high economic relevance [1, 2].
For the production of smoothies, drinks, and desserts, they
are mostly processed into purées. To enhance yields, mash
maceration using pectin-degrading commercial enzyme
preparations is usual. Besides flavor, consumers are pri-
marily attracted by the brilliant red color being considered
as an indicator of high product quality and freshness.
Anthocyanins, mainly pelargonidin 3-O-glucoside, pelar-
gonidin 3-O-malonyl-glucoside, and cyanidin 3-O-gluco-
side, are responsible for the appealing color shade of
fresh strawberries [3–5]. Unfavorably, the natural red
color of strawberries is considerably degraded during
processing and storage because of the instability of their
anthocyanins toward temperature, light, pH, and oxygen
[6]. Therefore, synthetic dyes exerting superior stability
are commonly applied to compensate for pigment losses
and retain initial color properties. However, some azo
dyes have been shown to negatively affect human health
and are thus rejected by an increasing number of con-
sumers. Moreover, according to a recent study [7], con-
sumption of beverages containing synthetic colorants and
preservatives was associated with an increase in hyper-
activity in children. Consequently, food industry aims at
substituting synthetic dyes by stabilizing natural pigments,
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the latter also contributing to consumer health and well-
being [8].
Anthocyanin stability is supported by intra- and inter-
molecular co-pigmentation, and self-association of
anthocyanins has also been shown to protect the pigments
from nucleophilic water attack [8]. Furthermore, inter-
molecular co-pigmentation in the presence of low-
molecular matrix compounds, such as organic acids,
flavonoids, amino acids, alkaloids, or nucleotides, may
stabilize the chromophoric flavylium cation [9]. However,
little attention has been paid so far to the interaction
between anthocyanins and macromolecular cell wall
compounds like pectin. Stabilizing electrostatic interac-
tions between dissociated carboxylic groups of the pectin
backbone and the positively charged flavylium cation
have been assumed [10, 11]. The intense color of blue
cornflower (Centaurea cyanus L.) was attributed to a high
molecular weight complex composed of the cyanidin 3,5-
O-diglucoside (cyanin) anhydro base and metal ions
associated with a macromolecular compound mainly
consisting of galacturonic acid [12]. Hydrophobic inter-
actions between methoxyl groups of the B-ring and pectin
methoxyl groups were suggested by Mazzarachio et al.
[13]. A stabilizing effect of pectin was also assumed by
the latter group to be a consequence of its ability to
immobilize water. In accordance with this assumption,
high sugar concentrations were also reported to enhance
anthocyanin stability due to water immobilization and the
reduction in water activity [8, 14–16].
Although pectin may have positive effects on anthocy-
anin stability, juice extraction and purée production are
mostly supported by enzymatic mash maceration using
pectin-degrading enzymes [17, 18]. In a previous study,
single strength juice prepared from fresh strawberries
exhibited greater anthocyanin stability than juice prepared
from concentrate [19]. Poorer pigment retention was
assumed to be due to the enzymatic degradation of pectin
prior to juice concentration indicating that depletion of
pectin is disadvantageous for pigment and color stability.
In contrast, Hartmann et al. [18] reported slightly improved
anthocyanin retention in purées after 11 weeks of storage
when strawberries were macerated using a pectinase
preparation exerting mainly PG activity. Thus, apart from
deleterious b-glucosidase side effects [20], only little is
known about the impact of enzymatic pulp maceration on
anthocyanin stability. Furthermore, no attention has so far
been paid to potential stabilizing interactions between cell
wall components resulting from enzymatic digestion and
strawberry anthocyanins. Therefore, the objective of the
present study was to analyze the pectin composition of
enzyme-treated strawberry purées to elucidate potential
correlations between pectin composition and pigment
stability.
123
Eur Food Res Technol (2012) 234:207–222
Many investigations into the impact of processing and
storage on the stability of strawberry anthocyanins have
been performed. In particular, harsh heat treatment has
been reported to significantly diminish anthocyanin con-
tents in strawberry products throughout processing [17, 18,
21–23]. Furthermore, genuine pigment-degrading enzymes,
mainly polyphenol oxidases and b-glucosidases, were
shown to significantly contribute to anthocyanin losses in
strawberries [23–26].
Therefore, the effect of an initial high-temperature
short-time heating step to inactivate crucial enzymes and
improve color stability during processing and storage
should also be assessed in the present study. Furthermore,
the impact of processing and storage conditions on antho-
cyanin stability, color retention, and antioxidant activities
of pasty strawberry products should be evaluated.
Materials and methods
Materials
Strawberries
Individually quick-frozen (IQF) strawberries (Fragar-
ia 9 ananassa Duch. cv. ‘‘Senga sengana’’) harvested 2008
in Poland were obtained from Odenwald Früchte (Breuberg,
Germany). Entire frozen fruits were stored at -18 °C.
Enzymes
The following preparations were used for the enzymatic
maceration of fruit pulp and were kindly provided by
Erbslöh (Geisenheim, Germany): Vegazym M (polygalac-
turonase activity, PG) and VP 0956/1 (highly purified
pectinesterase, PE).
Chemicals
All reagents and solvents were of analytical HPLC grade and
purchased from VWR (Darmstadt, Germany). For the char-
acterization and quantification of individual anthocyanins by
HPLC–DAD–MSn, cyanidin 3-O-glucoside chloride and
pelargonidin 3-O-glucoside chloride (Extrasynthèse, Lyon,
France) were applied as standards.
ABTS [2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfo-
nate)], trolox [6-hydroxy-2,5,7,8-tetra-methyl-chroman-2-
carboxylic acid], ABAP [2,20 -azobis(2-amidino-propane)
dihydrochloride], and TPTZ-Fe(II) [2,4,6-tri(2-pyridyl)-s-
triazine] were purchased from Sigma (St. Louis, MO,
USA). Sodium metabisulfite, potassium chloride, and sodium
acetate trihydrate were obtained from VWR (Darmstadt,
Germany).
Eur Food Res Technol (2012) 234:207–222
Methods
Preparation of strawberry pure´es
Strawberry purées were produced in quantities of 2.5 kg.
For this purpose, IQF strawberries were thawed overnight
at 4 °C and passed through a finisher (type PAP 0533,
Bertuzzi, Brugherio, Italy) fitted with a sieve of 1.5 mm
mesh size. Potassium sorbate (1 g/kg) was added to ensure
microbiological stability of the purées prior to enzymation
with PG and PE, respectively. The purées were heated to
25 and 45 °C, respectively, in a reaction vessel (EL 3,
ESCO-Labor, Riehen, Switzerland). After 90 min of
digestion, the mash was pasteurized at 85 °C for 15 min in
the double-walled vessel and subsequently filled in 100 g
glass jars. Purée variants were prepared by initial heating
(90 °C/10 s) prior to digestion to inactivate native
enzymes. Control samples were prepared without enzy-
matic digestion. After adding potassium sorbate (1 g/kg),
the strawberry mash was heated at 45 °C for 90 min prior
to pasteurization at 85 °C for 15 min to determine the
effect of thermal treatment.
Dry matter contents of the purées were determined using
an MA 40 electronic moisture analyzer (Sartorius,
Göttingen, Germany), pH values were measured with a
Metrohm 691 pH meter (Herisau, Switzerland), and water
activity was determined using an Aqua Lab CX-2 water
activity meter (Decagon, Hopkins, USA) (Table 1).
Sample storage
Strawberry purées were stored in transparent glass jars at
4 ± 2 and 20 ± 2 °C, respectively, in the dark over a
period of 24 weeks. Samples were drawn at regular inter-
vals of 1–4 weeks to quantify total and individual antho-
cyanins and for spectrophotometric and color analyses.
Determination of cell wall composition
Alcohol-insoluble residue Strawberry purées were freeze-
dried and ground with a Grindomix GM 200 knife mill
Table 1 Water activities, pH values, and dry matter contents of strawberry purées obtained by enzymatic mash maceration with polygalac-
turonase (PG) and pectinesterase (PE), respectively, compared to a pasteurized control prepared without enzyme addition
Water activity (aW)
Controla 1.001 ± 0.001 A
PGb 0.993 ± 0.002 A
PEb 1.002 ± 0.002 A
Different capital letters (vertical) illustrate significant differences (p \ 0.05)
a
Heated strawberry purée prepared without enzyme addition (45 °C, 90 min)
b
Strawberry purée treated with polygalacturonase (PG) and pectinesterase (PE), respectively (45 °C, 90 min)
209
(Retsch, Haan, Germany). The lyophilisates (5 g) were
homogenized in boiling aqueous ethanol (200 mL, 80%,
v/v) using an Ultra-Turrax blender. After boiling for 1 h,
insoluble solids were collected on a Büchner funnel. The
extraction was repeated five times until a colorless extract
was obtained. The residue was stirred overnight in acetone,
passed through a G1 glass filter and dried at 50 °C for 24 h.
Isolation of alcohol-insoluble residue (AIR) was performed
in triplicate. The resulting AIR preparations were weighed
and pooled for sequential extraction.
Sequential extraction of AIR The sequential extraction
was performed according to a method described previously
[27, 28]. AIR (0.5 g) was suspended in 50 mL of de-ion-
ized water and stirred at 40 °C for 30 min. After centri-
fugation at 6,600g for 10 min, the pellet was washed twice
with de-ionized water. The supernatants were pooled,
dialyzed exhaustively against de-ionized water for 48 h
using dialytic membranes (type 36/32, pore size 25–50 Å,
Roth, Karlsruhe, Germany). Subsequently, the water-solu-
ble pectin (WSP) fraction was freeze-dried using a Lyovac
GT4 (Finn-Aqua Santasalo-Sohlberg, Hürth, Germany).
Further extraction of the residue was performed with
50 mL of ammonium oxalate (0.5%, w/v) at 40 °C for
90 min. The suspension was centrifuged at 6,600g for
15 min and washed twice with de-ionized water. The
supernatants were pooled, dialyzed against de-ionized
water for 48 h, and freeze-dried to obtain the oxalate-sol-
uble fraction (OXP). The residue was subsequently
extracted with 50 mL of hydrochloric acid (0.05 mol/L) at
60 °C for 90 min. The homogenate was centrifuged at
6,600g for 15 min, and the remaining pellet washed twice
with de-ionized water. Dialyzation (48 h) and lyophiliza-
tion yielded the HCl-soluble pectin fraction (HSP). The
residue was then extracted with 50 mL of aqueous sodium
hydroxide (0.05 mol/L) at 5 °C for 180 min. After centri-
fugation at 6,600g for 15 min, the pellet was washed twice
with de-ionized water. The supernatants were pooled,
adjusted to pH 7.0 with HCl to yield the alkali-soluble
(OHP) fraction. This extract was finally treated as descri-
bed for the WSP, OXP, and HSP fractions. The final
pH value Dry matter content (%)
3.60 ± 0.01 AB 7.71 ± 0.11 B
3.62 ± 0.01 A 8.48 ± 0.07 A
3.58 ± 0.01 B 8.52 ± 0.12 A
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extraction was carried out with 50 mL of aqueous sodium
hydroxide solution (16%, w/w) at 30 °C for 5 h yielding
the hemicellulose fraction (HC), which was further treated
as described for the OHP fraction. The remaining pellet
consisted of insoluble lignin and cellulose (C fraction). The
pellet was suspended in 50 mL of de-ionized water, dia-
lyzed, and lyophilized. Sequential extraction of AIR was
performed in duplicate.
Extraction of anthocyanins
Aliquots of 2.5 g of the purées were weighed into Erlen-
meyer flasks and extracted with 25 mL of acidified meth-
anol (0.01% HCl, v/v) by stirring for 60 min. After
centrifugation at 4,000 rpm (3,309g) for 20 min, the
supernatant was collected and the insoluble residue was
re-extracted exhaustively with 25 mL of the same solvent
by stirring for 30 min. The suspension was filtered through
a Whatman No. 4 paper (Maidstone, UK). The combined
filtrates were evaporated to dryness in vacuo at 30 °C, and
the residue was dissolved in 10 mL of formic acid (5%,
v/v) and membrane-filtered (0.45 lm).
Determination of individual anthocyanins
by HPLC–DAD–MSn
HPLC system The determination of individual anthocya-
nins was performed using an Agilent HPLC series 1100
(Agilent, Waldbronn, Germany), a Phenomenex (Torrance,
CA, USA) Synergi 4u Hydro-RP 80A C18 column
(150 9 3.0 mm i.d.; 4 lm particle size) with a C18 ODS
guard column (4.0 9 2.0 mm i.d.) at a temperature of 25 °C
and a flow rate of 0.4 mL/min. Eluent A was 5% aqueous
formic acid, and acidified methanol (5% formic acid, v/v)
was used as eluent B. The gradient program was as follows:
10% B to 25% B (10 min), 25% B to 31% B (5 min), 31% B
to 40% B (5 min), 40% B to 50% B (10 min), 50% B to 100%
B (5 min), 100% B isocratic (5 min), 100% B to 10% B
(2 min), 10% B isocratic (3 min). Total run time was 50 min,
and the injection volume was 20 lL. Anthocyanins were
monitored at 520 nm.
HPLC–MSn system For peak assignment, LC–MS anal-
yses were performed with the HPLC system described
above coupled to a Bruker (Bremen, Germany) model
Esquire 3000? ion trap mass spectrometer fitted with
electrospray ionization (ESI) interface. Positive ion mass
spectra of the column eluate were recorded in the range of
m/z 50–1,500 at a scan speed of 13,000 m/z units/s.
Nitrogen was used both as drying gas at a flow rate of
9 L/min and as nebulizing gas at a pressure of 40 psi. The
nebulizer temperature was set at 365 °C. Helium was used
as collision gas at a pressure of 4.9 9 10-6 mbar.
123
Eur Food Res Technol (2012) 234:207–222
Quantification of individual anthocyanins by HPLC–
DAD Individual anthocyanins were quantified using cal-
ibration curves of the corresponding standard compounds.
In case of lacking standards, the calibration of structurally
related compounds was used and corrected by a molecular
weight factor [29].
Half-life values T1/2 of individual anthocyanins and total
anthocyanins calculated as sum of individually quantified
compounds were determined by monitoring the rate of
anthocyanin contents in purée samples directly after pro-
cessing (A0) and samples stored at 20 and 4 °C in the dark
for 7–168 days (At). The natural logarithm of At/A0 was
plotted against the storage period. The slope of this graph
was equated with k, from which half-life values (T1/2 = ln
2/k) were calculated.
Color analysis and determination of antioxidant activity
Color measurements of the strawberry purées were per-
formed using a Minolta Chromameter CR-300 (Konica
Minolta, Osaka, Japan). For monomeric anthocyanin
quantification and polymeric color measurements, a UV–
Vis photometer (Perkin-Elmer, Überlingen, Germany)
equipped with UV–Vis (UVWinLab V 2.85.04) and color
(Wincol V 2.05) software (Perkin Elmer Instruments,
Norwalk, CT, USA) and 1 cm path length disposable cells
were used. TEAC and FRAP assays were performed using
a Biotek microplate spectrophotometer (Biotek, Power
Wave XS, Bad Friedrichshall, Germany) equipped with
Gen5 software (Ver.1.04.5).
Monomeric anthocyanin contents Determination of
monomeric anthocyanin contents was based on the pH
differential method [30]. Data were expressed as pelar-
gonidin 3-O-glucoside equivalents and calculated accord-
ing to the following equation:
c½mg=L ¼ A  MW  DF  1; 000=eM  d ð1Þ
with A, absorption; MW, molecular weight of pelargonidin
3-O-glucoside [433 g/mol]; eM, molar extinction coefficient
of pelargonidin 3-O-glucoside at pH 1 [15,600 L/mol cm];
DF, dilution factor; and d, pathlength of the cuvette [1 cm].
Polymeric color measurements The proportion of poly-
meric anthocyanins was determined according to Giusti
and Wrolstad [30]. For the determination of polymeric
color P (Eq. 2), aliquots of the samples were mixed with
0.2 mL of a metabisulfite solution (200 g K2S2O5/L). For
color density measurements (Eq. 3), the metabisulfite
solution was substituted by 0.2 mL of de-ionized water.
After 20 min of equilibration, absorbance values were
measured at 420, 498 (kmax), and 700 nm, respectively, and
Eur Food Res Technol (2012) 234:207–222
the proportion of polymeric pigments was determined
according to Eq. 4.
Polymeric color
P ¼ ½ðA420 nm  A700 nm Þ þ ðA498 nm  A700 nm Þ  DF ð2Þ
Color density
K ¼ ½ðA420 nm  A700 nm Þ þ ðA498 nm  A700 nm Þ  DF ð3Þ
Percent polymeric color
PC ¼ P=K  100 ð%Þ ð4Þ
Antioxidant activity: TEAC assay A phosphate buffer (pH
7.4) was prepared by mixing 812 mL of a Na2HPO4
solution (0.066 mol/L) with 182 mL of a KH2PO4 solution
(0.066 mol/L) and 8.766 g of sodium chloride. For the
preparation of the radical solution, 0.5 mL of an ABTS
solution (20 mmol/L) in phosphate buffer (pH 7.4) and
100 mL of an ABAP solution (2.5 mmol/L) were mixed
and heated at 60 °C for 15 min in the dark. The reaction
was initiated by adding 200 lL ABTS? solution to 40 lL
of sample solution or 40 lL of water as a control. After
6 min at ambient temperature, the absorption of the sample
solution was measured at 734 nm. Aqueous trolox solu-
tions in a concentration range of 1.17–18.75 mg/L were
used as standards for calibration. The values were expres-
sed as lmol of trolox equivalents per 100 g of sample.
Antioxidant activity: FRAP assay The FRAP reagent was
prepared by mixing 25 mL of an acetate buffer (300 mmol/
L, pH 3.6) with 2.5 mL of a TPTZ solution (10 mmol/L) in
hydrochloric acid (40 mmol/L) and 2.5 mL of a FeCl3
solution (20 mmol/L). The reaction was started by adding
150 lL of FRAP reagent to 20 lL of sample solution.
After 4 min at ambient temperature, the absorbance was
measured at 593 nm. Aqueous trolox solutions in a con-
centration range of 9.38–150 mg/L were used as standards
for calibration. The values were expressed as lmol of
trolox equivalents per 100 g of sample.
Statistical analysis
Significant differences (a = 0.05) were determined using
the Tukey test for differences between independent sam-
ples. Data evaluation was performed using the SAS soft-
ware package (SAS Institute, Cary, NC, USA; v. 9.1).
Results and discussion
To evaluate the color-stabilizing effect of the complex
pectin matrix, the AIR composition of pasteurized straw-
berry purées produced with and without enzymatic mac-
eration using PG and PE preparations, respectively, has
211
been analyzed. Additionally, strawberry purées were ana-
lyzed prior to enzymatic and thermal treatment to assess
the influence of both process steps on AIR composition.
Isolation of AIR
The AIR contents determined prior to and after enzymatic
digestion of the strawberry purées are specified in Table 2.
Undigested IQF strawberries yielded about 13.5% of AIR.
These findings are in good agreement with those of pre-
vious studies reporting AIR contents of strawberries to vary
widely in a range of 12 and 37% (based on fresh fruit
weight) corresponding to 15.6 and 25.4% on a dry matter
base, respectively, which markedly depends on species,
cultivar, degree of ripeness, and storage conditions [27, 31,
32]. Total amounts of AIR following enzymatic treatment
with PG and PE preparations significantly differed. While
PG treatment significantly lowered the AIR content
(12.0%) due to the rapid degradation of pectins by endo-
activities, the PE treatment even resulted in significantly
higher values (14.7%). Higher AIR yields may be attrib-
uted to cross-linked calcium chelate complexes included in
the AIR, since de-esterified pectins may form high
molecular alcohol-insoluble chelate complexes in the
presence of calcium ions [33, 34].
Sequential extraction of AIR
To differentiate the impact of enzymatic treatment on cell
wall components, the AIR was sequentially extracted from
strawberry purées with water, ammonium oxalate, hot
diluted hydrochloric acid, cold aqueous sodium hydroxide
solution (0.05 mol/L), and aqueous sodium hydroxide
solution (16%, w/w). While pectin with a high degree of
esterification (DE), mainly located in the middle lamella of
the cell wall, is solubilized by water, low-esterified pectins
disposing of abundant free carboxylic groups are extracted
by ammonium oxalate. Subsequent treatment with hydro-
chloric acid allows the removal of protopectin by cleaving
glycosidic linkages. Alkali-soluble pectic compounds are
extracted with aqueous sodium hydroxide (0.05 mol/L).
Finally, hemicellulosic compounds are extracted with
aqueous sodium hydroxide (16%, w/w). The remaining
insoluble substance consists of minor amounts of cellulose
and lignified cell wall material [28].
The AIR composition of strawberry purées prior to and
after enzymatic maceration is specified in Table 2. Total
pectins accounted for *60% of the AIR in undigested IQF
strawberries, while previous studies revealed lower
amounts of pectic compounds for strawberries of the cul-
tivar ‘‘Senga sengana’’, ranging from 37.5 to 51.5% [27,
31]. These differences may be explained by differing
ripeness degrees [35, 36]. WSP was the predominant pectin
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212 Eur Food Res Technol (2012) 234:207–222

Table 2 Yields and composition of the alcohol-insoluble residue (AIR) from IQF strawberries and strawberry purées obtained by enzymatic mash maceration with polygalacturonase (PG) and

3.9 ± 1.1 A
3.7 ± 0.1 A
5.1 ± 0.5 A
3.5 ± 0.6 A
4.3 ± 0.4 A
4.7 ± 1.1 A
fraction with proportions ranging from 30.2 to 30.4%. In
agreement with a former study [27], OXP and HSP frac-
tions accounted for about 10% each, OHP fractions ranged
C from 7.1 to 10.2% of the AIR, and HC fractions were
*10%. C fractions from IQF strawberries were about
8.7 ± 0.5 AB
4.4%. In former studies, higher contents of the C fraction
11.2 ± 1.3 A

9.5 ± 0.9 A

10.3 ± 1.3 A
10.2 ± 0.2 A
5.7 ± 0.9 B
were reported for cv. ‘‘Senga sengana’’ ranging from 23.2
to 33.1% [27, 31]. This may also be attributed to different
maturity [36] or different cultivation methods, since
HC

strawberries grown under elevated salinity levels were

shown to have lower C and HC contents [37].
Compared to unheated IQF strawberries, thermal treat-
10.2 ± 0.2 A
7.9 ± 0.7 A
9.0 ± 0.1 A

7.1 ± 0.4 A
8.7 ± 2.0 A
2.0 ± 0.2 B

IQF strawberries used as raw materials for preparing the control and the strawberry purées obtained by enzymatic mash maceration, respectively
ment without enzymation did not affect the pectin com-

Strawberry purées obtained by enzymatic mash maceration with polygalacturonase (PG) and pectinesterase (PE), respectively (45 °C, 90 min)
position of strawberry purées. A slight but insignificant
OHP

WSP water-soluble pectin, OXP oxalate-soluble pectin, HSP acid-soluble pectin, OHP alkali-soluble pectin, HC hemicellulose, C cellulose

increase in WSP contents from 30.4 to 34.2% was observed

when heating at 45 °C for 90 min, whereas HSP and OHP
fractions were decreased in the macerated strawberry pu-
9.2 ± 0.3 AB

rée. This is presumably due to the enhanced solubilization

8.1 ± 0.2 BC
10.1 ± 0.3 A

9.4 ± 0.7 A
1.5 ± 0.2 D

7.4 ± 0.0 C

of protopectins by activated native macerating enzymes

forming water-soluble highly polymerized pectins [34]. In
HSP

contrast, the proportions of OXP, HC, and C remained

virtually unchanged upon moderate heating of the control
sample.
As expected, PG treatment (45 °C, 90 min) resulted in
18.3 ± 0.5 A
10.5 ± 0.1 B
11.5 ± 0.4 B
10.9 ± 0.1 B
2.2 ± 0.5 C
11.0 ± 1.2 B
pectinesterase (PE), respectively, compared to a pasteurized control prepared without enzyme addition

significant changes of the pectic fractions from the straw-

berry purée. The proportions of the OXP, HSP, and OHP
OXP

fractions decreased markedly. Moreover, an increase in

WSP due to enzymatic solubilization of protopectin, as
described for the control, was observed. However, since
34.2 ± 0.3 AB

high-esterified pectins of the WSP fraction were not

38.1 ± 1.9 A

35.7 ± 1.2 A
30.4 ± 0.9 B

30.2 ± 0.6 B

30.2 ± 0.2 B

degraded by this enzyme preparation, the PG used in the

present study was confirmed to exhibit selective PG
Different capital letters (vertical) illustrate significant differences (p \ 0.05)
WSP

activity as specified by the manufacturer. DE of pectins in

the AIR of strawberries was found to be 63% [33], whereas
Heated control prepared without enzyme addition (45 °C, 90 min)

for WSP, values ranging from 52.9 to 94.4% have been

70.1 ± 3.7 A
61.2 ± 1.4 B
61.6 ± 1.5 B
59.5 ± 1.5 B
43.8 ± 2.7 C
57.4 ± 2.0 B

reported, strongly depending on the cultivar [38, 39].

Total pectin
Composition of AIR (g/100 g AIR)

Proportions of the HC fraction slightly decreased from 9.5

to 5.7% upon treatment with PG. This might be attributed
to hemicellulosic side activities of the PG preparation,
while the C fraction was not affected by this enzyme
preparation.
AIR (g/100 g DM)

The application of PE (45 °C, 90 min) also resulted in

13.8 ± 0.6 AB
13.9 ± 0.2 AB
13.4 ± 0.7 AB

14.7 ± 0.2 A

significant changes of the pectin composition of strawberry

12.0 ± 0.3 C
13.3 ± 0.5 B

purées. As expected, due to de-esterification of strawberry

pectin, relatively high OXP proportions were observed
resulting in the formation of calcium reactive oxalate-sol-
uble polygalacturonic acids [33]. Again, WSP proportions
increased during enzymatic treatment as previously
IQF strawberriesa

IQF strawberriesa

IQF strawberriesa

observed for the control and PG -treated sample owing to

the solubilization of protopectins as a result of genuine
enzyme activities. Furthermore, the amounts of HSP were
Controlb
Sample

slightly decreased, while OHP, HC, and C fractions

PGc

PEc

remained unchanged following PE treatment.

b
a

123
Eur Food Res Technol (2012) 234:207–222 213

Characterization of strawberry anthocyanins mAU

800 2

Individual anthocyanins were tentatively assigned by

700
HPLC–DAD–MSn (Table 3) using the chlorides of
pelargonidin 3-O-glucoside and cyanidin 3-O-glucoside as 600
standards. The anthocyanin profile of the strawberry

Absorption
extracts (Fig. 1) and the fragmentation patterns of indi- 500

vidual compounds in mass spectrometric experiments were

in accordance with previous reports [4, 5]. Compound 1
was identified as cyanidin 3-O-glucoside (cya 3-O-glc, 3%)
exhibiting an [M?] ion at m/z 449 releasing cyanidin as
fragment ion in the MS2 experiment at m/z 287. The pre-
dominant anthocyanin (compound 2) accounting for
*77% of total anthocyanins was pelargonidin 3-O-gluco-
side (pel 3-O-glc) exhibiting an [M?] ion at m/z 433 and
releasing pelargonidin as fragment ion at m/z 271. Com-
pound 3 was assigned to pelargonidin 3-O-rutinoside (pel
3-O-rut, traces) based on its [M?] ion at m/z 579 and
fragment ion at m/z 271. Compounds 4 and 5 were tenta-
tively assigned to cyanidin 3-O-malonyl-glucoside (cya
3-O-mal-glc, 7%) and pelargonidin 3-O-malonyl-glucoside
(pel 3-O-mal-glc, 14%) on the basis of their molecular ions
and fragmentation patterns in the MS2 experiments
(Table 3).
Impact of processing on anthocyanin and color stability
of strawberry purées
Total anthocyanins
Strawberry purées were incubated at 45 °C for 90 min with
and without enzyme addition prior to pasteurization at
85 °C for 15 min in a reaction vessel. As can be seen from
Table 4, total anthocyanin amounts calculated as the sum
of the predominant compounds pel 3-O-glc, pel 3-O-mal-
glc, and cya 3-O-glc in IQF strawberries ranged from 538.4
to 632.6 mg/kg. The incubation at 45 °C caused a decrease
in total anthocyanins, which was assumed to result from
enzymatic degradation of pel 3-O-glc. Since pelargonidin
400
300
200
5
1
100
3 4
6
0
15 20 25 30
10 min
Retention time
Fig. 1 HPLC separation (520 nm) of anthocyanins from an extract
from strawberry purée prepared without enzyme addition. For peak
assignment see Table 3
was not detected in macerated strawberry purées, loss of its
glycosides could not be ascribed to de-glycosylation reac-
tions induced by endogenous b-glucosidase activities.
Therefore, it is supposed that pigment losses during mac-
eration resulted from the presence of polyphenol oxidases
(PPO), which are mainly made responsible for strawberry
anthocyanin degradation [23, 25].
Interestingly, major anthocyanin degradation (11.8%)
was observed in the control sample without enzyme addi-
tion, whereas the addition of PE and PG only resulted in
pigment losses of 2.8 and 9.3%, respectively. Thus,
anthocyanin degradation caused by native enzyme activi-
ties was overcompensated by the effects of PE and PG
activities, presumably due to stabilizing interactions
between the pigments and the enzymatically modified
pectin molecules. However, after pasteurization, total
anthocyanin contents and contents of pel 3-O-glc both in

Table 3 HPLC-DAD-MSn data of anthocyanins from strawberry purée extracts

Peak Retention kmax MS1 [M?] MS2 (m/z; % base peak) Peak assignment
time (min) (nm) (m/z)

1 15.5 516, 281 449 MS2 [449]: 287 (100) Cyanidin 3-O-glucosidea
2
2 17.1 500, 268 433 MS [433]: 271 (100), 272 (13) Pelargonidin 3-O-glucosidea
3 18.6 507, 246 579 MS2 [579]: 271 (100), 433 (18), 434 (14) Pelargonidin 3-O-rutinoside
4 21.4 517, 246 535 MS2 [535]: 287 (100), 449 (28), 288 (19), 450 (10) Cyanidin 3-O-malonyl-glucoside
5 22.8 505, 246 519 MS2 [519]: 271 (100) Pelargonidin 3-O-malonyl-glucoside
6 24.1 508, 246 461 MS2 [461]: 271 (100) Pelargonidin derivative
a
Identification with a standard compound

123
214
Table 4 Total anthocyanin contents (mg/kg fresh weight) of IQF strawberries and strawberry purées prepared with and without enzyme addition
including an initial heating for the inactivation of genuine enzymes
Enzymes Process parameters IQF strawberries
PG 25 °C/90 min 576.2 ± 6.9 AB
45 °C/90 min 587.2 ± 6.6 AB
PE 25 °C/90 min 632.6 ± 5.0 A
45 °C/90 min 611.4 ± 4.9 A
Control ? initial heating 45 °C/90 min 604.0 ± 23.6 A
90 °C/10 s; 45 °C/90 min 538.4 ± 25.7 B
Different capital letters (vertical) illustrate significant differences in anthocyanin contents of the strawberry purées (p \ 0.05)
PG polygalacturonase, PE pectinesterase
the control and enzyme-treated purées (45 °C) did not
differ significantly revealing losses of 24.1–26.6%. Again,
since the decrease in pel 3-O-glc was not accompanied by
the release of the aglycone, pigment integrity during pro-
cessing was obviously not affected by potential b-gluco-
sidase side activities of the enzyme preparations, which is
in accordance with results previously reported by Kam-
merer et al. [20].
To evaluate the impact of different enzymation tem-
peratures on anthocyanin stability, strawberry purées were
incubated at 25 and 45 °C for 90 min, respectively, prior to
pasteurization at 85 °C for 15 min. As can be deduced
from Table 4, incubation at 45 °C only caused minor
pigment losses amounting to 9.2 and 2.8% for PG and PE,
respectively, whereas maceration at 25 °C resulted in
higher pigment losses of 15.3% (PG) and 17.8% (PE).
According to the manufacturer, the optimum temperature
for PG and PE activity is *50 °C. Therefore, higher pig-
ment losses may be ascribed to reduced activities of the
added enzyme preparations at 25 °C, again supporting the
findings of beneficial effects of PE and PG treatment on
anthocyanin stability of strawberry purées. Moreover,
higher pigment losses at 25 °C might be attributed to
increased PPO activities at this temperature. In previous
studies [24, 40], the optimum temperature of activity for
strawberry PPO was found to vary in a wide range from
30 °C (cv. ‘‘Selva’’) to 65 °C (cv. ‘‘Madame Moutot’’).
After pasteurization, anthocyanin retention in the
strawberry purées did not significantly differ with contents
ranging from 75.7 to 78.9% of the initial values. Obvi-
ously, anthocyanin losses due to different maceration
temperatures were leveled following pasteurization.
In the present study, an initial high-temperature short-
time treatment (90 °C, 10 s) was applied to enable imme-
diate PPO inactivation and improve anthocyanin retention in
the course of further processing and storage. However, as can
be seen in Table 4, this measure significantly lowered
anthocyanin contents of the pasteurized purée (408.3 mg/kg)
123
Eur Food Res Technol (2012) 234:207–222
After enzymatic treatment After pasteurization (85 °C, 15 min)
488.1 ± 0.1 B 442.4 ± 24.4 BC
533.2 ± 6.1 B 444.3 ± 9.2 BC
519.9 ± 1.5 B 500.0 ± 2.2 A
594.4 ± 15.8 A 464.0 ± 12.8 AB
532.6 ± 27.7 B 443.4 ± 0.8 BC
500.6 ± 2.1 B 408.3 ± 10.3 C
as compared to the control sample, which was not subjected
to an initial heat treatment (443.4 mg/kg). According to
Lopez-Serrano et al. [25] and Gössinger et al. [26], PPO
plays an important role in strawberry anthocyanin and color
degradation during processing and storage. However, liter-
ature data concerning PPO stability in strawberries and
strawberry products are controversial. Whereas Seradell
et al. [41] reported almost complete inactivation of straw-
berry PPO through heating at 65 °C for 30 min, according to
Chisari et al. [24], thermal resistance of strawberry PPO was
strongly cultivar-dependent. PPO from ‘‘Elsanta’’ was
almost entirely inactivated upon heating at 60 °C for 20 min,
while in cv. ‘‘Madame Moutot’’ over 40% of initial PPO
activity was retained even after heating at 60 °C for 2 h [24].
In strawberry nectar, PPO inactivation was impossible, even
when heated at 90 °C for 60 min [26], thus supporting
extreme heat stability of PPO.
Monomeric anthocyanins and polymeric pigments
Strawberry purées were incubated at 45 °C for 90 min with
and without enzyme addition prior to pasteurization at 85 °C
for 15 min. In non-digested IQF strawberries, contents of
monomeric anthocyanins and proportions of polymeric
pigments ranged from 568.7 to 576.6 mg/kg and from 2.4 to
2.8%, respectively (Table 5). After pasteurization, maxi-
mum pigment retention was observed in the strawberry purée
mashed with PE (96.0%), whereas the control and PG-treated
purées showed inferior pigment retention (80.9 and 82.5%,
respectively). However, monomeric anthocyanin contents of
the strawberry purées did not statistically differ after heat
preservation. These findings are in agreement with the
studies of Hartmann et al. [18], revealing comparable
anthocyanin retentions of pasteurized strawberry purées with
and without maceration using the same commercial PG
preparation. Proportions of polymeric pigments of all pro-
cessed variants were independent of the enzyme applied for
mash maceration (Table 5).
Eur Food Res Technol (2012) 234:207–222 215

Table 5 Monomeric anthocyanin contents (mg pel 3-O-glc/kg fresh (control) enzyme addition (incubation at 45 °C for 90 min for all
weight) and proportions of polymeric pigments (%) of IQF straw- samples) during dark storage at 20 °C
berries and pasteurized strawberry purées prepared with and without
Storage (weeks) Control PG PE

Polymeric color
IQF strawberries 2.6 ± 0.5 A 2.8 ± 0.7 A 2.4 ± 0.6 A
After pasteurization 7.3 ± 1.8 A 3.6 ± 1.3 A 3.6 ± 0.5 A
4 9.3 ± 0.3 A 9.6 ± 0.8 A 6.7 ± 0.1 B
8 18.8 ± 2.8 A 17.5 ± 2.8 A 13.8 ± 1.9 A
12 24.5 ± 0.5 A 20.5 ± 1.7 A 19.3 ± 3.0 A
16 33.2 ± 1.7 A 31.4 ± 1.4 A 27.4 ± 0.9 A
20 43.3 ± 0.7 A 32.3 ± 1.7 B 25.9 ± 3.3 C
24 56.2 ± 0.7 A 40.4 ± 4.4 B 35.9 ± 3.5 B
Monomeric anthocyanin content
IQF strawberries 576.6 ± 31.1 A 568.7 ± 7.4 A 569.0 ± 2.4 A
After pasteurization 466.6 ± 8.8 A 469.3 ± 27.7 A 546.5 ± 26.2 A
4 307.9 ± 0.3 B 332.6 ± 6.8 B 389.0 ± 15.1 A
8 198.4 ± 6.1 B 195.5 ± 3.4 B 245.6 ± 18.0 A
12 132.6 ± 9.5 B 156.6 ± 11.6 AB 186.4 ± 3.9 A
16 90.4 ± 19.0 A 53.1 ± 5.6 A 95.5 ± 10.8 A
20 93.8 ± 5.5 A 49.4 ± 1.8 B 109.8 ± 12.4 A
24 38.3 ± 8.9 B 74.9 ± 1.1 A 100.9 ± 8.7 A

Different capital letters (horizontal) illustrate significant differences (p \ 0.05) in monomeric anthocyanin contents and polymeric color of the
strawberry purées
PG polygalacturonase, PE pectinesterase

Color properties Impact of storage on anthocyanin and color stability

Redness a* In agreement with literature data [23, 42],
redness values a* of IQF strawberries ranged from 33.5 to
35.8 (Fig. 2). Pasteurized strawberry purées exhibited sig-
nificantly lower a* values (30.2 and 30.4, respectively). As
previously reported [22], this can be attributed to thermal
degradation of anthocyanins, resulting in a loss of redness.
Lightness L* Lightness L* of IQF strawberries ranged
from 29.1 to 29.6 (Fig. 2). Markedly higher values of
strawberries have been reported in the literature [23, 42],
which is due to the use of the white-fleshed cv. ‘‘Elsanta’’,
whereas ‘‘Senga sengana’’ was used in the present study
being characterized by homogeneous pigment distribution
throughout the fruit [43]. After pasteurization, lightness L*
was significantly decreased (26.1 and 26.3, respectively),
which is probably due to the formation of Maillard prod-
ucts caused by thermal processing [42].
Yellowness b* Heat preservation caused a pronounced
decline of yellowness dropping from 15.9 to 11.2 and 17.2
to 10.5, respectively (Fig. 2). Again, this was presumably
an effect caused by the formation of Maillard reaction
products.
of strawberry purées
Total and individual anthocyanins
During storage of the pasteurized purée samples, total
anthocyanin contents decreased steadily following first-
order kinetics with coefficients of determination
(R2) C 0.95. After 24 weeks of storage at 20 °C, only
23.8 mg/kg of anthocyanins (5.4% of the initial pigment
content) were retained in the control strawberry purée pre-
pared at 45 °C (Table 6). Kammerer et al. [42] also reported
significant losses of strawberry anthocyanins upon light
storage at 16–18 °C of canned fruits accounting for about
90%. In strawberry purées, losses up to 93% were observed
after 16 weeks of storage at 22 °C in the dark [5]. In contrast,
enzymatic treatment with PG and PE at 45 °C resulted in
improved pigment retention as can be deduced from residual
anthocyanin contents of 45.5 mg/kg (10.2%) and 65.7 mg/
kg (14.2%), respectively (Table 6). Unexpectedly, total
anthocyanins were better retained when strawberry purées
were mashed enzymatically, which was confirmed by the
calculated half-life values. As illustrated in Table 6, enzy-
matic maceration with the PE led to an increment of half-life
values for total and individual anthocyanins. The former

123
216
Fig. 2 Lightness L* (a),
redness a* (b), and yellowness
b* (c) of IQF strawberries
(S) and pasteurized strawberry
purées prepared without enzyme
addition during storage at 20
and 4 °C in the dark
amounted to 52.9 days in PE-treated strawberry purées,
whereas the control sample exhibited a half-life value of only
38.9 days. A slight but insignificant increase in the total
anthocyanin half-life was also observed for the PG-treated
strawberry purée (48.0 days).
Thus, the PE treatment at 45 °C for 90 min improved
total and individual anthocyanin stability over 24 weeks of
storage at 20 °C in the dark. This may be attributed to the
formation of low-esterified pectin in such purées, thus
enhancing electrostatic interactions between the dissoci-
ated carboxylic groups of polygalacturonic acid and the
anthocyanin flavylium cation, which was previously sug-
gested by different authors [10, 11]. Higher stability of this
123
Eur Food Res Technol (2012) 234:207–222
association may be attributed to reduced hydration reac-
tions because of steric hindrance. The stabilizing effect of
co-pigments has also been ascribed to this effect [6, 8].
The pH values of the control sample, PG-, and PE-
treated strawberry purées were equally 3.6 (Table 1). At
pH \ 2, isolated anthocyanins mainly exist as red colored,
positively charged flavylium cation, whereas between pH 2
and 4, the colorless hemiacetal predominates [44]. Thus, at
the pH values of the strawberry purées, equilibrium
between the flavylium cation and the hemiacetal form is
achieved, allowing electrostatic interactions between
the flavylium cation and the carboxylic function of the
de-esterified pectin.
Eur Food Res Technol (2012) 234:207–222 217

Table 6 Total and individual anthocyanin contents (mg/kg fresh different enzyme preparations throughout processing (incubation at
weight) and anthocyanin half-life (T1/2) values (days) of strawberry 45 °C for 90 min for all samples) and storage at 20 °C in the dark
purées prepared with and without (control) enzyme addition using
Control PG PE

IQF strawberries
Cya 3-O-glc 40.8 ± 3.2 A 41.0 ± 0.9 A 39.6 ± 1.4 A
Pel 3-O-glc 473.3 ± 14.7 A 468.4 ± 6.9 A 481.5 ± 1.2 A
Pel 3-O-mal-glc 89.9 ± 5.7 A 77.8 ± 0.7 A 90.3 ± 2.3 A
Sum 604.0 ± 23.6 A 587.2 ± 6.6 A 611.4 ± 4.9 A
After enzymatic treatment
Cya 3-O-glc 41.1 ± 3.7 A 37.6 ± 0.1 A 36.0 ± 1.0 A
Pel 3-O-glc 405.0 ± 24.2 B 420.3 ± 4.0 AB 473.6 ± 10.0 A
Pel 3-O-mal-glc 86.5 ± 0.4 A 74.7 ± 2.3 A 84.7 ± 4.8 A
Sum 532.6 ± 27.7 A 532.6 ± 7.3 A 594.4 ± 15.8 A
After pasteurization
Cya 3-O-glc 26.7 ± 0.2 C 33.7 ± 0.9 A 31.3 ± 0.1 B
Pel 3-O-glc 356.5 ± 0.2 A 354.1 ± 11.4 A 372.6 ± 12.8 A
Pel 3-O-mal-glc 60.3 ± 0.4 A 56.4 ± 1.3 B 60.1 ± 0.1 A
Sum 443.4 ± 0.8 A 444.3 ± 9.2 A 464.0 ± 12.8 A
1 week
Cya 3-O-glc 14.7 ± 0.2 B 21.2 ± 0.7 A 22.3 ± 0.4 A
Pel 3-O-glc 276.3 ± 18.6 B 276.9 ± 10.6 B 347.1 ± 2.8 A
Pel 3-O-mal-glc 44.2 ± 2.7 A 45.4 ± 7.1 A 56.8 ± 0.4 A
Sum 335.3 ± 21.4 B 343.5 ± 18.4 B 426.2 ± 2.8 A
2 weeks
Cya 3-O-glc 12.5 ± 0.6 B 20.3 ± 0.8 A 19.0 ± 0.6 A
Pel 3-O-glc 241.7 ± 15.4 A 266.7 ± 18.2 A 293.3 ± 14.4 A
Pel 3-O-mal-glc 37.1 ± 1.4 A 45.0 ± 3.2 A 45.3 ± 2.7 A
Sum 291.3 ± 17.4 A 332.0 ± 22.2 A 357.6 ± 17.7 A
4 weeks
Cya 3-O-glc 7.9 ± 0.3 B 14.9 ± 0.1 A 15.8 ± 0.4 A
Pel 3-O-glc 186.5 ± 11.4 B 212.1 ± 5.5 AB 238.6 ± 7.4 A
Pel 3-O-mal-glc 24.8 ± 1.4 B 32.1 ± 0.6 A 33.6 ± 1.3 A
Sum 219.2 ± 13.1 B 259.1 ± 6.1 AB 288.0 ± 8.3 A
8 weeks
Cya 3-O-glc ND 8.7 ± 0.0 A 8.1 ± 0.0 B
Pel 3-O-glc 110.7 ± 13.4 A 127.9 ± 3.1 A 146.6 ± 7.0 A
Pel 3-O-mal-glc 11.6 ± 3.2 B 20.0 ± 0.5 A 23.0 ± 0.8 A
Sum 122.4 ± 16.6 B 156.6 ± 3.6 AB 177.8 ± 7.9 A
12 weeks
Cya 3-O-glc ND 6.5 ± 0.1 A 6.0 ± 0.2 A
Pel 3-O-glc 89.5 ± 0.3 B 99.5 ± 5.7 AB 115.1 ± 3.8 A
Pel 3-O-mal-glc 9.0 ± 0.0 B 15.8 ± 0.9 A 17.6 ± 0.5 A
Sum 98.6 ± 0.3 B 121.8 ± 6.6 A 138.7 ± 4.5 A
16 weeks
Cya 3-O-glc ND 1.2 ± 0.2 B 2.6 ± 0.2 A
Pel 3-O-glc 48.9 ± 6.6 B 64.0 ± 0.1 AB 73.5 ± 2.0 A
Pel 3-O-mal-glc 3.9 ± 0.7 B 11.8 ± 0.2 A 13.2 ± 0.1 A
Sum 52.9 ± 7.3 B 77.0 ± 0.2 A 89.4 ± 1.9 A

123
218 Eur Food Res Technol (2012) 234:207–222

Table 6 continued
Control PG PE

20 weeks
Cya 3-O-glc ND 1.0 ± 0.1 B 2.6 ± 0.2 A
Pel 3-O-glc 42.8 ± 1.5 C 54.8 ± 0.2 B 63.3 ± 1.2 A
Pel 3-O-mal-glc 2.9 ± 0.2 C 10.5 ± 0.1 B 11.3 ± 0.1 A
Sum 45.7 ± 1.7 C 66.3 ± 0.2 B 77.2 ± 1.1 A
24 weeks
Cya 3-O-glc ND ND 2.5 ± 0.2 A
Pel 3-O-glc 23.1 ± 8.4 B 36.7 ± 0.7 AB 52.9 ± 5.1 A
Pel 3-O-mal-glc 0.7 ± 0.9 B 8.8 ± 0.1 A 10.3 ± 0.5 A
Sum 23.8 ± 9.3 B 45.5 ± 0.7 AB 65.7 ± 5.9 A
Half-life T1/2 (days)
Cya 3-O-glc 7.8 ± 0.1 26.5 ± 1.1 31.7 ± 0.7
Pel 3-O-glc 41.7 ± 2.0 48.8 ± 1.0 53.8 ± 0.0
Pel 3-O-mal-glc 29.5 ± 0.4 54.6 ± 0.6 56.6 ± 1.0
Sum 38.9 ± 1.7 B 48.0 ± 0.7 AB 52.9 ± 0.0 A

Different capital letters (horizontal) illustrate significant differences in anthocyanin contents of the strawberry purées
Cya cyanidin, pel pelargonidin, mal malonyl, glc glucoside, PG polygalacturonase, PE pectinesterase, ND not detectable

Astonishingly, a stabilizing effect was also observed
upon application of the PG containing preparation. This
was really surprising, because pectin has been shown to
enhance color stability in model systems [10, 45]. More-
over, in a previous study, strawberry juices prepared from
fresh material showed higher pigment stability than juice
from concentrate, which was assumed to result from pectin
decomposition prior to concentrate production [19].
Therefore, the enzymatic degradation of matrix compounds
was expected to affect anthocyanin stability. Higher
anthocyanin contents in the PG-treated purée may also be
attributed to co-pigmentation effects resulting from the
enhanced release of pigments and co-pigments due to
enzymatic cell wall degradation [8].
Anthocyanin stability during storage was inferior when
enzymation was performed at lower temperature (25 °C),
as can be seen from the half-life values specified in
Table 7. However, differences between the samples
digested at 25 and 45 °C were insignificant.
Applying an initial heating step insignificantly increased
half-life values of total anthocyanins (Table 7). This is
supposedly due to partial thermal inactivation of color-
degrading enzymes such as PPO, as previously described.
However, systematic optimization of the time–temperature
regime of an initial heating step is still required also con-
sidering residual PPO activities and potential reactivation of
the enzyme.
Independent of treatment, cya 3-O-glc exerted the lowest
stability among individual anthocyanins as demonstrated by
their specific half-life values (R2 C 0.85). This pigment was
completely degraded within 24 weeks of storage at 20 °C in
Table 7 Half-life (T1/2) values (days) of strawberry purées stored in
the dark at 4 and 20 °C, respectively, with and without (control)
enzyme addition using different enzymes and incubation temperatures
Half-life T1/2 (days)
Storage 20 °C
PG (25 °C) 45.8 ± 1.5 B, b
PG (45 °C) 48.0 ± 0.7 AB, b
PE (25 °C) 48.8 ± 0.5 AB, b
PE (45 °C) 52.9 ± 0.0 A, b
Control (45 °C) 38.9 ± 1.7 B, b
? Initial heating step 45.8 ± 2.8 AB, b
Storage 4 °C
Control (45 °C) 169.5 ± 11.7 a
Different capital letters illustrate significant differences of anthocya-
nin half-life values of strawberry purées stored at 20 °C (p \ 0.05).
Lower case letters illustrate significant differences between antho-
cyanin half-life values of strawberry purées stored at 20 and 4 °C,
respectively (p \ 0.05)
PG polygalacturonase, PE pectinesterase
the control strawberry purées and following PG treatment,
respectively. Similarly, only minor amounts were found in
the PE-treated purée (Table 6). In all samples, pel 3-O-glc
(R2 C 0.95) was found to be more stable, which is not sur-
prising, considering that a higher degree of hydroxylation in
the B-ring results in increased sensitivity toward PPO going
along with reduced half-life values [46, 47]. Following
enzymatic mash treatment, pel 3-O-mal-glc (R2 C 0.85)
exhibited highest stability in strawberry purées, which may
be ascribed to the acylation of the sugar moiety [3, 46].
However, the half-life value of this pigment in the control

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Eur Food Res Technol (2012) 234:207–222
Table 8 Total anthocyanin contents (mg/kg fresh weight), mono-
meric anthocyanin contents (mg pel 3-O-glc/kg fresh weight), and
proportions of polymeric pigments (%) of pasteurized strawberry
Total anthocyanin contents
Storage 20 °C 23.8 ± 9.3 B
Storage 4 °C 237.7 ± 1.0 A
Different capital letters (vertical) illustrate significant differences (p \ 0.05)
Fig. 3 Temperature-dependent degradation of total anthocyanins in
pasteurized strawberry purées prepared without enzyme addition
during storage at 20 and 4 °C as a function of time. The regression
lines represent a first-order kinetic model. A0 total anthocyanin
content (mg/kg fresh weight) after processing
sample was lower compared to that of its non-acylated
counterpart (Table 6).
Strawberry purées were stored at 20 and 4 °C in the dark
to assess the impact of storage temperature on total antho-
cyanin degradation. During 24 weeks of storage at 20 °C,
total anthocyanin contents decreased steadily to 23.8 mg/kg
in the strawberry purée control sample, which is equivalent
to relative pigment retention of 5.4%. In contrast, up to
42.6% (237.7 mg/kg) of anthocyanins were retained when
strawberry purées were stored at 4 °C (Table 8). During
storage, anthocyanin degradation followed first-order
kinetics irrespective of storage temperature. The tempera-
ture-dependent decrease in total anthocyanins is illustrated
in Fig. 3. The anthocyanin half-life value T1/2 of the purée
stored at 4 °C was about four times higher than the values
determined for the samples stored at 20 °C (Table 7).
Retention of pel 3-O-glc, pel 3-O-mal-glc, and cya 3-O-glc
amounted to 54.1, 54.3, and 42.8% at the end of the storage at
4 °C, again indicating that cya 3-O-glc is more prone to
degradation than the pelargonidin glycosides. These findings
clearly support previous observations that storage tempera-
ture is a key factor to improve anthocyanin retention in
strawberry products [5, 48, 49]. Since they are mostly filled
219
purées prepared without (control) enzyme addition stored at 20 and
4 °C for 24 weeks in the dark
Monomeric anthocyanin contents Polymeric pigments
38.3 ± 8.9 B 56.2 ± 0.7 A
306.0 ± 7.0 A 9.2 ± 0.6 B
into transparent glass jars, the impact of light on anthocyanin
storage stability is subject of our current research.
Monomeric anthocyanins and polymeric pigments
Expectedly, monomeric anthocyanin contents decreased
during storage, whereas the proportions of polymeric pig-
ments increased (Table 5). After 24 weeks of storage at
20 °C in the dark, only 8.2% of the initial monomeric
anthocyanins were retained in the control, while the
application of the PG and PE preparations resulted in better
pigment retention (16.0 and 18.5%, respectively). This is
consistent with our results from HPLC–DAD–MSn mea-
surements. Retention of monomeric anthocyanins was
significantly improved when purées were stored at 4 °C. As
an example, 68.5% of the monomeric anthocyanins were
retained in the control purée after 24 weeks of storage
(Table 8).
Polymeric anthocyanin contents increased significantly
during storage with the control showing the highest
increment at the end of storage at 20 °C. After 24 weeks,
relative amounts of polymeric pigments of this variant
were considerably higher (56.2%) than those of the purées
macerated with PG and PE (40.4 and 35.9%, respectively).
The lowest proportions of polymeric anthocyanins
amounting to 9.2% were determined in the purées produced
without mash enzymes, which were stored at 4 °C
(Table 8). Thus, in agreement with previous reports [21],
the formation of polymeric pigments was strongly
enhanced at elevated storage temperatures.
Color properties
Color changes of strawberry purées are exemplified in
Fig. 2 for the control sample without enzyme addition.
Enzymatic maceration did not show notable effects on
color properties of the purées. Therefore, color parameters
proved to be unsuitable for evaluating the impact of dif-
ferent enzyme preparations on color.
Redness a* During 24 weeks of storage at 20 °C, a
constant but comparatively slight decline of a* was
observed in the strawberry purées (a*24 weeks/20 °C = 18.7),
whereas total anthocyanin contents dropped to about 5% of
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220 Eur Food Res Technol (2012) 234:207–222

their initial values. Thus, relative anthocyanin losses better
reflected pigment degradation than the decrease in redness.
These findings are in agreement with the data of Ngo et al.
[21] and Gössinger et al. [26], who reported the impact of
storage to be more pronounced on anthocyanin degradation
than on color loss. Lowering the storage temperature to
4 °C resulted in higher a* values after 24 weeks of storage
(a*24 weeks/4 °C = 24.1), which is in accordance with for-
mer investigations [48, 50].
Lightness L* Consistent with previous findings [23],
lightness slightly increased during 24 weeks of storage at
20 °C (L*24 weeks/20 °C = 31.1) (Fig. 2), which may be
attributed to the formation of colorless anthocyanin deg-
radation products. In contrast, lightness L* of the straw-
berry purées remained constant when stored at 4 °C in the
dark (L*24 weeks/4 °C = 26.9).
Yellowness b* On the contrary, an increase in
yellowness was observed during storage at 20 °C
(b*24 weeks/20 °C = 14.5), which is in accordance with the
results of a previous study [23]. This might again be due to
the formation of browning pigments of the aged products.
In contrast, cold storage of the strawberry purées markedly
lowered yellowness values (b*24 weeks/4 °C = 8.7).
Antioxidant activity of stored pure´es as determined
by the FRAP and TEAC assays
Strawberries are known to be valuable sources of com-
pounds with high antioxidant capacities [5, 18, 22].
Therefore, the effect of enzymatic treatment on antioxi-
dant activities of strawberry purées was evaluated during
storage at 20 °C using the FRAP and TEAC assays
(Fig. 4). For all samples, antioxidant values of the FRAP
assay were markedly higher than those determined using
the TEAC assay, which is in accordance with former
investigations [51]. After pasteurization, differences
between samples with and without enzymatic maceration
were insignificant, ranging from 665.6 to 735.4 lmol
TAE/100 g FW in the FRAP assay and from 291.8 to
292.5 lmol TAE/100 g FW in the TEAC assay. The
former assay revealed a slight increase in antioxidant
activity within the first 4 weeks of storage. This may be
attributed to the formation of oligomeric and polymeric
compounds displaying enhanced reactivity in this system.
After 24 weeks of storage, the antioxidant activities of
all samples came close to the initial values of both
assays. Altogether, antioxidant activities only underwent
minor changes, which have frequently been reported for
such storage trials [5, 18, 50].

Fig. 4 Antioxidant activities of a 1000

pasteurized strawberry purées control pectinesterase polygalacturonase
900
(µmol T A E /100 g FW)

obtained by enzymatic mash

maceration compared to a 800
Antioxidant activity

pasteurized control prepared 700

without enzyme addition as
600
determined by the FRAP (a) and
TEAC (b) assay 500
400
300
200
100
0
0 4 8 10 12 16 20 24
storage (weeks)
b 350
control pectinesterase polygalacturonase
(µmol T A E /100 g FW)

300
Antioxidant activity

250

200

150

100

50

0
0 4 8 10 12 16 20 24
storage (weeks)

123
Eur Food Res Technol (2012) 234:207–222
Conclusions
In contrast to earlier reported pigment degradation by
enzymatic maceration such as hydrolytic cleavage of
anthocyanidin glycosides, the present study clearly dem-
onstrates that specific mash enzymes predominantly
exerting PE and PG activity, respectively, may even sta-
bilize anthocyanins in strawberry purées. Supportive ionic
interactions of the enzymatically modified pectin matrix
with the anthocyanin flavylium cations are assumed to be
mainly responsible for the improved color retention. An
early inactivation of genuine pigment-degrading enzymes
such as PPO through initial high-temperature short-time
heating also proved to be beneficial with respect to pigment
retention. However, lowering the storage temperature from
20 to 4 °C was found to be by far the most effective
measure to improve pigment retention over time, whereas
the influence of light on anthocyanin and color stability of
strawberry products during storage is subject of our current
work. Moreover, further investigations are currently per-
formed aiming at the minimization of pigment losses by
genuine enzyme activities to further improve pigment
retention of pasty strawberry products treated with mac-
eration enzymes.
Acknowledgments This research project was supported by the
German Ministry of Economics and Technology (via AiF) and the
FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn). Project
AiF 16005 N.
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