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R E S E A R C H Open Access
Refrigerated and frozen storage impact
aronia berry quality
Erica S. King
1
, Andrea Noll
1
, Susan Glenn
2
and Bradley W. Bolling
1*
Abstract
Postharvest storage of many freshly picked berries affects polyphenol and sugar content. However, little is known
about the impact of refrigerated and frozen storage on aronia berry composition. Therefore, the objective of this
study was to characterize how storage at 4 ± 2 °C and −20 ± 2 °C, and temperature cycles affect aronia berry
polyphenols, total solid content, pH, titratable acidity, polyphenol oxidase (PPO) activity, sugar content, acid
content, color, and cell structure. Refrigerated storage reduced proanthocyanidins (21%), anthocyanins (36%), and
total phenols (21%) after 12 weeks. Frozen storage increased polyphenols in the first 6 mo. of frozen storage but
then decreased polyphenols at mo. 8 to levels similar to initial values. Frozen temperature cycling reduced
anthocyanins 18% but did not affect total phenols or proanthocyanidins. Scanning electron microscopy analysis
indicated temperature cycling induced cell damage, shrinking, and fusion. This disruption led to the release of
anthocyanins inside the berry tissue. PPO activity did not significantly correlate with the decrease in polyphenol
content during storage. °Brix did not significantly change during refrigeration and frozen storage but did during the
12th temperature cycle. Aronia berries’pH and titratable acidity were affected more by refrigeration than frozen
and temperature storage. The pH increased by 4% during refrigeration, and titratable acidity decreased by 17% at
12 weeks. In conclusion, refrigerated storage results in a modest reduction of aronia berry polyphenols, but absolute
extractable polyphenols are stable for up to 8 months of frozen storage.
Keywords: Aronia berry, Polyphenols, Storage, Refrigeration, Sugars, Acids, Frozen
Introduction
Berries have polyphenols and nutrients that promote hu-
man health (Diaconeasa 2018). Unlike climacteric fruit,
berries are non-climacteric and harvested at full maturity
because they will not continue to ripen (Kahramanoglu
2017). However, because they are harvested fully rip-
ened, fresh berries have short shelf lives because of high
respiration rates, water loss, and susceptibility toward
decay and damage. Thus, postharvest storage changes
berry composition, including soluble solids, pH, titrat-
able acidity, polyphenol content, vitamins, and minerals
(Kahramanoglu 2017). Therefore, it is essential to
characterize and optimize the postharvest storage of ber-
ries to limit polyphenol and nutrient loss.
Berries are preserved by refrigeration, freezing, drying,
canning, or processing into jams, jellies, and juices. Berry
polyphenols are sensitive to light, oxygen, increased pH,
and heat, so refrigeration and freezing are used for short
and long-term storage (Diaconeasa 2018). Lowering the
temperature reduces mold growth, respiration rate, pig-
ment degradation, chemical changes in flavor, acid de-
terioration, sugar conversions, and enzymatic reactions
with polyphenols and the cell structure (Yahia et al.
2017). Prior studies have illustrated how freezing im-
pacts berry polyphenols over extended periods. The re-
sults from all the different studies indicate, the rates of
change of polyphenols, nutrients, sugars, and acids dur-
ing storage and temperature depends on the berry type,
cultivar, and fruit maturity (Khattab et al. 2015; Rickman
et al. 2007;Šamec & Piljac-Žegarac 2014).
When fruit is stored in the freezer, most water be-
comes ice, reducing microbial, oxidation, and enzymatic
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* Correspondence: bwbolling@wisc.edu
1
Department of Food Science, University of Wisconsin-Madison, 1605 Linden
Dr., Madison, WI 53706, USA
Full list of author information is available at the end of the article
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King et al. Food Production, Processing and Nutrition (2022) 4:3
https://doi.org/10.1186/s43014-021-00080-y
reactions (Šamec & Piljac-Žegarac 2014). Consequently,
cell damage occurs from the ice-crystal formation during
freezing, resulting in alterations of a fruit’s structure and
composition. Additionally, the fruit will go through
temperature fluctuations due to storage (refrigeration
temperature change), transportation, and consumer
treatment, leading to temperature abuse that cause a re-
duction in nutrients and physical deterioration (Hajji
et al. 2019).
Aronia berries contain significant amounts of antho-
cyanin, proanthocyanidin, phenolic acids and flavonol
polyphenols. Anthocyanins are the predominant poly-
phenol in aronia berries and are mainly cyanidin glyco-
sides. Taheri et al. (2013) reported the order of
anthocyanin abundance as: cyanidin-3-O-galactoside >
cyanidin-3- O-arabinoside > cyanidin-3- O-glucoside.
The proanthocyanidins in aronia berries are mainly
highly-polymerized B-type procyanidins (Taheri et al.
2013; Wu et al. 2004). The primary proanthocyanidins
in aronia berries are polymers with degrees of
polymerization > 10 and contain 1–19% with degrees of
polymerization < 10 (Taheri et al. 2013; Wu et al. 2004).
Aronia flavonols are largely quercetin galactosides, glu-
cosides, or rutinosides attached to the molecule, while
its phenolic acids are primarily hydroxcinnamic acids
(Taheri et al. 2013). The abundance of these polyphe-
nols, particularly the anthocyanins, contribute to the po-
tential health benefits of aronia berry consumption
(King & Bolling 2020; Valdez & Bolling 2019).
Less is known about the impact of refrigerated and
frozen storage on aronia berry quality. Prior studies re-
ported how storage affects the phytochemicals in aronia
berry juice, jam, and puree (Wilkes et al. 2014; Georgiev
and Ludneva 2009; Yuan et al. 2018). Nevertheless, the
stability of heat-treated and fresh fruit is not comparable
due to the deactivation of enzymes in processed prod-
ucts (Piljac-Žegarac & Šamec 2011). To our knowledge,
there are currently no reports on how refrigerated and
frozen storage affects aronia berry quality. Therefore,
this study was created to increase our understanding of
how aronia berries change in composition during long-
term storage. This study evaluates the effects on aronia
berries in different storage conditions (refrigeration 4 ±
2 °C, freezer −20 ± 2 °C, and temperature cycles) by
examining polyphenols, total solid content, pH, titratable
acidity, polyphenol oxidase (PPO), sugar content, acid
content, color, and cell structure.
Materials and methods
Reagents
(+)-Catechin hydrate (98% purity) was from Cayman
Chemical (Ann Arbor, MI, U.S.A.). 4- (Dimethylamino)
cinnamaldehyde, TRIZMA buffer (primary Standard and
Buffer, ≥99.9%), TRIZMA hydrochloride (reagent grade,
≥99.0%), catechol (99% purity),calcium chloride dihy-
drate (ACS reagent grade, 99% pure), sodium dibasic
(ACS reagent grade), sodium phosphate monobasic
(ACS reagent grade), Titron x-100 (laboratory grade),
Folin & Ciocalteu’s phenol reagent, formic acid (reagent
grade ≥95% pure)), sodium bicarbonate (BioReagent),
were from Sigma-Aldrich (St. Louis, MO, U.S.A). Acet-
one (HPLC grade), hydrochloric acid (ACS reagent
grade), potassium acid phthalate, potassium chloride
(ACS reagent grade) from Thermo Fisher Scientific
(Waltham, MA, U.S.A). Ethanol (anhydrous, USP stand-
ard) was from Decon Labs (King of Prussia, PA, U.S.A.).
Gallic acid monohydrate (ACS reagent grade) was from
Acros Organic morris plains NJ, USA. Sodium Hydrox-
ide (1.0007 N) was from La-Mar-Ka (Baton Rouge, LA.
U.S.A). Ultrapure water was filtered at > 18.1 M Ωcm
using a Barnstead water filtration system (Thermo Fisher
Scientific).
Storage conditions
For experiments evaluating storage on aronia berry qual-
ity, ‘Viking’aronia berries were collected from 6 different
plants in Madison, equivalent to week 4 of harvest, were
analyzed as n= 4 composite samples representing berries
from 1 to 3 different plants each. For these experiments,
~ 125 g of freshly harvested berries were sealed in a
sealed plastic bag and subjected to different storage con-
ditions. These included fresh berries (extracts and juices
were prepared on the same day of harvest), refrigeration
(4 ± 2 °C for 2, 4, 6, 8, 10, 12 weeks), freezer (−20 ± 2 °C
for 1, 2, 4, 6, 8 mo.), and temperature cycles. To
minimize temperature change, the berries were stored in
drawers during the refrigeration, freezer, and
temperature cycles experiments.
Frozen temperature cycling experiments
Fresh aronia berries were placed in 125 g aliquots in
sealed plastic bags and stored in a −20 ± 2 °C freezer
drawer for 4 mo., using a frost-free freezer (Insignia -
17.0 Cu. Ft. Frost-Free Upright Convertible Freezer/Re-
frigerator; Model: NS-UZ17WH0). The freezing point of
aronia berry juice was determined to be −2 °C using The
Advanced™Osmometer Model 3250 (Thermo Fisher Sci-
entific, Waltham, MA, U.S.A). Next, the frozen berries
were subjected to temperature cycles making sure the
temperature of berries did not rise above the freezing
point of aronia berry juice. Each cycle started with ber-
ries at −20 ± 2 °C, followed by placing the berries in
open bags in a 60 Hz microwave on defrost setting for 5
s. The bag of berries were taken out of the microwave
shaken up and placed back in the microwave for 5 s.
Then, a temperature probe was used to measure the
temperature of three different berries. Berries with tem-
peratures −3to−2 °C were placed back in the freezer.
King et al. Food Production, Processing and Nutrition (2022) 4:3 Page 2 of 12
However, if the berries were below −3 °C, they were
microwaved for another 5 or 10 s until they reached −3
or −2 °C. Every other day, the procedure was repeated to
ensure the aronia berries were frozen before warming
them up again to −2 °C. Berries went through 24
temperature cycles, the composition of the aronia berry
was evaluated every 6th temperature cycle. Experiments
were repeated in 4 separate batches for each of the four
conditions. After each temperature cycle was completed,
the berries were analyzed for their polyphenol, sugar,
acid, and PPO content. Additionally, they were lyophi-
lized to examine the cell structure by microscopy and
color change.
SEM analysis
Representative aronia samples were taken from each
temperature cycle (6, 12, 18, and 24) with additional
control aronia samples that did not go through the
temperature cycles. The samples were cut in half to
show the cells and tissue structure under a scanning
electron microscope (SEM). The fractions were placed in
a freeze-dryer for 48 h to ensure no moisture was left in
the aronia samples. Samples were mounted on a
12.7X11MM pin mount (Ted Pella Inc., Redding, CA,
U.S.A) fracture side up and coated with platinum 10 um
thick, using a Prep-Leica ACE600 Deposition. The sam-
ples were with an SEM Zeiss GeminiSEM 450. Pictures
were taken using the magnification of 50x. Two samples
from control and each treatment were examined and
used for results.
Polyphenol content
To extract polyphenols, aronia berries were placed under
liquid nitrogen and immediately ground to a powder
with a blender. The frozen berry powder (100 mg) was
placed into a 25 mL centrifuge tube with 10 mL of acet-
one: water (70:30) and extracted for 24 h on a test tube
rocker at ambient conditions. The polyphenol profile
aronia berries has been well established elsewhere (King
& Bolling 2020; Taheri et al. 2013). Spectrophotometric
methods for analysis of anthocyanins, proanthocyani-
dins, and total phenols correlate with LC-MS and
HPLC-DAD methods for quantitation of aronia pheno-
lics (Taheri et al. 2013; Bolling et al. 2015). Thus, stan-
dardized spectrophotometric methods adapted for 96-
well microplates were used for aronia berry phenolic
analysis. Extracts were centrifuged, and total phenols
were assessed by the Folin-Ciocalteu assay as gallic acid
equivalents (Singleton et al. 1999); anthocyanins by the
pH differential method as cyanidin-3-glucoside equiva-
lents (Lee et al. 2005); and proanthocyanidins by the 4-
dimethylaminocinnamaldehyde (DMAC) method as
(+)-catechin equivalents (Prior et al. 2010).
Acid and °brix content
Aronia berries (45 g) were juiced using a hand press
prior to the determination of pH, titratable acidity,
and °Brix. Briefly, juice was centrifuged and pH deter-
mined with a Seven Compact pH/Ion meter S220
(Mettler Toledo, Columbus, OH, U.S.A.); titratable
acidity by titration with potassium acid phthalate to
pH 7.0 (Nielsen 2003); and °Brix with an Abbe re-
fractometer (Thermo-Spectronic, U.S.A).
Polyphenol oxidase (PPO) activity
The method for extraction and analysis of PPO activ-
ity was modified from Siddiq & Dolan (2017). First,
15 g of aronia berries were submerged in liquid nitro-
gen and blended to a powder, from which 12.5 g was
transferred to a 50 mL centrifuge tube. To lyse any
intact cells, 25 mL of 0.1 M pH 9.5 TRIZMA buffer
and 0.5% of Triton X-100 was added to the centrifuge
tube, vortexed for 30 s, and placed on a rocker for 10
min. The sample was centrifuged for 2465 ⨯gfor20
min at 4 °C and the supernatant was discarded. To
wash the residue, 50 mL of acetone was added and
then vortexed for 30 s, centrifuged at 2465 ⨯gfor10
min at 4 °C. After the wash, the supernatant was
again discarded. To extract PPO and precipitate pec-
tin, 6 mL of 0.1 M pH 7.0 sodium phosphate buffer
and 2 mL of 0.3 M of calcium chloride was added to
the residue. This solution was vortexed for 30 s,
placed on a rocker for 10 min, and centrifuged at
2465 ⨯gfor20minat4°C.Thesupernatantwasre-
served for analysis of PPO activity. For this, 15 μL
supernatant of PPO extract was aliquoted into a 96-
well plate in quadruplicate. Then, 285 μLof0.3M
catechol and 0.1 M pH 7.0 sodium phosphate buffer
were added to wells with the extract. The microplate
was quickly placed in a spectrophotometer, and the
change in absorbance was measured at 420 nm for 3
min every 5 s. PPO activity was defined by a 0.001
change in absorbance per min (△A420/ min).
Statistical analysis
Results were expressed by the means ± standard devia-
tions of four different composite berry samples, with
triplicate analysis of each aliquot. Results were analyzed
using one-way ANOVA, with significance of P< 0.05.
Tukey’s multiple comparisons were performed where
ANOVA determined the data was significantly different.
Data were analyzed using Rstudio (Rstudio, Boston, MA,
U.S.A) and SAS (SAS Institute, Cary, NC, U.S.A) soft-
ware, using total phenols, monomeric anthocyanins,
proanthocyanidins, PPO, pH, titratable acidity, °Brix, and
°Brix: acid as variables.
King et al. Food Production, Processing and Nutrition (2022) 4:3 Page 3 of 12
Results and discussion
Refrigeration and aronia berry quality
Aronia berry polyphenols
Initial values of total phenols, monomeric anthocyanins,
and proanthocyanidins from fresh aronia berries were
slightly lower than Bolling et al. (2015) (Fig. 1A-C). The
difference may be due to weather conditions, geograph-
ical location, and other pre-harvest factors. Aronia berry
polyphenol content fluctuated throughout storage at 4 ±
2 °C. Total phenols slightly increased at 2 weeks and de-
clined for the rest of the 10 weeks (Fig. 1A). At 12 weeks,
total phenols decreased by 21%. Fluctuation occurred at
the first 6 weeks and then reduced slightly until week 12.
Monomeric anthocyanins followed a similar pattern as
total phenols, as it had a slight increase at week 2, and
then decreased 36% by week 10 (Fig. 1B). Proanthocyani-
dins decreased from 1.95 at 0 weeks to 1.52 mg CE/g at
12 weeks (Fig. 1C). Former studies observed a similar
trend with strawberries as total phenolics and anthocya-
nins increased at 2 weeks of storage (Šamec & Piljac-
Žegarac 2011). Free polyphenols are more sensitive to
light, PPO, and other enzymes, leading to reduced con-
centrations in plant tissue. The increased polyphenol
content may also be attributed to phenylalanine
ammonia-lyase (PAL) activated from cold-temperature
stress (Lattanzio 2003). The anthocyanin decrease may
Fig. 1 Storage at 4 °C affects fresh aronia berry polyphenols. After refrigerated storage, aronia berry was analyzed for (A) extractable total phenols
by Folin–Ciocalteu method as gallic acid equivalents (GAE); (B) extractable monomeric anthocyanins by pH differential method as cyandin-3-
glucoside equivalents (C3GE); (C) extractable proanthocyanins by 4-dimethylaminocinnamaldehyde (DMAC) method as catechin equivalents (CE);
(D) extractable polyphenol oxidase (PPO) activity; (E) juice °Brix, (F) juice titratable acidity; (G) juice pH; and (E) juice °Brix: acid ratio. Week 0
represents analysis of unfrozen fresh berries picked within 6 h. A unit of PPO activity is a 0.001 change in absorbance per min (△A420/ min). Data
are means ± standard deviation of n= 4 aliquots aronia plants, with triplicates for each plant. Data were analyzed by one-way ANOVA with
Tukey’s multiple comparison test vs. the fresh sample, * P< 0.05 vs. time 0
King et al. Food Production, Processing and Nutrition (2022) 4:3 Page 4 of 12
be explained by increased pH, which destabilizes antho-
cyanins (Wu et al. 2010). In contrast, refrigerated storage
increases anthocyanins and total phenols in blackberries,
cherries, plums, and other fruits (Šamec & Piljac-Žegarac
2011). Nevertheless, polyphenols’reaction patterns differ
during refrigerated storage due to phenolic composition,
ripeness, and enzyme activity (Wu et al. 2010).
Aronia berry PPO activity
During 4 ± 2 °C storage of fresh aronia berry, its PPO ac-
tivity increased by 49% at week 2, then decreased at
week 4, remaining consistent for the next 4 weeks, and
then increased by 65% at week 10 (Fig. 1D). Visual in-
spection over the 12 weeks did not reveal the obvious
color or other physical changes. However, at week 13,
mold growth was observed. The PPO trend for aronia
berry was similar to that reported in pear, as PPO activ-
ity increased at 2 weeks (Yan et al. 2013). This increase
may be from damage from cooling the berries, thus in-
creasing the expression of PPO activity.
Although PPO enzymes did not significantly affect or
correlate with the polyphenol concentrations during
storage, other enzymes may have caused changes in
polyphenols. The phenylalanine ammonia-lyase (PAL),
an enzyme that acts as a bridge between the primary
pathway (shimiki pathway) and the secondary pathway
(phenylpropanoid pathway) in the phenolic metabolism,
may increase in berries due to low-temperature stress
(Lattanzio 2003). In one study, cherries, sour cherries,
strawberries, and red currents increased in their antho-
cyanin and or flavonoid content during storage at 4 °C
(Piljac-Žegarac & Šamec 2011). Wu et al. (2010) demon-
strated blackberries’anthocyanin concentration also rose
during storage at 2 °C.
Conversely, enzymes, including peroxidase (POD) and
PPO, are linked to polyphenol metabolism, causing a re-
duction in anthocyanins and other polyphenols (Lattan-
zio 2003). POD enzymes are highly thermostable that
cause an oxidation reaction when hydrogen peroxide is
present (Nokthai et al. 2010). Studies concluded that
PPO’s involvement in oxidizing polyphenols is hydrolyz-
ing a monophenol to an o-diphenol, creating an o-
quinone, causing a brown color to form or lose pigment
in fruits (Nokthai et al. 2010). Future studies need to
examine the correlation of polyphenol concentrations to
other enzymes during storage studies.
Aronia berry °brix and acid
During refrigerated storage, aronia berry juice °Brix fluc-
tuated with no significant difference between the 12
weeks (Fig. 1E). Titratable acidity declined 17% during at
week 12 of storage (Fig. 3F). However, after week 6, ti-
tratable acidity stayed relatively consistent. Aronia berry
pH increased at weeks 2, 6, 8, 10, and 12 (Fig. 3G). A
similar trend was observed for the °Brix: acid content,
increasing by 24% at the end of 12 weeks (Fig. 3H).
Changes in °Brix and acid during storage can
change the taste of fruit (Yanyun Zhao 2007,pp.
213–214). Refrigeration can reduce titratable acidity
loss by slowing down the primary metabolism (Briz-
zolara et al. 2020). However, the sugar-acid metabol-
ism is not entirely stopped and can still impact
acidity and °Brix. Previous studies reported refriger-
ated storage increases pH and decreases titratable
acidy in blackberries (Kim et al. 2015; Perkins-Veazie
et al. 1999;Wuetal.2010) and mangoes (Hossain
et al. 2014).
Long-term frozen storage of aronia berry
Aronia berry polyphenols
Storage of aronia berry at −20 ± 2 °C changed total phe-
nols at only one time point. At 6 mo. total phenols were
increased, but then declined again at 8 mo. (Fig. 2A).
Thus, at 8 mo., total phenols remained the same as that
as the fresh aronia berries. Anthocyanins (Fig. 2B) and
proanthocyanidins (Fig. 2C) were also increased only at
6 mo. of frozen storage.
The effect of frozen storage on fruit polyphenols de-
pends on the type of fruit, its protein/ sugar content
(both protein and sugar have a cryoprotective effect),
phenolic composition, biosynthesis during post-harvest,
enzyme concentration, preharvest factors (ripeness, loca-
tion, weather, cultivation method, season), and cultivar
(Neri et al. 2020;Šamec & Piljac-Žegarac 2014). Past
studies have described little change to total phenols dur-
ing storage of raspberries, blueberries, cherries, black-
berries, and grapes (González et al. 2003; Cocetta et al.
2015;Šamec & Piljac-Žegarac 2014). Šamec & Piljac-
Žegarac (2014) observed the most fluctuation in straw-
berries during frozen storage, increasing 13% at 3 mo.,
followed by a 40% decrease at 6 mo., and a 19% increase
at 12 mo. In contrast, other studies report decreased
total phenolic changes during frozen storage. After 10
mo. at −20 °C, total phenols decreased by 28% for blue-
berries, 42% for blackberries, and 47% for raspberries
(Poiana et al. 2010). However, results are inconsistent
between different studies as polyphenol content of vari-
ous berries may decrease (Chaovanalikit & Wrolstad
2004), increase (de Ancos et al. 2000; Urbanyi & Horti
1992), or remain unchanged (Cocetta et al. 2015; Khat-
tab et al. 2015). The increase in polyphenol content is
mainly attributed to cellular degradation, causing a re-
lease of polyphenols from the cell structure and increas-
ing extractability (Sablani 2015). This may explain the
observed increase and subsequent decrease of aronia
berry phenolics as free polyphenols’degradation rates
are increased.
King et al. Food Production, Processing and Nutrition (2022) 4:3 Page 5 of 12
Fig. 2 Storage at −20 °C affects aronia berry polyphenol content. After frozen storage, aronia berry was analyzed for (A) extractable total phenols
by Folin–Ciocalteu method as gallic acid equivalents (GAE); (B) extractable monomeric anthocyanins by pH differential method as cyandin-3-
glucoside equivalents (C3GE); (C) extractable proanthocyanins by 4-dimethylaminocinnamaldehyde (DMAC) method as catechin equivalents (CE);
(D) extractable polyphenol oxidase (PPO) activity; (E) juice °Brix, (F) juice titratable acidity; (G) juice pH; and (E) juice °Brix: acid ratio. Week 0
represents analysis of unfrozen fresh berries picked within 6 h. A unit of PPO activity is a 0.001 change in absorbance per min (△A420/ min). Data
are means ± standard deviation of n= 4 aliquots aronia plants, with triplicates for each plant. Data were analyzed by one-way ANOVA with
Tukey’s multiple comparison test vs. the fresh sample, * P< 0.05 vs. time 0
King et al. Food Production, Processing and Nutrition (2022) 4:3 Page 6 of 12
Frozen aronia berry PPO activity
Prolonged frozen storage increased aronia berry PPO ac-
tivity (Fig. 2D). Aronia berry PPO activity was reduced
at 1 mo. of frozen storage, but at 4 mo., PPO activity in-
creased by 111% compared to the fresh aronia berries.
At subsequent months, PPO activity was decreased from
the peak 4 mo. activity. The decrease is to be expected
as PPO activity eventually decreases during storage
(Nokthai et al. 2010). The increase in activity in the first
4 months is likely a result of tissue damage from freezing
the aronia berries (Concellón et al. 2004; Khattab et al.,
2015; Lattanzio 2003). The formation of large ice crystals
can damage cells, releasing PPO and increasing reactivity
with polyphenols. Similar results, with an increase in
PPO activity during long-term frozen storage, were seen
in dates (Alhamdan 2016) and papaya (Cano et al. 1998).
However, the contribution of increased PPO to aronia
polyphenol levels in frozen berries is expected to be low
because PPO activity is reduced below 7 °C (Singh, B.,
2018).
Frozen aronia berry °brix and acid
Frozen storage had a limited impact on aronia berry
°Brix, acid, pH, and °Brix: acid (Fig. 2E-H). At 6 mo., ti-
tratable acidity was increased by 14% from initial values,
but at 8 mo., °Brix: acid was similar to initial values.
Studies have indicated that when frozen, acids in ber-
ries and citric fruits are more stable than other fruits
such as peaches (Dawson et al. 2020; Skrede G 1996).
Additionally, berries contain different respiration rates:
blueberries, currants, and cranberries have slower respir-
ation rates and a longer storage life than blackberries
and raspberries (Yanyun Zhao 2007, pp. 207–223). Aro-
nia berries may contain lower respiration and acid me-
tabolism rates, causing only slight changes in titratable
acidity during storage.
Frozen temperature cycling of frozen aronia berry
Aronia berry polyphenols during temperature cycling
Temperature cycling initially decreased aronia berry
total phenols from 6 to 12 cycles, but values normalized
to baseline after 18 and 24 cycles (Fig. 3A). Aronia berry
anthocyanins were decreased 24 to 33% after 12 and 18
cycles (Fig. 3B). In contrast, aronia berry proanthocyani-
dins were not affected by up to 24 temperature cycles
(Fig. 3C). The increase in total phenols can be explained
by ice-induced cellular destruction, thus increasing rates
of polyphenol extraction during analysis. Apparently,
these changes are independent of anthocyanins and
proanthocyanidins, indicating that low molecular weight
phenolics are impacted more than large molecular
weight proanthocyanidins. Therefore, anthocyanin ex-
traction may increase but be destabilized during
redistribution.
Holzwarth et al. (2012) described strawberries sub-
jected to temperature cycling and reported increased
total phenols but reduced anthocyanins. The recovery
of strawberry anthocyanins in microwaved berries was
higher than those thawed at 4 or 25 °C (Holzwarth
et al. 2012). Anthocyanins are degraded as the
temperature cycles increase damage to cell structures,
allowing anthocyanins to be released and exposed to
a higher pH, leading to the formation of less-stable
quinoidal bases, carbinol pseudo-bases, or chalcones
(Sablani 2015). Additionally, exposed anthocyanins are
more susceptible to enzymes, ascorbic acid, metal
ions, oxygen, and light (Holzwarth et al. 2012;Sablani
2015).
Aronia berry PPO activity after temperature cycling
At 18 cycles, aronia berry PPO activity increased 31%
from baseline but returned to baseline at 24 cycles
(Fig. 3D). PPO activity and anthocyanin content were
not statistically significant (r = −0.880, p= 0.49).
Temperature cycling had a limited impact on aronia
berry PPO activity relative to refrigerated and frozen
storage.
The temperature-cycling method used a microwave
on the defrost setting to obtain homogeneous defrost-
ing time for all berries (Li & Sun, 2002). Former
studies used a microwave as a technique for
temperature-cycling experiments and concluded no
deterioration in polyphenols (Holzwarth et al. 2012).
However, there is no certainty the microwave did not
affect the PPO enzymes in the aronia berries, causing
lower PPO activity than frozen and refrigeration
storage.
Aronia berry °brix and acid after temperature cycling
Aronia berry °Brix decreased by 7% at cycle 12, but
gradually returned to baseline by 24 cycles (Fig. 3E).
The increase in °Brix can also be attributed to the
cell damage during freezing and thawing, allowing the
sugars to be released from the cells, increasing extrac-
tion from berries when juiced. Additionally, the fluc-
tuation of °Brix may be due to a decrease in titratable
acidy, anthocyanin content, and the pectin cell wall
starting to dissolve (Yanyun Zhao 2007,pp.213–214).
However, temperature cycling did not affect titratable
acidity and pH (Fig. 3F, G). °Brix: acid decreased at
cycle 12, due to the decrease in °Brix (Fig. 3H).
Aronia berry color after temperature cycling
Frozen temperature cycling significantly modulated
aronia berry color, despite the limited impact on
polyphenol content (Table 1,Fig.4,Fig.5A-B). The
average of lightness (L*), redness (a*), and blueness
(b*) of fresh berries decreased progressively, leading
King et al. Food Production, Processing and Nutrition (2022) 4:3 Page 7 of 12
to increased total color change (ΔE*). The aronia
berry reached the darkest color at 24 cycles, with
9.99 L*. Temperature cycling caused aronia berries to
become bluer (less yellow) and more green (less red)
with more induced temperature cycle treatments. The
increase in darkness may be attributed to the increase
in ice crystal formation during the temperature cycles,
causing the cells to rupture, losing water from the
Fig. 3 Temperature cycling affects aronia berry polyphenols. Aronia berry was subjected to repeated frozen temperature cycling and analyzed for
(A) extractable total phenols by Folin–Ciocalteu method as gallic acid equivalents (GAE); (B) extractable monomeric anthocyanins by pH
differential method as cyandin-3-glucoside equivalents (C3GE); (C) extractable proanthocyanins by 4-dimethylaminocinnamaldehyde (DMAC)
method as catechin equivalents (CE); (D) extractable polyphenol oxidase (PPO) activity; (E) juice °Brix, (F) juice titratable acidity; (G) juice pH; and
(E) juice °Brix: acid ratio. Week 0 represents analysis of unfrozen fresh berries picked within 6 h. A unit of PPO activity is a 0.001 change in
absorbance per min (△A420/ min). Data are means ± standard deviation of n= 4 aliquots aronia plants, with triplicates for each plant. Data were
analyzed by one-way ANOVA with Tukey’s multiple comparison test vs. the fresh sample, * P< 0.05 vs. cycle 0
King et al. Food Production, Processing and Nutrition (2022) 4:3 Page 8 of 12
vacuole, thus compressing cell walls (Dawson et al.
2020). The color changes observed here support the
hypothesis that anthocyanins and other phenolics are
redistributed by temperature cycling.
Scanning electron microscopy (SEM) of aronia berry
subjected to temperature cycling
Temperature cycling during frozen storage led to cellu-
lar deformation in the aronia berries (Fig. 5C, D). Berries
that were not subject to temperature cycling had more
predominant and intact cell structures. Cell damage was
observed at 6 cycles and increased progressively through
the 24 cycles. Temperature cycling damaged cell walls
and led to cell shrinkage. Cell wall fusion is predomin-
antly observed at cycles 18 and 24. The thick white tis-
sue in the images are deformed cell walls and enlarged
weak intercellular spaces (Delgado & Rubiolo 2005;Li
et al. 2018). When the cell wall is damaged, the pectin
and hemicellulose connect to other cell walls, attributing
to the enlargement of the intercellular space (Li et al.
2018). The cellular deformation induced by freezing has
also been reported for strawberries (Delgado & Rubiolo
2005), apples (Chassagne-Berces et al. 2009), and blue-
berries (Nowak et al. 2018).
There were minor variations in the extent of dam-
age between berries from the same number of
temperature cycles, especially in cycles 18 and 24.
Thedifferenceindamagecouldbeduetoberrysize,
position in the bag during thawing, and the berry
positioninthebagduringstorage.Thesizeofthe
berry can impact the temperature of the berry during
thawing. Furthermore, the position in the bag during
storage and thawing affects the berry because the en-
ergy required to cool down or heat up the berry is
more accessible to berries located on the outer perim-
eter. Thus, more significant variation in damage is
likely to occur in the cells when berries are exposed
to more temperature cycles.
When the berries were in the freezer, not all the
water was frozen due to the sugar content in aronia
berries (Allan-Wojtas et al. 2006). In Poiana et al.
2010, berries stored at −18 °C included 89% of total
water frozen, and at −30 °C, 91% were completely
frozen. The unfrozen water migrates to outer cells,
containing less water, generating larger ice crystals
and recrystallization (Allan-Wojtas et al. 2006). The
crystal growth provokes punctured cells and cellular
shrinkage, causing deformation. Recrystallization was
also created by the temperature cycles. After being
thawed, berries placed in the freezer may have gained
larger ice crystals replacing smaller ice crystals and
caused damage and shrinkage to the cells of the aro-
nia berry.
Aronia berries in our study were exposed to slow
freezing. A slower rate causes large ice crystals with less
nucleation because the water has more time to migrate
out of the cell, causing large ice crystals in one area. The
large sharp crystals, formed during slow freezing, may
have punctured the aronia berry cell organelles (vacu-
ole), causing a release of water and other contents from
the cell (De Ancos et al. 2006; Phothiset & Charoenrein
2013).
Conclusions
Prolonged refrigeration of fresh aronia berry at 4 ±
2 °C for up to 12 weeks led to more significant poly-
phenolslossthanfrozenstorageat−20 ± 2 °C and up
to 24 temperature cycles. However, 12 weeks of 4 °C
refrigeration only led to modest losses in polyphenols,
with anthocyanins having the most loss among phe-
nolics, at 36% of initial values. Reducing temperatures
closer to the freezing point of berries would be ex-
pected to lower rates of degradation further. Freezing
and temperature cycling led to a redistribution of
Table 1 Colorimetric analysis of halved aronia berries after
temperature cycling
1
No. of temperature cycles L* a* b*
0 22.0 ± 4.3
a
20.2 ± 0.1
a
8.79 ± 0.96
a
6 16.2 ± 2.3
b
13.1 ± 1.2
b
5.56 ± 0.82
b
12 13.2 ± 1.9
b,c
11.3 ± 1.1
c
4.66 ± 0.91
c
18 12.1 ± 0.6
c
10.8 ± 1.1
c
3.87 ± 0.37
c,d
24 9.99 ± 0.56
c
9.59 ± 0.73
d
3.30 ± 0.18
d
1
Data are means ± standard deviation of n= 4 aliquots aronia plants, with
duplicate analysis for each plant. Data were analyzed by one-way ANOVA with
Tukey’s multiple comparison test P< 0.05. Identical letters in the same column
indicate values are not significantly different
Fig. 4 Temperature cycling increases ΔE of halved aronia berries.
Data are means ± standard deviation of n= 4 aliquots aronia plants,
with duplicate analysis of for each aliquot. Data were analyzed by
one-way ANOVA with Tukey’s multiple comparison test (P< 0.05). All
cycles were significantly different from other observations
King et al. Food Production, Processing and Nutrition (2022) 4:3 Page 9 of 12
polyphenols, which increased extractability and likely
contributed to some degradation during prolonged
frozen storage. As with refrigerated storage, aronia
berry anthocyanins were most susceptible to degrad-
ation than other phenolics. Thus, refrigeration and
frozen storage are viable means to preserve aronia
berry polyphenol content and quality.
Acknowledgements
Not applicable.
Authors’contributions
Erica King: Conceptualization; Methodology; Investigation; Writing –Original
Draft; Andrea Noll: Methodology, Investigation; Susan Glenn: Formal analysis;
Bradley Bolling: Conceptualization; Supervision; Funding acquisition; Writing
–Review & Editing. The final document was read and approved by the
authors.
Authors’information
Not applicable.
Funding
Supported by the Fritz Friday Chair for Vegetable Processing Research,
College of Agriculture to BWB.
Availability of data and materials
Please contact Dr. Bolling for data requests.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Food Science, University of Wisconsin-Madison, 1605 Linden
Dr., Madison, WI 53706, USA.
2
Department of Statistics, Medical Sciences
Center, University of Wisconsin-Madison, 1300 University Ave Rm 1220,
Madison, WI 53706, USA.
Fig. 5 Frozen storage temperature cycling affects aronia berry structure. (A) Visual Appearance of lyophilized halved aronia berries after
temperature cycling (B) SEM images of aronia berries subjected to temperature cycles. SEM magnification 50x. Images are N= 2 different berries
from each temperature cycle
King et al. Food Production, Processing and Nutrition (2022) 4:3 Page 10 of 12
Received: 26 October 2021 Accepted: 22 November 2021
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