Food Research International 48 (2012) 241–248

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Food Research International

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Evaluation of the effects of different freezing and thawing methods

on color, polyphenol and ascorbic acid retention in strawberries
(Fragaria × ananassa Duch.)
Melanie Holzwarth, Sabine Korhummel, Reinhold Carle, Dietmar R. Kammerer ⁎
Hohenheim University, Institute of Food Science and Biotechnology, Chair Plant Foodstuff Technology, Garbenstrasse 25, 70599 Stuttgart, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Strawberries were frozen conventionally (− 20 °C) and using liquid nitrogen, respectively, and subsequently
Received 22 February 2012 thawed at various temperatures (+ 4, + 20 and + 37 °C, respectively) and in a microwave oven. Drip loss was
Accepted 12 April 2012 determined by weighing the exudates, color measurements were carried out applying the CIE L*a*b* system,
ascorbic acid was monitored spectrophotometrically, and individual anthocyanins and non-anthocyanin
Keywords:
phenolics were assessed by HPLC-DAD-MS n.
Strawberries
Freezing
While anthocyanin and ascorbic acid retentions after thawing were independent of the freezing technology,
Thawing different thawing procedures significantly affected fruit quality. Anthocyanins were best retained when
Polyphenols strawberries were thawed at 20 °C and in a microwave oven, respectively. Maximum ascorbic acid retention
Ascorbic acid was observed when strawberries were thawed in a microwave oven (10 min). Usual thawing at 4 °C (24 h)
Color caused the most pronounced pigment and ascorbic acid losses. Thus, the thawing regime proved to be the
key parameter for color and vitamin C retention of strawberry products.
© 2012 Elsevier Ltd. All rights reserved.

1. Introduction Accordingly, freezing results in cell decompartmentalization allowing

reactions between genuine enzyme activities and their corresponding
Strawberries (Fragaria×ananassa Duch.) are perishable soft fruits substrates (Tomás-Barberán & Espín, 2001). Therefore, anthocyanins
exhibiting an extremely short postharvest shelf-life (Wills & Kim, and ascorbic acid may already be degraded during thawing due to their
1995), and firm texture is a highly valued attribute which has so far interaction with oxidative enzyme activities such as polyphenoloxidases
resisted most attempts at its retention in processed fruits (Carle, (PPO) and ascorbic acid oxidases (AAO). Polyphenoloxidases are mainly
Borzych, Dubb, Siliha, & Maier, 2001). Furthermore, color plays a major made responsible for anthocyanin degradation in strawberries (Chisari,
role in quality assessment of food significantly determining consumers' Barbagallo, & Spagna, 2007). In accordance with this assumption, consid-
choice (Stintzing & Carle, 2004). Accordingly, strawberries are commonly erable pigment losses were reported after freezing and thawing of
frozen, thus allowing a year-round production of jams, juices, fruit prep- strawberries (Larsen & Poll, 1995; Oszmiański et al., 2009). However, in
arations, purées and concentrates (Oszmiański, Wojdylo, & Kolniak, the aforementioned studies, frozen fruits have been stored over a period
2009; Skrede, 1996). Although freezing is an efficient preservation mea- of several months at −20 °C prior to thawing and analytical investigation.
sure (Singh & Wang, 1977), it is inevitably accompanied by irreversible Therefore, systematic evaluation of the impact of the different freezing
structural damage of the cell wall, middle lamella, and protoplast, result- and thawing conditions on anthocyanin retention independent of any
ing in textural quality losses (Van Buggenhout, Sila, Duvetter, Van Loey, & storage effects is still lacking. Furthermore, only little is known about
Hendrickx, 2009). Microscopic studies have demonstrated quick freezing, the influence of different freeze–thaw treatments on non-anthocyanin
i.e. the production of individually quick-frozen (IQF) fruit using cryogenic phenolics.
gasses, to better retain cell integrity of strawberries than slower block Since strawberries are known to be valuable sources of ascorbic acid
freezing. This can be attributed to the formation of small intracellular with contents amounting to about 60 mg/100 g, however, strongly
ice crystals, whereas during slow freezing large ice crystals are formed depending on cultivar (Bardonaba & Terry, 2010; Kawanobu, Wajima,
in the extracellular space leading to increased drip loss after thawing. Zushi, Mori, & Matsuzoe, 2010), freezing and thawing processes should
Cell integrity of strawberries was also found to be influenced by thawing be evaluated with respect to vitamin C retention. Ascorbic acid is very
regimes. Fast thawing was reported to better retain fruit quality (Delgado susceptible toward heat, light and oxygen, and ascorbate oxidases
& Rubiolo, 2005; Skrede, 1996). have also been reported to contribute to ascorbic acid degradation in
fruits and vegetables (Davey et al., 2000). Accordingly, severe losses of
ascorbic acid have been reported upon freezing and thawing of
strawberries (Hartmann, Patz, Andlauer, Dietrich, & Ludwig, 2008;
⁎ Corresponding author. Tel.: +49 711 45922995; fax: +49 711 45924110. Larsen & Poll, 1995). Nevertheless, a systematic evaluation of different
E-mail address: [email protected] (D.R. Kammerer). freeze–thaw regimes is still needed.

0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2012.04.004
242 M. Holzwarth et al. / Food Research International 48 (2012) 241–248

In summary, the present study aimed at investigating the effects of ~8 h at 20 °C and ~2 h at 37 °C). Additionally, samples were thawed
different freezing and thawing conditions on color, polyphenol and in a microwave oven (700 W) for 10 min. Drip losses were deter-
ascorbic acid retention in strawberries. For this purpose, strawberries mined by weighing the exudates immediately after the freezing–
were conventionally block frozen at −20 °C and thawed at +20 °C, thawing process. Subsequently, thawed samples including the exu-
and quality parameters were compared to fruits obtained from further dates were lyophilized. Finally, lyophilized samples were ground
freeze–thaw treatments such as freezing at ~−196 °C using liquid ni- with a knife mill (Retsch, Haan, Germany).
trogen and thawing at ambient temperature of +37 °C and +4 °C, re-
spectively, and in a microwave oven. 2.2.3. Ascorbic acid
Aliquots of 1 g of the lyophilized samples were weighed into
2. Materials and methods Erlenmeyer flasks, and 25 mL of meta-phosphoric acid (1.5%, w/v;
pH 3.5) was added. The suspension was homogenized for 20 s using
2.1. Materials an Ultra-Turrax blender and centrifuged at 1600 rpm (573 g) for
5 min. The supernatant was used for ascorbic acid analyses using a
2.1.1. Strawberries commercial enzyme test kit (R-Biopharm, Darmstadt, Germany).
Red-fleshed strawberries (Fragaria× ananassa Duch.) of the cultivars
‘Candonga’ and ‘Sabrosa’ harvested in 2011 in Spain were obtained from 2.2.4. Extraction of phenolic compounds
the local market (Stuttgart, Germany). IQF strawberries (Fragaria × Aliquots of 1 g of the lyophilized samples were weighed into Er-
ananassa Duch. cv. ‘Senga sengana’) harvested in 2008 in Poland were lenmeyer flasks and extracted with 25 mL of acidified methanol
a gift from Odenwald Früchte (Breuberg, Germany). (0.1% HCl, v/v) by stirring for 60 min. Subsequently, the suspension
was filtered through a Whatman No. 4 paper (Maidstone, England).
2.1.2. Chemicals The filtrate was evaporated to dryness in vacuo at 30 °C, and the res-
All reagents and solvents were of analytical HPLC grade and idue was dissolved in 10 mL of formic acid (5%, v/v) and membrane-
purchased from VWR (Darmstadt, Germany). The following standards filtered (0.45 μm). The extract obtained by this procedure was used
were used for characterization and quantitation with HPLC-DAD-MSn: for HPLC and color measurements.
pelargonidin 3-O-glucoside chloride, cyanidin 3-O-glucoside chloride,
quercetin 3-O-glucoside, isorhamnetin 3-O-glucoside, cinnamic acid 2.2.5. Color analyses
(Extrasynthèse, Lyon, France), p-coumaric acid (Roth, Karlsruhe, Color measurements of frozen and thawed strawberries were per-
Germany) and ellagic acid (Serva, Heidelberg, Germany). Di-sodium formed with a Minolta Chromameter CR-300 (1.5 cm reading head,
hydrogen phosphate (Na2HPO4), sodium dodecyl sulfate (SDS) and L- Konica Minolta, Osaka, Japan) using D65 as illuminant and a 10° ob-
proline were obtained from Merck (Darmstadt, Germany). 4- server angle. The instrument was calibrated with a standard white
Methylcatechol was purchased from Fluka (Steinheim, Germany). De- tile (L* = 97.43, a* = −0.01, b* = 1.64). Determinations (n = 5) were
ionized water was used throughout. carried out on the surface of the fruits immediately after thawing.
Chroma [C* = (a* 2 + b* 2) 0.5] and hue angle [h° = (arctan b* / a*)]
2.2. Methods were calculated from CIE a* and b* values. Color properties of the
strawberry extracts were determined with a Perkin Elmer spectro-
2.2.1. Polyphenoloxidase (PPO) extraction and assay photometer also using illuminant D65 and a 10° observer angle.
PPO extraction was carried out according to a method described pre- Total color differences (ΔE*) were calculated according to the follow-
viously with some modifications (Chisari et al., 2007). Aliquots of 20 g ing equation:
of strawberry purée were weighed into Erlenmeyer flasks and stirred
h


2

2 i1=2
continuously for 10 min with 40 mL of cold acetone (−20 °C). After

2
ΔE ¼ ΔL þ Δa þ Δb : ð1Þ
centrifugation at 4000 rpm (3309 g) for 10 min, the residue was
extracted with 25 g of McIlvaine buffer (pH 6.5) at 4 °C for 120 min.
The suspension was centrifuged again (4000 rpm/10 min), and the su-
pernatant was filtered through a Whatman No. 4 paper (Maidstone, 2.2.6. Determination of individual phenolic compounds by HPLC-DAD-MSn
UK) and adjusted to 35 g with McIlvaine buffer. The clear supernatant
was kept cool until the PPO activity assay. All extraction protocols 2.2.6.1. HPLC-DAD system. The determination of individual phenolic
were carried out in duplicate. The PPO assay was performed according compounds was performed using an Agilent HPLC series 1100
to a method described earlier (Schweiggert, Schieber, & Carle, 2005). (Agilent, Waldbronn, Germany), a Waters (Milford, MA, USA) Sunfire
Product formation was recorded by absorption measurements of the C18 column (250 × 4.6 mm i.d.; 5 μm particle size) with a C18 ODS
pink L-proline-catechol adduct at 525 nm (ε = 1550 L/mol × cm) in in- guard column (4.0 × 2.0 mm i.d.) at a temperature of 25 °C and a
tervals of 15 s for 10 min. PPO activity was calculated from the slope flow rate of 0.8 mL/min. Eluent A was a 5% aqueous formic acid, and
of the linear absorption–time curve obtained after a short lag-phase. acidified methanol (5% formic acid, v/v) was used as eluent B. The
For density measurements of the PPO extracts, a DMA 80 density gradient program was as follows: 10% B to 25% B (10 min), 25% B to
meter was used (Anton Paar GmbH, Graz, Austria). 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
2.2.2. Freezing and thawing of strawberries (2 min), and 10% B isocratic (3 min). Total run time was 45 min,
Fresh strawberries were washed with tap water and dried on a and the injection volume was 20 μL. Simultaneous monitoring was
paper towel before sepals were removed with a kitchen knife. Dry performed at 280, 320, 370 and 520 nm.
matter contents were determined using an MA 40 electronic moisture
analyzer (Sartorius, Göttingen, Germany), and pH values were mea- 2.2.6.2. HPLC-DAD-MS n system. For peak assignment, LC–MS analyses
sured with a Metrohm 691 pH meter (Herisau, Switzerland). Freezing were performed with the HPLC system described above coupled to a
was carried out in portions of ~250 g in 500 mL plastic containers in a Bruker (Bremen, Germany) model Esquire 3000+ ion trap mass spec-
deep freezer (−20 °C) or using liquid nitrogen (120 s). After 24 h at trometer fitted with an electrospray ionization (ESI) interface. Positive
−20 °C, samples were thawed in plastic containers at 4 °C, 20 °C (anthocyanins) and negative (other polyphenolic compounds) ion
and 37 °C, respectively. Thawing was considered complete when the mass spectra of the column eluate were recorded in a range of m/z
temperature in the center of the fruit reached + 4 °C (~24 h at 4 °C, 50–1500 at a scan speed of 13,000 m/z units/s. Nitrogen was used
 M. Holzwarth et al. / Food Research International 48 (2012) 241–248 243

both as a drying gas at a flow rate of 9 L/min and as a nebulizing gas at a Buendía et al., 2010). This compound showed an [M−H]− ion at m/z
pressure of 40 psi. The nebulizer temperature was set at 365 °C. Helium 325 and produced a fragment ion at m/z 163, indicating the loss of a
was used as collision gas at a pressure of 4.9 × 10− 6 mbar. hexose moiety. Compounds 3 and 11 were tentatively identified as
coumaroyl hexoside derivatives exhibiting a fragment ion at m/z 325.
2.2.6.3. Quantitation of individual polyphenols by HPLC-DAD. Individual p-Coumaric acid (compound 12) and cinnamic acid (compound 19)
polyphenols were quantitated using calibration curves of the correspond- were also detected in cultivars ‘Candonga’, ‘Sabrosa’ and ‘Senga
ing standard compounds. In case of lacking standards, the calibration of sengana’. These compounds were tentatively identified by comparing
structurally related compounds was used and corrected by a molecular their retention times and UV spectra with those of the reference sub-
weight factor (Chandra, Rana, & Li, 2001). Considering non-anthocyanin stances. Compound 1 was tentatively assigned to 4-caffeoylquinic acid
phenolics, only the major compounds were quantitated. showing an [M−H]− ion at m/z 353 and a fragment ion at m/z 173. A
further component was characterized as hydroxybenzoyl hexose
2.2.7. Statistical analysis based on its [M−H]− ion at m/z 299 and the fragment ion at m/z 137
Significant differences (α = 0.05) were determined using the (compound 8). Moreover, ellagic acid (compound 16) was found
Tukey test for differences between independent samples. Data evalu- being characterized by an [M−H]− ion at m/z 301 and its typical UV/
ation was performed using the SAS software package (SAS institute, Vis spectrum (λmax: 253 and 368 nm). In cv. ‘Senga sengana’, ellagic
Cary, NC, USA; v. 9.1). acid was even found to be the predominant non-anthocyanin phenolic
compound. Moreover, two ellagitannins were detected with retention
3. Results and discussion times of 11.0 and 15.2 min and tentatively characterized as galloyl-
hexahydroxydiphenoyl (HHDP) hexose (compound 6) and galloyl-
3.1. Characterization of strawberry polyphenols bis-HHDP hexose (compound 10). The latter showed [M−H]− ions at
m/z 633 and m/z 935 and produced fragment ions at m/z 301, being typ-
3.1.1. Anthocyanins ical for ellagic acid. Another ellagic acid derivative was detected show-
Individual anthocyanins were tentatively assigned by HPLC-DAD-MSn ing an [M−H]− ion at m/z 447 and yielding a fragment ion at m/z 301
(Table 1), as previously reported (Holzwarth, Korhummel, Carle, & (compound 14). This compound was characterized as ellagic acid
Kammerer, 2012). The anthocyanin profile (Fig. 1A) of strawberry ex- deoxyhexoside, as previously reported (Aaby, Ekeberg & Skrede, 2007;
tracts and the fragmentation patterns of individual compounds in Buendía et al., 2010; Kajdžanoska, Gjamovski, & Stefova, 2010). In the
mass spectrometric experiments were in general accordance with pre- present study, (+)-catechin was detected in the cultivars ‘Candonga’
viously published data (Aaby, Ekeberg, & Skrede, 2007; Aaby, Wrolstad, and ‘Sabrosa’ exhibiting an [M−H]− ion at m/z 289 and a fragment
Ekeberg & Skrede, 2007; Shikov, Kammerer, Mihalev, Mollov, & Carle, ion at m/z 245 (compound 7). In cv. ‘Senga sengana’, (+)-catechin
2008). Compound A1 was assigned to cyanidin 3-O-glucoside (cya 3- could not be detected, although its occurrence has previously been
O-glc) exhibiting an [M+] ion at m/z 449 releasing cyanidin as reported in this cultivar (Aaby, Ekeberg, & Skrede, 2007). Compound 5
fragment ion in the MS 2 experiment at m/z 287. The predominant was tentatively assigned to proanthocyanidin B1 exhibiting an [M−
anthocyanin (compound A2) in ‘Candonga’, ‘Sabrosa’ and ‘Senga H]− ion at m/z 577 and a fragment ion at m/z 425. Consistent with pre-
sengana’ strawberries was pelargonidin 3-O-glucoside (pel 3-O-glc) vious findings (Aaby, Ekeberg, & Skrede, 2007; Buendía et al., 2010;
exhibiting an [M+] ion at m/z 433 and releasing pelargonidin as frag- Kajdžanoska et al., 2010; Simirgiotis, Theoduloz, Caligari, & Schmeda-
ment at m/z 271. Compound A3 was assigned to pelargonidin 3-O-ruti- Hirschmann, 2009), non-anthocyanin flavonoid glycosides were
noside (pel 3-O-rut) based on its [M+] ion at m/z 579 and fragment ion detected in cvs. ‘Candonga’, ‘Sabrosa’ and ‘Senga sengana’, respectively.
at m/z 271. Compound A4 was only found in ‘Senga sengana’ straw- Compound 15 displayed an [M−H]− ion at m/z 477 and produced a
berries and was tentatively assigned to pelargonidin 3-O-malonyl-gluco- fragment ion at m/z 301 and was assigned to quercetin 3-O-glucuronide.
side (pel 3-O-mal-glc) based on its molecular ion and fragmentation Compound 18 was characterized as kaempferol 3-O-glucuronide
pattern in the MS 2 experiment. Compound A5 revealed an [M+] ion at exhibiting an [M−H]− ion at m/z 461 and a fragment ion at m/z 285.
m/z 461 releasing pelargonidin as fragment ion at m/z 271, and was Compound 17 showed an [M−H]− ion at m/z 491 and a fragment
therefore characterized as a pelargonidin derivative. Compound A6 ion at m/z 301 and was tentatively assigned to isorhamnetin 3-O-
showed an [M+] ion at m/z 475 and a fragment ion at m/z 271, and glucuronide. Fragmentation patterns of individual non-anthocyanin
was therefore assigned to pelargonidin 3-O-acetyl-glucoside. In the pre- compounds in mass spectrometric experiments were consistent with
sent study, this compound was only detected in the ‘Senga sengana’ cul- the results of previous studies (Aaby, Ekeberg, & Skrede, 2007; Aaby,
tivar (Table 1). Wrolstad, Ekeberg, & Skrede, 2007; Buendía et al., 2010; Clifford,
Johnston, Knight, & Kuhnert, 2003; Kajdžanoska et al., 2010; Määttä-
3.1.2. Non-anthocyanin phenolics Riihinen, Kamal-Eldin, & Törrönen, 2004; Simirgiotis et al., 2009).
Individual non-anthocyanin phenolics were characterized by HPLC-
DAD-MSn (Table 2; Fig. 1). In accordance with previous studies on the 3.2. Drip loss
phenolic profile of strawberries, p-coumaroyl hexose (compound 9)
was found to be the predominant phenolic acid derivative in ‘Candonga’ As can be seen from Table 3, drip loss strongly depended on freez-
and ‘Sabrosa’ strawberries (Aaby, Wrolstad, Ekeberg, & Skrede, 2007; ing velocity. As expected, block freezing at −20 °C resulted in higher

Table 1
HPLC-DAD-MSn data of anthocyanins in three red-fleshed strawberry cultivars (C — Candonga; S — Sabrosa; SS — Senga sengana).

Peak Rt (min) λmax MW MS1 [M+] MS2 Peak assignment C S SS

(m/z) (m/z, % base peak)

A1 17.0 281, 519 449 449 MS2 [449]: 287 (100), 288 (16) Cyanidin 3-O-glucoside x x x
A2 18.8 277, 502 433 433 MS2 [433]: 271 (100), 272 (17) Pelargonidin 3-O-glucoside x x x
A3 20.1 278, 505 579 579 MS2 [579]: 271 (100), 272 (17) Pelargonidin 3 O-rutinoside x x x
A4 25.0 280, 504 519 519 MS2 [519]: 271 (100), 272 (18) Pelargonidin 3-O-malonyl-glucoside x
A5 26.1 282, 506 461 461 MS2 [461]: 271 (100), 272 (23) Pelargonidin derivative x x x
A6 27.2 274, 505 475 475 MS2 [475]: 271 (100), 272 (25), 270 (14) Pelargonidin 3-O-acetyl-glucoside x
244 M. Holzwarth et al. / Food Research International 48 (2012) 241–248

1400 A2 A2
A 1000 B
Absorption (mAU)

Absorption (mAU)
1200
1000
520 nm 800 280 nm
800 600
600 10
A5 400 A4 19
400 11 12 A5
200 2 3 A6
200 A1
A3 A4
A6 1 4 8 9 A1 A3 16 17
6
0 0
0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40
Retention time (min) Retention time (min)

160 A2
300 12

140
C D
Absorption (mAU)

Absorption (mAU)
250 9 A2
120 370 nm 320 nm
200
100
80 16 150
17
60 13 100
A5 A6
40 18 A4
A4 19 A1
A1 A6 15 50 4 11 A5 16 17 19
20 9 A3 15
0 0
0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40
Retention time (min) Retention time (min)

Fig. 1. Reversed phase HPLC-DAD-MSn separation of polyphenolic compounds at 520 nm (A), 280 nm (B), 370 nm (C) and 320 nm (D), respectively, extracted from cv. ‘Senga sengana’. For
peak assignment see Tables 1 and 2.

values for drip loss after thawing (5.7 and 20.4% for cvs. ‘Candonga’ Lightness L* of ‘Senga sengana’, ‘Candonga’ and ‘Sabrosa’ strawberries fro-
and ‘Sabrosa’, respectively) than quick-freezing with liquid nitrogen zen at −20 °C amounted to 30.7, 30.4 and 29.2 units, respectively. After
(0.0 and 9.4% for ‘Candonga’ and ‘Sabrosa’, respectively). This is prob- thawing, L* values were slightly decreased. Compared to conventionally
ably due to the formation of large ice crystals in the extracellular frozen fruits, cryogenically frozen strawberries exhibited markedly higher
space in the former case leading to cell wall disruption. In contrast, L* values, which might be a result of small ice crystal formation on the
quick-freezing causes less cell wall damage because only small ice fruit surface. However, after thawing, L* values were comparable inde-
crystals are formed intracellularly (Delgado & Rubiolo, 2005; Van pendent of the freezing process. Conventionally frozen strawberries
Buggenhout et al., 2009). exhibited b* values ranging from 6.3 to 8.7 units, whereas b* values
After thawing at 20 °C for 8 h, drip loss of ‘Senga sengana’ were slightly higher, when fruits were frozen with liquid nitrogen
strawberries was found to be 9.1%. Increased values were observed for (~14 units). After thawing, b* values of conventionally frozen samples
the samples thawed for 2 h at 37 °C and for the samples thawed within were increased, whereas b* values of cryogenically frozen samples were
10 min in the microwave oven (17.4 and 16.8%, respectively). In found to be slightly decreased.
contrast, strawberries thawed at 4 °C for over 24 h suffered from
lower drip loss (11.6%). However, further storage (48 h) of thawed
strawberries resulted in increased values accounting for 22.6% (20 °C) 3.4. Effect of freezing and thawing on anthocyanin, non-anthocyanin
and 16.8% (4 °C), respectively. However, this trend could not be ob- phenolic and ascorbic acid contents in strawberries
served considering the samples thawed at 37 °C, where values for drip
loss were not markedly different after 2 h and 24 h, respectively 3.4.1. Anthocyanins
(~18%). Thus, at 37 °C, loss of cell sap was obviously completed after Total anthocyanins were calculated as the sum of the contents of
2 h (Table 3). cya 3-O-glc, pel 3-O-glc, pel 3-O-rut, pel 3-O-mal-glc (only detected
in cv. ‘Senga sengana’), an unidentified pel derivative and pel 3-O-
acetyl-glc (only detected in cv. ‘Senga sengana’). Thus, anthocyanin
3.3. Color properties of strawberries during the freezing and thawing patterns in strawberries were shown to be cultivar dependent, as pre-
processes viously reported (Lopes da Silva, Escribano-Bailón, Alonso, Rivas-
Gonzalo, & Santos-Buelga, 2007). In contrast to the results obtained
Chromatic changes of strawberries during freezing and thawing in the present study, pel 3-O-acetyl-glc has previously been found
under various conditions are illustrated in Fig. 2. Conventionally block in the strawberry cv. ‘Candonga’ (Buendía et al., 2010) and further
frozen (−20 °C) strawberries exhibited a* values ranging from 22.3 to pigments, e.g. cya 3-O-mal-glc and pel diglc, respectively, have been
25.3 units. After thawing at 20 °C for 8 h, a* values were markedly in- detected in cv. ‘Senga sengana’ (Shikov et al., 2008). These findings
creased to 31.8 (cv. ‘Senga sengana’), 41.3 (cv. ‘Candonga’) and 41.2 indicate that anthocyanin patterns may even vary within the same
(cv. ‘Sabrosa’) units, respectively. This might be attributed to pigment strawberry cultivar. The unidentified pel derivative has previously
diffusion from the center of the fruit to the outmost cell layers because been detected in the strawberry cv. ‘Camarosa’ (Cerezo, Cuevas,
of disrupted cell walls, as previously reported (Ngo, Wrolstad, & Zhao, Winterhalter, Garcia-Parrilla, & Troncoso, 2010).
2007). Differences of a* values between strawberries thawed at 20 °C, As can be seen from Table 4, total anthocyanin contents in frozen
37 °C, 4 °C and in the microwave oven could not be observed (Fig. 2A). In- ‘Candonga’ and ‘Sabrosa’ strawberries were comparable amounting to
terestingly, a* values were not increased after thawing, when liquid nitro- 332.7 and 325.4 mg/100 g DM, respectively, whereas markedly higher
gen was used for freezing. This may be due to reduced tissue damage values were found for ‘Senga sengana’ (559.4 mg/100 g DM). This may
resulting in lowered pigment diffusion (Delgado & Rubiolo, 2005). be attributed to the more homogenous pigment distribution throughout
 M. Holzwarth et al. / Food Research International 48 (2012) 241–248 245

Table 2
HPLC-DAD-MSn data of non-anthocyanin phenolics in three red-fleshed strawberry cultivars (C — Candonga; S — Sabrosa; SS — Senga sengana; HHDP — hexahydroxydiphenoyl).

Compound Rt λmax MW MS1 [M-H]− HPLC-ESI(−)-MSn experiment Peak assignment C S SS

(min) (m/z) (m/z, % base peak)

1 3.5 252 354 353 MS2 [353]: 173 (100), 111 (53), 293 (11) 4-Caffeoylquinic x x x
MS3 [173]: 111 (100) acid
2 7.4 248 420 419 MS2 [419]: 231 (100), 169 (44), 230 (26) Unknown x x x
3 7.9 250, 524 523 MS2 [523]: 325 (100) Coumaric acid x x
293sh MS3 [325]: 145 (100) derivative
4 8.5 284 382 381 MS2 [381]: 169 (100) Unknown x
5 9.9 278 578 577 MS2 [577]: 425 (100), 407 (45), 559 (30), 451 (26), 408 (21), 289 (19) Proanthocyanidin x x
MS3 [425]: 407 (100), 408 (14) B1
6 11.0 267 634 633 MS2 [633]: 301 (100), 302 (17), 463 (12) Galloyl-HHDP x x x
hexose
2
7 12.0 280 290 289 MS [289]: 245 (100), 205 (35), 179 (23) (+)-Catechin x x
8 13.5 278 300 299 MS2 [299]: 137 (100) Hydroxybenzoyl x x x
MS3 [137]: 93 (100) hexose
9 14.9 312 326 325 MS2 [325]: 163 (100), 145 (90), 187 (41), 146 (18), 119 (18) p-Coumaroyl x x x
MS3 [163]: 119 (100) hexose
10 15.2 315 936 935 MS2 [935]: 633 (100), 301 (10) Galloyl-bis HHDP x x x
MS3 [633]: 301 (100), 302 (12) hexose
11 15.7 314 362 361 MS2 [361]: 325 (100), 326 (19) Coumaric acid x x x
MS3 [325]: 163 (100), 145 (87), 187 (35), 119 (19), 164 (18), 117 (14), 265 (12), 235 (12) derivative
12 23.6 302 164 163 Identification with a standard p-Coumaric acid x x x
13 25.9 283 346 345 MS2 [345]: 197 (100), 248 (20), 147 (11) Unknown x x x
14 27.5 251, 448 447 MS2 [447]: 301 (100), 300 (29) Ellagic acid x x x
376 MS3 [301]: 257 (100) deoxyhexoside
15 29.1 255, 478 477 MS2 [477]: 301 (100) Quercetin 3-O- x x x
356 MS3 [301]: 151 (100), 273 (44), 179 (42), 107 (41), 240 (39) glucuronide
16 30.5 253, 302 301 Ellagic acid x x x
368
17 32.5 257, 492 491 MS2 [491]: 301 (100), 300 (54), 315 (36), 319 (13), 473 (11) Isorhamnetin 3- x x x
354 MS3 [301]: 107 (100) O-glucuronide
18 33.1 268, 462 461 MS2 [461]: 285 (100), 286 (11) Kaempferol 3-O- x x x
292, glucuronide
346
19 36.1 277 Identification with a standard Cinnamic acid x x x

the fruit tissues of this cultivar resulting in higher pigment contents, as Presumably, this pigment was degraded to pel 3-O-glc during the
previously reported (Skupień & Oszmiański, 2004). thawing process (Table 4).
Independent of treatment, pel 3-O-mal-glc was found to be the Compared to frozen fruits, the total anthocyanin contents in cv.
most stable pigment in cv. ‘Senga sengana’, being completely retained ‘Senga sengana’ were significantly decreased after thawing under
after thawing which may be ascribed to the acylation of the sugar each of the conditions. Pigment losses during thawing are assumed
moiety (Holzwarth et al., 2012). Since pel 3-O-mal-glc could not be to result from enzymatic degradation of anthocyanins by PPO activi-
detected in the modern cvs. ‘Candonga’ and ‘Sabrosa’, respectively, ties. PPO and peroxidase (POD) have previously been reported to be
pel 3-O-glc and pel 3-O-rut exhibited the highest relative retentions still active at lower temperatures (Cano, De Ancos, & Lobo, 1995;
in these cultivars. Independent of treatment, the unidentified pel de- Chisari et al., 2007). In cv. ‘Senga sengana’, PPO activities amounted
rivative was found to be the least stable pigment in all cultivars. to 36.6 U/100 g DM even after thawing. This is in accordance with

Table 3
Ascorbic acid contents, drip loss and color properties of frozen and thawed strawberries.

Cultivar Freezing Thawing Ascorbic acid Drip loss L*extract a*extract b*extract Hue angle Chroma ΔEextract
parameters parameters (mg/100 g DM) (%) h°extract C*extract

Senga IQF – 205.6 ± 1.8 a – 76.0 ± 0.7 d 35.8 ± 1.7 a 39.0 ± 2.2 a 47.5 ± 0.4 a 52.9 ± 2.8 a –
sengana IQF 20 °C/8 h 160.8 ± 13.3 bc 9.1 ± 0.0 76.8 ± 0.4 cd 35.3 ± 0.2 ab 38.4 ± 0.8 a 47.4 ± 0.5 a 52.2 ± 0.7 a 1.0
IQF 20 °C/48 h 14.8 ± 1.4 e 22.6 ± 0.1 78.8 ± 0.3 a 31.7 ± 0.6 c 30.9 ± 1.0 bc 44.3 ± 0.4 c 44.2 ± 1.1 b 9.5
IQF 37 °C/2 h 170.8 ± 15.7 ab 17.4 ± 0.4 78.7 ± 0.7 ab 30.8 ± 0.4 c 30.4 ± 0.5 bc 44.7 ± 0.2 bc 43.3 ± 0.6 b 10.3
IQF 37 °C/24 h 40.7 ± 3.8 e 19.2 ± 0.4 78.5 ± 0.7 ab 31.5 ± 0.9 c 31.1 ± 1.6 bc 44.6 ± 0.7 c 44.3 ± 1.7 b 9.3
IQF Microwave/10 min 197.4 ± 4.1 a 16.8 ± 0.2 77.3 ± 0.6 bcd 33.0 ± 1.2 bc 34.1 ± 1.6 b 46.0 ± 0.4 b 47.5 ± 2.0 b 5.7
IQF 4 °C/24 h 134.1 ± 9.9 cd 11.6 ± 1.1 79.3 ± 0.7 a 30.9 ± 1.2 c 30.1 ± 1.9 c 44.3 ± 0.7 c 43.2 ± 2.1 b 10.6
IQF 4 °C/48 h 104.5 ± 10.4 d 16.8 ± 0.4 78.0 ± 0.7 abc 32.6 ± 1.2 c 33.0 ± 3.2 bc 45.3 ± 1.0 bc 46.4 ± 2.5 b 7.0
Candonga −18 °C – 400.6 ± 28.4 a – 83.3 ± 1.0 a 23.1 ± 2.6 ab 20.6 ± 3.3 a 41.7 ± 1.3 a 30.9 ± 4.1 a –
−18 °C 20 °C/8 h 364.2 ± 12.1 a 5.7 ± 0.5 84.9 ± 0.8 a 20.0 ± 1.3 b 17.6 ± 1.7 a 41.3 ± 1.2 a 26.7 ± 2.0 a 4.6
N2 – 334.7 ± 11.6 a – 83.1 ± 1.1 a 24.0 ± 2.1 a 21.4 ± 2.5 a 41.7 ± 0.8 a 32.1 ± 3.2 a –
N2 20 °C/8 h 331.0 ± 16.6 a 0 84.4 ± 0.9 a 20.9 ± 1.0 ab 18.5 ± 1.6 a 41.4 ± 1.3 a 27.9 ± 1.8 a 4.4
Sabrosa −18 °C – 327.0 ± 30.4 a – 82.2 ± 0.8 a 25.5 ± 1.8 a 23.6 ± 2.3 a 42.7 ± 0.8 a 34.8 ± 2.9 a –
−18 °C 20 °C/8 h 259.5 ± 10.2 ab 20.4 ± 0.0 82.8 ± 0.6 a 24.1 ± 1.0 a 22.2 ± 1.3 a 42.7 ± 0.5 a 32.8 ± 1.6 a 2.1
N2 – 189.4 ± 15.8 b – 82.2 ± 1.3 a 23.0 ± 0.1 a 20.5 ± 2.4 a 41.6 ± 0.8 a 30.8 ± 3.2 a –
N2 20 °C/8 h 262.2 ± 13.9 ab 9.4 ± 0.4 83.5 ± 0.8 a 23.0 ± 1.7 a 20.7 ± 1.9 a 41.9 ± 0.5 a 31.0 ± 2.5 a 0.3

Different lower case letters (vertical) indicate significant differences (p b 0.05) between the strawberry cvs. ‘Senga sengana’, ‘Candonga’ and ‘Sabrosa’, respectively.
246 M. Holzwarth et al. / Food Research International 48 (2012) 241–248

L* a* b* determination temperatures and methods (Chisari et al., 2007;

50 A López-Serrano & Ros Barceló, 2001).
Astonishingly, the highest pigment losses (21.4%) were observed
40
when strawberries were thawed at 4 °C within 24 h which might be
30 attributed to the prolonged thawing period at this temperature. Con-
sequently, thawing strawberries for 10 min in a microwave oven and
20 thawing at 20 °C for 8 h resulted in lowered pigment losses account-
ing for 12.4 and 6.8%, respectively. However, although thawing at
10 37 °C was already completed after 2 h, pigment losses were compara-
bly high accounting for 19.4%. Presumably, the positive effects of the
0 short thawing period were annihilated by increased PPO activities at
IQF IQF/ T20 IQF/ T37 IQF/ Tmicro IQF/ T4
elevated temperatures. In previous studies, the optimum temperature
for PPO activity was reported to vary considerably from 30 °C (cv.
50 B L* a* b* ‘Selva’) to 65 °C (cv. ‘Mme Moutot’) (Chisari et al., 2007). When
thawed strawberries were further stored at 4 °C, 20 °C and 37 °C, re-
40 spectively, anthocyanin degradation was found to proceed (Table 4).
At slow freezing rates (e.g. conventional block freezing at −20 °C), big
30 ice crystals are formed mainly in the extracellular space of fruits leading to
irreversible textural damage of the cell wall, middle lamella and proto-
20
plast. In contrast, quick-freezing (e.g. flash freezing with liquid nitrogen)
is associated with the formation of tiny ice crystals in the intracellular
10
space thus leading to less cell rupture (Van Buggenhout et al., 2009).
0 Accordingly, pigment retentions were expected to be improved after
CF CF/ T20 NF NF/ T20 thawing, when fruits were frozen with liquid nitrogen. Considering cv.
‘Sabrosa’, this assumption could be confirmed, since pigments were
L* a* b* completely retained after thawing. However, for cv. ‘Candonga’, pigment
50 C losses during thawing were ~19%, independent of the freezing procedure.
40

30 3.4.2. Non-anthocyanin phenolics

Consistent with the results of previous studies (Cao et al., 2011),
20 total contents of non-anthocyanin phenolics in frozen and lyophilized
strawberries cvs. ‘Candonga’, ‘Sabrosa’ and ‘Senga sengana’ ranged
10 from 82.3 to 114.4 mg/100 g DM (Table 5). In contrast to anthocya-
nins, the total amounts of non-anthocyanin phenolics did not decline
0
upon thawing under each of the conditions. Most interestingly, con-
CF CF/ T20 NF NF/ T20
tents of p-coumaroyl hexose were even significantly increased after
Fig. 2. Color properties of cvs. ‘Senga sengana’ (A), ‘Candonga’ (B) and ‘Sabrosa’ (C) conventional freezing (− 20 °C) and thawing (20 °C/8 h), whereas
during thawing. IQF — individually quick-frozen, CF — conventionally frozen at contents of p-coumaric acid were found to be slightly decreased.
− 20 °C, NF — frozen with liquid nitrogen, TX — thawed at X °C, micro — microwave.
This tendency was observed for all cultivars investigated and was as-
sumed to result from enzymatic glycosylation of p-coumaric acid dur-
ing thawing. Accordingly, in a recently published study, a cinnamate
the results of previous studies, where PPO activities in strawberries glucosyltransferase isolated from Fragaria×ananassa was shown to con-
were reported to vary in a wide range from 20 to 800 U/100 g DM. vert cinnamic acids into their corresponding hexosides (Lunkenbein et
Differences may be explained by various cultivars, ripeness degrees, al., 2006).

Table 4
Anthocyanin contents (mg/100 g DM) in frozen and thawed strawberries.

Cultivar Freezing Thawing Cya 3-O-glc Pel 3-O-glc Pel 3-O-rut Pel 3-O-mal- Pel Pel 3-O-acetyl- Total content
parameters parameters glc derivative glc

Senga IQF – 38.3 ± 0.6 a 402.6 ± 9.6 a 29.7 ± 3.6 a 17.7 ± 2.4 c 29.0 ± 3.4 a 42.1 ± 0.6 a 559.4 ± 19.4 a
sengana IQF 20 °C/8 h 31.7 ± 0.2 bc 386.1 ± 2.3 a 25.6 ± 0.2 a 36.6 ± 2.1 a 14.4 ± 1.8 b 26.6 ± 1.1 bcd 521.1 ± 3.0 b
IQF 20 °C/48 h 27.5 ± 0.5 cd 340.6 ± 3.6 bc 16.6 ± 0.5 de 24.3 ± 5.4 abc 8.1 ± 0.2 cd 32.4 ± 4.1 b 449.5 ± 3.1 c
IQF 37 °C/2 h 27.0 ± 1.1 d 336.1 ± 2.0 bcd 20.4 ± 0.8 cd 32.4 ± 5.0 abc 7.9 ± 0.3 d 27.3 ± 4.0 b 451.0 ± 1.3 c
IQF 37 °C/24 h 27.6 ± 2.8 cd 316.9 ± 10.6 d 25.2 ± 1.6 ab 38.3 ± 2.9 a 12.1 ± 1.0 bc 17.8 ± 2.3 d 438.5 ± 14.3 c
IQF Microwave 33.3 ± 3.2 b 357.8 ± 15.1 b 24.6 ± 1.8 bc 38.9 ± 7.7 a 12.5 ± 1.3 b 23.0 ± 5.0 cd 490.0 ± 22.5 b
IQF 4 °C/24 h 28.6 ± 1.3 cd 328.4 ± 2.6 cd 22.7 ± 0.8 bc 20.5 ± 7.1 c 7.0 ± 0.4 d 33.2 ± 2.2 b 439.9 ± 7.3 c
IQF 4 °C/48 h 25.6 ± 0.6 d 333.8 ± 4.2 cd 14.6 ± 1.4 e 23.2 ± 7.3 bc 8.2 ± 0.1 cd 22.0 ± 2.6 cd 427.4 ± 4.8 c
Candonga −18 °C – 17.5 ± 2.3 a 266.0 ± 42.7 a 33.7 ± 1.7 a n.d. 14.8 ± 4.8 a n.d. 332.7 ± 45.0 a
−18 °C 20 °C/8 h 14.3 ± 2.5 a 217.6 ± 4.9 a 27.8 ± 3.0 b n.d. 9.5 ± 1.0 ab n.d. 269.2 ± 8.5 a
N2 – 16.4 ± 2.4 a 277.8 ± 31.5 a 37.3 ± 5.3 a n.d. 9.0 ± 1.0 ab n.d. 340.5 ± 40.7 a
N2 20 °C/8 h 14.8 ± 0.4 a 222.9 ± 11.7 a 31.4 ± 0.7 ab n.d. 6.9 ± 0.6 b n.d. 275.9 ± 11.6 a
Sabrosa − 18 °C – 17.5 ± 2.2 a 254.8 ± 15.3 ab 38.8 ± 3.9 a n.d. 14.4 ± 1.8 a n.d. 325.4 ± 19.7 ab
−18 °C 20 °C/8 h 11.5 ± 1.3 a 226.0 ± 16.4 b 31.3 ± 1.7 b n.d. 9.8 ± 0.2 b n.d. 278.6 ± 19.1 b
N2 – 14.3 ± 1.5 ab 251.2 ± 15.8 ab 37.3 ± 1.1 ab n.d. 6.1 ± 0.6 c n.d. 308.8 ± 15.1 ab
N2 20 °C/8 h 14.8 ± 1.0 ab 271.9 ± 16.8 a 40.9 ± 2.8 a n.d. 6.3 ± 0.2 c n.d. 333.9 ± 20.0 a

Different lower case letters (vertical) indicate significant differences (p b 0.05) between the strawberry cvs. ‘Senga sengana’, ‘Candonga’ and ‘Sabrosa’, respectively.
 M. Holzwarth et al. / Food Research International 48 (2012) 241–248 247

Table 5
Contents of non-anthocyanin phenolics (mg/100 g DM) in frozen and thawed strawberries.

Cultivar Freezing `Thawing p-Coumaroyl p-Coumaric Quercetin 3- Ellagic acid Isorhamnetin 3- Cinnamic Total content
parameters parameters hexose acid O-glucuronide O-glucuronide acid

Senga sengana IQF – 17.6 ± 0.9 d 21.9 ± 0.8 a 8.4 ± 0.4 c 27.6 ± 2.1 b 21.2 ± 0.6 b 10.8 ± 0.6 a 107.4 ± 3.3 b
IQF 20 °C/8 h 57.4 ± 1.6 a 9.8 ± 0.4 c 14.9 ± 0.7 b 24.8 ± 0.4 bcd 14.6 ± 0.5 cd 5.3 ± 0.3 c 126.9 ± 1.5 a
IQF 20 °C/48 h 13.6 ± 2.1 d 21.3 ± 2.0 a 6.1 ± 0.2 c 27.9 ± 1.8 b 13.2 ± 1.8 d 10.6 ± 0.6 ab 92.6 ± 2.8 b
IQF 37 °C/2 h 24.3 ± 4.3 cd 15.8 ± 1.4 b 9.7 ± 0.4 bc 23.5 ± 0.4 cd 26.6 ± 0.4 a 7.3 ± 0.3 c 107.2 ± 5.5 b
IQF 37 °C/24 h 44.9 ± 3.8 ab 11.9 ± 1.6 bc 21.7 ± 4.0 a 32.5 ± 0.6 a 11.2 ± 1.2 d 6.4 ± 0.2 c 128.7 ± 6.1 a
IQF Microwave 32.7 ± 4.0 bc 10.8 ± 1.7 c 14.8 ± 3.3 b 25.1 ± 1.4 bc 17.5 ± 2.5 bc 5.6 ± 0.7 c 106.5 ± 5.5 b
IQF 4 °C/24 h 16.3 ± 7.1 d 20.3 ± 1.0 a 8.5 ± 1.8 c 21.0 ± 0.6 d 14.1 ± 1.0 cd 12.9 ± 0.8 a 93.1 ± 8.7 b
IQF 4 °C/48 h 23.6 ± 9.5 cd 19.7 ± 1.5 a 11.7 ± 1.3 bc 23.4 ± 2.2 d 12.3 ± 2.4 d 10.2 ± 0.1 b 100.9 ± 8.5 b
Candonga −18 °C – 64.9 ± 12.6 b 11.8 ± 1.2 a 10.7 ± 1.8 a 8.9 ± 0.8 a 13.4 ± 2.7 a 4.8 ± 0.2 a 114.4 ± 10.1 b
−18 °C 20 °C/8 h 90.2 ± 8.7 a 11.6 ± 0.9 a 13.7 ± 2.6 a 8.8 ± 2.2 a 9.2 ± 1.7 ab 4.4 ± 0.5 a 137.4 ± 6.6 a
N2 – 95.5 ± 4.4 a 5.7 ± 1.3 b 15.5 ± 2.2 a 10.1 ± 1.3 a 8.2 ± 1.1 b 1.9 ± 0.7 b 136.8 ± 4.4 a
N2 20 °C/8 h 75.3 ± 6.4 ab 11.7 ± 2.8 a 13.6 ± 0.6 a 9.6 ± 2.0 a 10.1 ± 1.0 ab 4.8 ± 0.6 a 125.0 ± 10.5 ab
Sabrosa −18 °C – 29.9 ± 3.5 c 21.2 ± 1.9 a 4.2 ± 0.3 b 9.8 ± 1.0 a 8.1 ± 0.6 a 9.2 ± 0.8 a 82.3 ± 1.5 b
−18 °C 20 °C/8 h 50.9 ± 4.3 b 16.9 ± 1.4 ab 4.5 ± 0.8 b 10.0 ± 0.4 a 4.5 ± 0.3 b 7.8 ± 1.0 ab 94.6 ± 1.1 a
N2 – 58.3 ± 3.3 ab 13.9 ± 4.5 bc 8.8 ± 1.3 a 8.3 ± 1.0 ab 4.7 ± 0.2 b 4.1 ± 3.0 b 98.1 ± 5.4 a
N2 20 °C/8 h 69.6 ± 6.4 a 7.8 ± 0.9 c 9.2 ± 0.6 a 6.7 ± 0.3 b 4.9 ± 0.2 b 3.9 ± 0.8 b 102.1 ± 5.1 a

Different lower case letters (vertical) indicate significant differences (p b 0.05) between the strawberry cvs. ‘Senga sengana’, ‘Candonga’ and ‘Sabrosa’, respectively.

3.4.3. Ascorbic acid 4. Conclusion

Ascorbic acid contents in conventionally frozen and lyophilized straw-
berries cvs. ‘Senga sengana’, ‘Candonga’ and ‘Sabrosa’ amounted to 205.6, The present study mainly focused on investigating the influence of dif-
400.6 and 327.0 mg/100 g DM, respectively (Table 3). Presumably, ascor- ferent freezing and thawing procedures on color, polyphenol and ascorbic
bic acid has already partially been degraded during the foregoing lyophi- acid retention in strawberries. Whereas the effects of differing freezing
lization (Asami, Hong, Barrett, & Mitchell, 2003). Expectedly, contents technologies were negligible, different thawing regimes significantly af-
decreased during thawing under each of the conditions. This is probably fected the a. m. quality parameters. Thawing at 20 °C and in a microwave
due to the enzymatic (via AAO) and non-enzymatic oxidation of ascorbic oven, respectively, was found to be favorable with respect to anthocyanin
acid in the presence of oxygen (Larsen & Poll, 1995; Liao & Seib, 1988; retention. Ascorbic acid was also best retained when strawberries were
Skrede, 1996). Most interestingly, thawing at 4 °C caused the most pro- thawed in a microwave oven. Interestingly, thawing the fruits at 4 °C,
nounced losses (34.8%) which might be attributed to the prolonged expo- which is common practice, caused most pronounced pigment and ascor-
sure to oxygen during thawing at this temperature (24 h). In contrast, bic acid losses. This was assumed to result from the prolonged thawing
only 4.0% was lost when strawberries were thawed within 10 min in a mi- period at 4 °C leading to increased oxidation of anthocyanins and ascorbic
crowave oven. Thawing at 20 °C (8 h) and 37 °C (2 h) resulted in losses acid by genuine enzyme activities such as PPOs and AAO, respectively. Ac-
accounting for 21.8% and 16.9%, respectively. This is consistent with the cordingly, anthocyanin degradation proceeded during further storage of
results of Hartmann et al. (2008), who reported losses of ~20% during thawed fruits, and ascorbic acid loss was almost completely annihilated
thawing of ‘Senga sengana’ at 19 °C for 15 h. Ascorbic acid loss was almost after 48 h at 20 °C. Therefore, it was concluded that thawing regime is a
complete when thawed strawberries were further stored at 20 °C and key factor to improve pigment and ascorbic acid retention in strawberries.
37 °C, respectively, indicating again that thawing time is a key factor to
improve ascorbic acid retention in strawberries, while ascorbic acid reten- Acknowledgments
tion was not determined by the previous freezing technology, i.e. block
and cryogenic freezing. We are grateful to Odenwald Früchte (Breuberg, Germany) for provid-
ing the strawberries. This research project was supported by the German
3.5. Impact of freezing and thawing on color properties of strawberry Ministry of Economics and Technology (via AiF) and the FEI (Forschung-
extracts skreis der Ernährungsindustrie e.V., Bonn), Project AiF 16005N.

As can be seen in Table 3, thawing the fruits at 20 °C for 8 h resulted in References

a slight increase of L* values for all cultivars. However, a considerable
Aaby, K., Ekeberg, D., & Skrede, G. (2007). Characterization of phenolic compounds in
lightening (ΔL*extract =3.3 units) was observed, when strawberries were strawberry (Fragaria × ananassa) fruits by different HPLC detectors and contribu-
thawed at 4 °C for 24 h. In contrast, a* values were found to decrease tion of individual compounds to total antioxidant capacity. Journal of Agricultural
which may be probably attributed to the enzymatic degradation of antho- and Food Chemistry, 55, 4395–4406.
Aaby, K., Wrolstad, R. E., Ekeberg, D., & Skrede, G. (2007). Polyphenol composition and
cyanins during thawing, as described earlier. While thawing at 20 °C for antioxidant activity in strawberry purées; impact of achene level and storage.
8 h only resulted in a slight decrease of the a* value (Δa*extract =0.5 units Journal of Agricultural and Food Chemistry, 55, 5156–5166.
for ‘Senga sengana’), severe color losses were observed for thawing at Asami, D. K., Hong, Y. -J., Barrett, D. M., & Mitchell, A. E. (2003). Comparison of the total
phenolic and ascorbic acid content of freeze-dried and air-dried marionberry,
37 °C (2 h) and 4 °C (24 h) amounting to 5.0 and 4.9 units, respectively. strawberry, and corn using conventional, organic, and sustainable agricultural
Accordingly, such detrimental thawing conditions were associated with practices. Journal of Agricultural and Food Chemistry, 51, 1237–1241.
the most pronounced pigment losses (Table 4). In most samples, b* values Bardonaba, J. G., & Terry, L. A. (2010). Manipulating the taste-related composition of
strawberry fruits (Fragaria × ananassa) from different cultivars using deficit irriga-
were also lowered after thawing. The highest declines of the b* values tion. Food Chemistry, 122, 1020–1026.
(Δb*extract ~8.7 units) were determined for the samples thawed at 37 °C Buendía, B., Gil, M. I., Tudela, J. A., Gady, A. L., Medina, J. J., Coria, C., et al. (2010). HPLC-
(2 h) and 4 °C (24 h). Total color differences ΔE* were also highest in MS analysis of proanthocyanidin oligomers and other phenolics in 15 strawberry
cultivars. Journal of Agricultural and Food Chemistry, 58, 3916–3926.
those samples. As can be seen from Table 3, only minor differences
Cano, M. P., De Ancos, B., & Lobo, G. (1995). Peroxidase and polyphenoloxidase activi-
were observed for hue angle values h° of strawberry extracts, which is ties in papayas during postharvest ripening and after freezing/thawing. Journal of
presumably due to the concomitant decrease of a* and b* values upon Food Science, 60, 815–817.
thawing. Chroma C* was found to decrease upon thawing independent Cao, X., Zhang, Y., Zhang, F., Wang, Y., Yi, J., & Liao, X. (2011). Effects of high hydrostatic
pressure on enzymes, phenolic compounds, anthocyanins, polymeric color and
of the thawing regime. Interestingly, applying different freezing methods color of strawberry pulps. Journal of the Science of Food and Agriculture, 91,
did not affect the color properties of the extracts. 877–885.
248 M. Holzwarth et al. / Food Research International 48 (2012) 241–248

Carle, R., Borzych, P., Dubb, P., Siliha, H., & Maier, O. (2001). A new process for firmer Lunkenbein, S., Bellido, M., Aharoni, A., Salentijn, E. M. J., Kaldenhoff, R., Coiner, H. A.,
canned cherries and strawberries. Food Australia, 53, 343–348. et al. (2006). Cinnamate metabolism in ripening fruit. Characterization of a UDP-
Cerezo, A. B., Cuevas, E., Winterhalter, P., Garcia-Parrilla, M. C., & Troncoso, A. M. glucose: Cinnamate glucosyltransferase from strawberry. Plant Physiology, 140,
(2010). Isolation, identification, and antioxidant activity of anthocyanin com- 1047–1058.
pounds in Camarosa strawberries. Food Chemistry, 123, 574–582. Määttä-Riihinen, K., Kamal-Eldin, A., & Törrönen, A. R. (2004). Identification and quan-
Chandra, A., Rana, J., & Li, Y. (2001). Separation, identification, quantification and meth- tification of phenolic compounds in berries of Fragaria and Rubus species (Family
od validation of anthocyanins in botanical supplement raw materials by HPLC and Rosaceae). Journal of Agricultural and Food Chemistry, 52, 6178–6187.
HPLC–MS. Journal of Agricultural and Food Chemistry, 49, 3515–3521. Ngo, T., Wrolstad, R. E., & Zhao, Y. (2007). Color quality of Oregon strawberries — Impact
Chisari, M., Barbagallo, R. N., & Spagna, G. (2007). Characterisation of polyphenol oxi- of genotype, composition, and processing. Journal of Food Science, 72, C25–C32.
dase and peroxidase and influence on browning of cold stored strawberry fruit. Oszmiański, J., Wojdylo, A., & Kolniak, J. (2009). Effect of L-ascorbic acid, sugar, pectin
Journal of Agricultural and Food Chemistry, 55, 3469–3476. and freeze–thaw treatment on polyphenol content of frozen strawberries. Food
Clifford, M. N., Johnston, K. J., Knight, S., & Kuhnert, N. (2003). Hierarchical scheme for LC– Science and Technology, 42, 581–586.
MSn identification of chlorogenic acids. Journal of Agricultural and Food Chemistry, 51, Schweiggert, U., Schieber, A., & Carle, R. (2005). Inactivation of peroxidase, polyphenol-
2900–2911. oxidase, and lipoxygenase in paprika and chili powder after immediate thermal
Davey, M. W., Van Montagu, M., Inzé, D., Sanmartin, M., Kanellis, A., Smirnoff, N., et al. treatment of the plant material. Innovative Food Science and Emerging Technologies,
(2000). Plant L-ascorbic acid: Chemistry, function, metabolism, bioavailability and 6, 403–411.
effects of processing. Journal of the Science of Food and Agriculture, 80, 825–860. Shikov, V., Kammerer, D. R., Mihalev, K., Mollov, P., & Carle, R. (2008). Heat stability of
Delgado, A. E., & Rubiolo, A. C. (2005). Microstructural changes in strawberry after strawberry anthocyanins in model solutions containing natural copigments
freezing and thawing processes. Lebensmittelwissenschaft und -Technologie, 38, extracted from rose (Rosa damascena Mill.) petals. Journal of Agricultural and
135–142. Food Chemistry, 56, 8521–8526.
Hartmann, A., Patz, C. -D., Andlauer, W., Dietrich, H., & Ludwig, M. (2008). Influence of Simirgiotis, M. J., Theoduloz, C., Caligari, P. D. S., & Schmeda-Hirschmann, G. (2009).
processing on quality parameters of strawberries. Journal of Agricultural and Food Comparison of phenolic composition and antioxidant properties of two native
Chemistry, 56, 9484–9489. Chilean and one domestic strawberry genotype. Food Chemistry, 113, 377–385.
Holzwarth, M., Korhummel, S., Carle, R., & Kammerer, D. R. (2012). Impact of enzymatic Singh, R. P., & Wang, C. Y. (1977). Quality of frozen foods — A review. Journal of Food
mash maceration and storage on anthocyanin and color retention of pasteurized Process Engineering, 1, 97–127.
strawberry purées. European Food Research and Technology, 234, 207–222. Skrede, G. (1996). Effect of freezing on fruits. Food Science and Technology, 72, 183–245.
Kajdžanoska, M., Gjamovski, V., & Stefova, M. (2010). HPLC-DAD–ESI–MSn identification of Skupień, K., & Oszmiański, J. (2004). Comparison of six cultivars of strawberries
phenolic compounds in cultivated strawberries from Macedonia. Macedonian Journal (Fragaria × ananassa Duch.) grown in northwest Poland. European Food Research
of Chemistry and Chemical Engineering, 29, 181–194. and Technology, 219, 66–70.
Kawanobu, S., Wajima, T., Zushi, K., Mori, T., & Matsuzoe, N. (2010). Seasonal variations Stintzing, F. C., & Carle, R. (2004). Functional properties of anthocyanins and betalains
in the maturation period, anthocyanin content, and ascorbic acid content in in plants, food, and in human nutrition. Trends in Food Science and Technology, 15,
strawberry fruits. Environmental Control in Biology, 48, 175–184. 19–38.
Larsen, M., & Poll, L. (1995). Changes in the composition of aromatic compounds and Tomás-Barberán, F. A., & Espín, J. C. (2001). Phenolic compounds and related enzymes
other quality parameters of strawberries during freezing and thawing. Zeitschrift as determinants of quality in fruits and vegetables. Journal of the Science of Food and
für Lebensmitteluntersuchung und -Forschung, 201, 275–277. Agriculture, 81, 853–876.
Liao, M. -L., & Seib, P. A. (1988). Chemistry of L-ascorbic acid related to foods. Food Van Buggenhout, S., Sila, D. N., Duvetter, T., Van Loey, A., & Hendrickx, M. (2009). Pectins in
Chemistry, 30, 289–312. processed fruits and vegetables: Part III — Texture engineering. Comprehensive Reviews
Lopes da Silva, F., Escribano-Bailón, M. T., Alonso, J. J. P., Rivas-Gonzalo, J. C., & Santos-Buelga, in Food Science and Food Safety, 8, 105–117.
C. (2007). Anthocyanin pigments in strawberry. LWT- Food Science and Technology, 40, Wills, R. B. H., & Kim, G. H. (1995). Effect of ethylene on postharvest life of strawberries.
374–382. Postharvest Biology and Technology, 6, 249–255.
López-Serrano, M., & Ros Barceló, A. (2001). Histochemical localization and develop-
mental expression of peroxidase and polyphenol oxidase in strawberries. Journal
of the American Society for Horticultural Science, 126, 27–32.