Journal of Food Process Engineering ISSN 1745-4530

IMPACT OF COMBINATION TREATMENTS OF MODIFIED

ATMOSPHERE PACKAGING AND REFRIGERATION ON
THE STATUS OF ANTIOXIDANTS IN HIGHLY
PERISHABLE STRAWBERRIES
RAJEEV BHAT1,3 and RAINER STAMMINGER2
1
Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, Penang 11800, Malaysia
2
Institut fur Landtechnik, Sektion Haushaltstechnik, Universität Bonn, Bonn, Germany

3
Corresponding author. ABSTRACT
TEL: +604-653-5212;
FAX: +604-657-3678; Strawberry fruits are highly appreciated for their distinctive aroma, mouthfeel and
EMAIL: rajeevbhat@usm.my; exceptional organoleptic properties. However, owing to their perishable nature,
rajeevbhat1304@gmail.com preservation and extension of shelf life of strawberries has been a major challenge.
In this study, for the first time, combination effects of modified atmosphere pack-
Received for Publication October 28, 2014
Accepted for Publication February 5, 2015
aging (combination of N2 and CO2 with 1% O2) and refrigeration (0, 5 and 10C,
with 25C as control) on antioxidant activity and level of antioxidant compounds
doi:10.1111/jfpe.12205 were evaluated. Encouraging results were obtained, which were all depended on
the packaging, temperature used and storage days.

PRACTICAL APPLICATIONS
The present research work is expected to add database to the existing knowledge
on various challenges incurred for safe preservation and shelf life extension of
strawberries. Depending on the requirements, it is envisaged that the information
generated in this study will be of practical help to comprehend the effectiveness of
employing combination treatments of modified atmosphere packaging and cold
storage treatments on handling freshly harvested strawberries and can be
explored commercially for preservation purposes, provided more standardization
is undertaken.

available on this aspect. Among a wide array of techniques

INTRODUCTION
Strawberry fruits (Fragaria × ananassa Duch.) are highly
appreciated by consumers worldwide for its pleasant and
distinctive aroma, mouthfeel and exceptional organoleptic
properties. However, owing to the smooth texture and
elevated sensitivity to attack by molds, they are considered
as one of the fastest perishable fruits (Blanda et al. 2009;
Bhat and Stamminger 2014, 2015). Strawberries are meta-
bolically active after harvest and can rapidly deteriorate
even in the absence of any spoilage microorganisms. Straw-
berry fruits encompass high amounts of bioactive com-
pounds (natural antioxidants such as phenolic compounds,
flavonoids, vitamin C) and exhibit good in vitro antioxidant
activities (Hannum 2004). Preservation and extension of
shelf life of strawberry fruit has been a major challenge for
researchers, and several works and research reports are
available, modified atmosphere packaging (MAP) has been
reported to have some positive impacts on extension of
shelf life. This technique provides a suitable protective
gaseous atmosphere around the product (Van der Steen
et al. 2002; Zheng et al. 2007, 2008; Nielsen and Leufven
2008; Shin et al. 2008). However, there are certain draw-
backs in using MAP, some of which include monitoring of
gas combinations (O2 and CO2 are most vital) and tempera-
ture in the package. Use of CO2 augmented atmosphere is
reported to be successful in enhancing the shelf life of
strawberries. Conversely, various disadvantages related to
off-odor development, browning, physiological decay as
well as loss of texture have been observed when CO2 accu-
mulates inside the package in the presence of low O2 levels
(Gil et al. 1997; Allende et al. 2004). MAP when used singly
may not be effective in controlling the respiration rate,

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PRESERVATION OF STRAWBERRIES
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microbial proliferation, enzymatic activity, oxidation and
others. Hence, attempts have been made to use combination
of MAP with other preservation techniques (Ulukanli et al.
2012; Aday et al. 2013; Jouki and Khazaei 2014).
Use of cold or refrigerated storage can prolong the shelf
life of fruits (Wang 2010). Storing of strawberries at 0C
within 1 h of harvest as well as maintaining them at the
same temperature throughout the transport and marketing
outlet can retain the highest quality (Kader 1992; Pérez et al.
1999). Applying low temperature not only reduces the tem-
perature in a foodstuff but also, at a certain range, can lead
to formation of ice crystals. Reductions in water activity
level, enzymatic and microbial activity are some of the
common phenomena associated with refrigerated storage.
These changes are capable of extending the shelf life of a
fresh produce or a food product (Chevalier et al. 2000;
Fellows 2000). With regard to strawberries, cold storage is
one of the most commonly employed methods at farm level
as well as at household.
Based on this backdrop, the key objective to undertake
this study was to investigate the effects of combining MAP
and refrigeration (at 0, 5, 10C with room temperature of
25C maintained as control) on some of the vital antioxidant
compounds and activity (percentage inhibition of DPPH
[1,1-diphenyl-2-picryl hydrazyl] radical, total phenolics,
flavonoids and ascorbic acid) in strawberries, which were
stored over a time period of up to 4 days. It is envisaged that
the information generated will be of practical help to com-
prehend the effectiveness of employing combination treat-
ments of MAP and cold storage treatments on handling
harvested strawberries and it can be explored commercially
for preservation purposes.
MATERIAL AND METHODS
Sample Collection, Packaging and
Storage Experiments
Freshly harvested strawberry samples were collected from a
local farm in Troisdorf region of Germany. Strawberries
were screened to check for commercial ripeness (approxi-
mately three-fourths of the fruits need to retain the red
color) and were transported directly to the laboratory at
Sektion Haushaltstechnik, Universität Bonn, Germany.
Strawberries were packed (200 g) in individual polypropyl-
ene boxes (Fearch Plast, Holstebro, Denmark) with care
taken to remove injured, defective and over-ripe strawber-
ries. For MAP, Tray-Sealer packaging machine was
employed with gas mixture being 75% of N2, 24% of CO2
with 1% O2 (Tray-Sealer T-200, Multivac Sepp.
Haggenmüller GmbH and Company, Wolfertschwenden,
Germany). Separate batches (in triplicate) were maintained,
wherein the first batch of samples was without packaging
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and the second batch was with packaging (MAP). For
samples with MAP, a covering lid of polyethylene film
having a low gas and water vapor permeability was used
(Top Tray 50 LAF, Sudpack Verpackungen GmbH and
Company, Ochsenhausen, Germany). Packed samples were
stored in cold conditions at 0, 5 and 10C (in energy-efficient
refrigerators with electronic temperature control; 220–
240 V, KGFF33a [Siemens, Munich, Germany] for 0C and
10C, and KSR 38a for 5C [Robert Bosch GmbH, Stuttgart,
Germany]) with maintenance of a control batch at room
temperature of 25C. Samples without packaging were con-
sidered as “open,” whereas those samples with packaging
were termed as “closed” or MAP.
Antioxidant Compounds and Activity
Sample Extraction. Sample extraction to evaluate the
antioxidant activity and concentration levels of the antioxi-
dant compounds was based on the previously available
reports (Xu and Chang 2007; Emmy et al. 2009). In this
study, we used ethanol as an extracting solvent, mainly
owing to their proved safety and being a food-grade solvent.
Concisely, freeze-dried strawberry powder was extracted
with ethanol (1:10, two to three times), and the obtained
mixture was pooled together. Following this, centrifugation
and agitation was performed using an orbital shaker
(at 200 rpm, for 2–3 h) (optimal care was taken to check for
minimal light exposure). The obtained supernatant was col-
lected and the extraction procedure was repeated for each
analysis undertaken.
Percentage Inhibition of DPPH. The antioxidant
capacity was determined using DPPH radical method (De
Ancos et al. 2002), with slight modifications. A known
volume of the strawberry fruit extract (10 μL) was mixed
with 90 μL of distilled water and 3.9 mL of DPPH
methanolic solution (25 mM). Further, the mixture was
vortex-mixed thoroughly for 1–2 min and was left intact for
30 min in the dark at room temperature (25C). Subse-
quently, the absorbance was measured (at 515 nm) against a
blank. Results were determined as percentage inhibition of
DPPH radical and calculated using the following equation:
( Abs control − Abs sample)
% inhibition of DPPH = ×100
Abs control
where Abs control is the absorbance of the DPPH solution
without fruit extracts.
Total Polyphenolics. Determination of total poly-
phenolic content (TPC) was based on the Folin–Ciocalteu
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STAMMINGER
(FC) assay method (Singleton and Rossi 1965), with slight
modifications. Appropriately diluted (until absorbance unit
as measured in spectrophotometer was found to be <1.2)
freeze-dried strawberry fruit extracts (450 mL + 2.25 mL of
FC reagent, 10-fold dilution with distilled water) and
1.8 mL of sodium carbonate (7.5% w/v) were mixed
together and the contents were allowed to stand for 30 min
at room temperature (25 ± 1C). Following this, the
absorbance was recorded at 765 nm (using UV-visible
spectrophotometer, model Shimadzu UV-160A PC,
Shimadzu Corporation, Kyoto, Japan). The results obtained
were expressed as mg gallic acid equivalent (GAE)/100 g of
sample weight.
Flavonoid Content. The total flavonoid content (TFC)
in the strawberry fruit extracts was determined by the
method described by Sakanaka et al. (2005). Briefly, 250 μL
of strawberry fruit extract was mixed with distilled water
(1.25 mL) in a test tube, followed by addition of 5% sodium
nitrite solution (75 μL) and 150 μL of 10% aluminum chlo-
ride solution with 6-min time interval. This mixture was
kept undisturbed for 5 min and finally followed by addition
of 1 M sodium hydroxide (0.5 mL). The mixture was made
up to 2.5 mL with distilled water and was mixed thoroughly.
Further, the absorbance was measured at 510 nm using
UV-visible spectrophotometer (Shimadzu UV-160A) and
the results were interpolated by drawing a calibration curve
of the catechin solution. The results obtained for TFC were
expressed as mg catechin equivalent (CE)/100 g of extract.
Ascorbic Acid Content. The ascorbic acid (vitamin C)
content of strawberry fruit extracts was determined using
titrimetric method of 2,6-dichloroindophenol (DCPIP)
(AOAC 1995). Titration was performed using the standard
ascorbic acid solution with a standard dye solution until a
pink colored end point was obtained. To an identified
volume of the extract (10 mL), dilution was carried out (up
to 100 mL) using 3% metaphosphoric acid. This was fol-
lowed by filtering using Whatman (No. 1) filter paper and
titrating a known volume of the filtrate (5 mL) with DCPIP
indicator until an end point of changed color appeared. The
results were expressed as mg ascorbic acid (AA) equivalent/
100 g of sample using the following equation:
Ascorbic acid (mg 100 g or mL ) = ( Titer × Dye factor
× Concentration × 100) (Extract aliquot used for
estimation × Volume of sample used for esstimation )
Freeze Drying of Samples. Freeze drying of strawberry
samples was performed individually using a commercial
freeze drier (model ALPHA 1–2 LD-Plus, Martin Christ
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Gefriertrocknungsanlagen GmbH, Osterode am Harz,
Germany) with temperature fixed at −27C, pressure of
0.52 mbar and freezing time of 70–90 h.
Chemicals and Reagents
All of the chemicals were purchased from Sigma-Aldrich
(St. Louis, MO), except for hydrochloric acid, ethanol, alu-
minum chloride, sodium hydroxide, sodium acetate, alu-
minium trichloride, potassium persulfate, which were
purchased from R and M Chemicals (Essex, U.K.).
Statistical Analysis
All of the results attained in this study are presented as
mean values (three independent replicates, n = 3) ± stan-
dard deviation (SD). In addition, analysis of variance (one-
way ANOVA) was performed with the level of significance
being determined at P < 0.05, by employing Duncan’s
new multiple range tests (SPSS 14.0 software, SPSS, Inc.,
Chicago, IL).
RESULTS AND DISCUSSION
Freeze Drying of Strawberry Samples
Providing data on antioxidant compounds and their activity
in fruit juice or in fresh fruit extracts might not be of much
practical use in the food industry, as majority of the perish-
able fruits (including those of strawberries) need to be pro-
cessed into their respective products. Hence, furnishing data
on the level of antioxidants followed by the application of
preservation techniques commends higher importance. This
holds true in the present study wherein combination treat-
ments such as MAP and refrigeration were used for
preservation/shelf life extension.
In this study, we evaluated antioxidant compounds and
their activity in freeze-dried strawberry samples. Freeze
drying involves elimination of water by dehydration (via ice
sublimation) and the technique is recommended for drying
plant resources that contain heat-sensitive antioxidant com-
pounds (such as that of polyphenols and ascorbic acid).
Besides, freeze-dried products can retain comparable char-
acteristic features as that of fresh ones (Asami et al. 2003;
Chang et al. 2006; Michalczyk et al. 2009; Shofian et al.
2011; Alfaro et al. 2014). Reports indicate that freeze drying
is a vital technique that can be employed to preserve
polyphenols in berry fruits (Michalczyk et al. 2009; Alfaro
et al. 2014). Previous studies in tomatoes revealed insignifi-
cant changes in ascorbic acid (vitamin C) content between
fresh and freeze-dried “Sheng-Neu” and “I-Tien-Hung” vari-
eties (Chang et al. 2006). Likewise, an insignificant change
in ascorbic acid content is also reported in freeze-dried
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papaya (Hawlader et al. 2006). Further, nonsignificant dif-
ferences in antioxidant compounds were noticed in fresh
and freeze-dried muskmelon and watermelon. Also, nonsig-
nificant difference in the ferric reducing antioxidant power
values (FRAP) was revealed between fresh and freeze-dried
papaya, muskmelon and watermelon (Shofian et al. 2011).
Wojdyło et al. (2009); while investigating on the influence of
drying treatments (such as freeze drying, vacuum drying,
vacuum-microwave drying and convection drying), freeze-
dried strawberry samples were observed to exhibit have
high antioxidant effects compared with all other drying
treatments. Freeze drying has been reported to preserve
high level of total polyphenolic compounds in strawberries
compared with air drying (Asami et al. 2003). In one of the
sensory studies reporting on the effects of freeze drying and
convectional drying of strawberries, majority of the panel-
ists preferred freeze-dried strawberries (Gamboa-Santos
et al. 2014). In addition, these authors found that in
“Elsanta” strawberries, freeze drying to enhance the
polyphenols (fresh, 2,405.9 and freeze-dried 2,411.5 mg/
100 g dry weight [d.w.], respectively) and (+)-catechin
(fresh, 42.3 and freeze-dried 49.0 mg/100 g d.w., respec-
tively). Also, freeze-dried/dehydrated strawberries, peaches
and apples contained higher level of antioxidants, such as
total phenolics and anthocyanins, compared with the
respective fresh fruit (Rababah et al. 2005; Marques et al.
2010). Moreover, Marques et al. (2010) indicated that
freeze-dried strawberries can be a credible alternative for
fresh strawberries to enhance the antioxidant intake by con-
sumers. As an outcome of one of the studies (Marques et al.
2010) pertaining to comparisons made with regard to anti-
oxidant levels in fresh, frozen and freeze-dried strawberries
and strawberry jam, it was suggested that supplementing
freeze-dried strawberries in the human diet can render
health benefits.
Apart from all of these, our experimental design included
investigating the level of antioxidant compounds and their
activity on a daily basis (up to 4 days), which incurred large
volume of strawberry samples that required to be analyzed
in a short span of time (all in triplicate), which was practi-
cally very difficult. Hence, we strategized to employ freeze
drying of the samples for analysis.
Antioxidant Compounds and Activity
Antioxidants in fruits encompass those essential
phytochemicals (or bioactive compounds) that are capable
of inducing protective effects in humans against the ill-
effects of free radical-induced damage. Strawberry is
reported to contain amounts of antioxidant compounds
(e.g., phenolic acids, flavonoids, anthocyanins, ascorbic
acids), and available reports indicate them to be a good
scavenger of hydroxyl, peroxyl and superoxide radicals
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(Zheng et al. 2007; Giampieri et al. 2012, 2013). Besides,
storage of strawberries at high temperature of 10C is
reported to exhibit higher activity of antioxidant enzymes
and antioxidant capacities compared with those stored at
low temperatures of 0 or 5C, in organic and conventional
cultural systems (Jin et al. 2011). Reports available indicate
that use of controlled or modified atmospheres with high
CO2 levels (12–20%) is capable of extending the shelf life of
strawberries (Fernández-Trujillo et al. 2007). Nevertheless,
in this study, for the first time, we used a combination of N2
and CO2 (with 1% O2), along with a combination of
refrigerated storage conditions, to evaluate the antioxidant
qualities.
The results obtained for percentage inhibition of
DPPH• radical, TPC and TFC are depicted in Table 1.
Percentage Inhibition of DPPH• Radical
Evaluating the antioxidant activity in a given sample by
DPPH• assay is considered to be one of the most reliable,
sensitive and rapid methods (Sánchez-Moreno 2002; Bhat
and Stamminger 2014; Shin et al. 2014). In the present
study, strawberry samples used as control (freeze-dried)
showed higher inhibition of DPPH• radical (95.96%), and
this can be attributed to the presence of elevated amounts of
antioxidant compounds in strawberries (Hannum 2004;
Bhat and Stamminger 2014).
With regard to strawberry samples stored at room tem-
perature (25C), samples without MAP showed significant
reduction in the percentage inhibition (compared with
control) during storage (day 1, 94.48%; day 2, 93.73% and
day 3, 92.50%) with no inhibition/activity recorded on day
4. This can be attributed to rapid degradation of strawber-
ries in the presence of air (effect of light exposure, mold
attack, enhanced respiration rate, etc), which, in turn, is
expected to reduce the overall level of antioxidant com-
pounds and activities. Regarding samples stored under same
conditions at 25C and with MAP, significant reduction was
recorded only on day 4 of storage (94.64%). Our observa-
tions can be supported by the fact that storage of small
fruits at 25C as compared to refrigeration (4C) can lead to
rapid spoilage (Piljac-Žegarac and Šamec 2011), thus affect-
ing the overall quality.
Further, pertaining to the samples stored at 0C, no sig-
nificant changes were recorded in samples without MAP,
whereas samples with MAP + refrigeration at 0C showed
significant reductions on day 4 (93.40%) compared with
control samples. Concerning samples stored at 5C, signifi-
cant reduction was recorded in samples without MAP,
whereas samples with MAP + 5C showed significant
enhancement on day 4 (96.21%). Storage of strawberries
at 5C was chosen, as this is a direct representative of
common household storage temperature by majority of the
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TABLE 1. RESULTS OBTAINED FOR INHIBITION

Total polyphenol Total flavonoid
OF DPPH RADICAL-SCAVENGING ACTIVITY,
Samples DPPH assay (%) content (GAE g/100 g) content (CE g/100 g)
TOTAL POLYPHENOLIC CONTENT AND
FLAVONOIDS IN STRAWBERRY SAMPLES Control 95.96 ± 0.1s 1919.11 ± 43.0p 912.42 ± 1.3z
SUBJECTED TO COMBINATION TREATMENTS Day 1
OF MODIFIED ATMOSPHERE PACKAGING RT Open 94.48 ± 0.1fg 653.43 ± 6.0a 81.34 ± 1.0b
AND REFRIGERATION FOLLOWED BY RT MAP 95.06 ± 0.1d 1917.48 ± 10.9e 70.01 ± 0.9a
STORAGE 0C Open 94.97 ± 0.1lm 1807.15 ± 33.3f 260.51 ± 1.3g
0C MAP 95.05 ± 0.1mn 1915.78 ± 32.2hi 296.73 ± 1.0j
5C Open 96.13 ± 0.1t 1723.28 ± 7.8kl 508.52 ± 1.3x
5C MAP 95.47 ± 0.1p 1523.75 ± 1.0h 348.34 ± 1.5o
10C Open 95.80 ± 0.1r 1926.42 ± 21.6h 335.36 ± 1.4l
10C MAP 94.45 ± 0.0efg 1534.26 ± 65.9h 438.36 ± 1.3v
Day 2
RT Open 93.73 ± 0.1c 1848.57 ± 8.1c 140.83 ± 1.2d
RT MAP 95.22 ± 0.1o 1752.89 ± 4.7lmn 559.98 ± 1.5y
0C Open 94.56 ± 0.1gh 1747.84 ± 21.6lm 557.16 ± 1.3z
0C MAP 95.71 ± 0.1qr 1868.27 ± 32.3o 342.52 ± 1.2n
5C Open 95.71 ± 0.1qr 1804.77 ± 69.3n 471.25 ± 1.3h
5C MAP 96.29 ± 0.1u 1564.36 ± 4.4g 341.28 ± 1.3m
10C Open 94.81 ± 0.1jk 1784.06 ± 12.0q 559.58 ± 1.4y
10C MAP 94.89 ± 0.1kl 746.69 ± 14.2b 201.26 ± 1.3f
Day 3
RT Open 92.50 ± 0.1a 1566.93 ± 42.9hi 363.70 ± 1.4p
RT MAP 95.71 ± 0.1qr 1741.40 ± 10.2d 278.19 ± 1.2i
0C Open 94.72 ± 0.1ij 2139.10 ± 20.0r 515.51 ± 1.3y
0C MAP 94.48 ± 0.1fg 1682.43 ± 38.5jk 429.36 ± 1.4u
5C Open 95.05 ± 0.1mn 2019.11 ± 33.2q 428.08 ± 1.3u
5C MAP 95.96 ± 0.1s 1560.03 ± 37.4hi 346.20 ± 1.4t
10C Open 94.89 ± 0.1kl 1801.43 ± 52.1mn 619.27 ± 1.5#
10C MAP 94.81 ± 0.1e 942.43 ± 57.2d 157.39 ± 1.3e
Day 4
RT Open D4 ND ND ND
RT MAP D4 94.64 ± 0.1hi 1601.49 ± 27.4i 312.99 ± 1.4k
0C Open D4 94.39 ± 0.1ef 1684.25 ± 33.7jk 347.17 ± 1.2o
0C MAP D4 93.40 ± 0.1b 669.65 ± 8.0a 122.46 ± 1.3c
5C Open D4 95.14 ± 0.1no 1660.75 ± 16.6j 366.24 ± 1.4q
5C MAP D4 96.21 ± 0.1tu 1548.80 ± 6.3lm 346.93 ± 1.5o
10C Open D4 94.48 ± 0.1fg 635.02 ± 9.6a 383.54 ± 1.3s
10C MAP D4 95.63 ± 0.1q 789.07 ± 17.3mn 147.06 ± 1.4r

Mean of triplicate determinations (n = 3 ± SD). Values followed by the same letter within the
same column are not significantly different from each other (P > 0.05).
RT, room temperature; MAP, modified atmosphere packaging; ND, not determined; Open,
samples without MAP.

consumers with respect to perishable fruits. Enhancement
in the antioxidant activities in guava fruit is reported during
extended storage at 5C (Patthamakanokporn et al. 2008).
Nevertheless, a decrease in the antioxidant capacity (based
on the DPPH• method) in “Salustiana” oranges (stored at
4C) was also reported (Del Caro et al. 2004). Finally, regard-
ing samples stored at 10C, no significant changes were
recorded in either nonpacked or packed samples even on
day 4 of storage. Details are available wherein treatment
with ozone gas is reported to extract and accumulate higher
level of antioxidants in strawberries (Perez et al. 1999).
Besides, in one of the studies on blueberries, Kalt et al.
(1999) reported nonsignificant changes in the antioxidant
activity during storage (for up to 8 days) when various tem-
perature range was employed (0, 10, 20 and 30C). Report by
Rapisarda et al. (2008) indicates enhancement in the anti-
oxidant capacity of “blood” and “blond” oranges after cold
storage (at 6C) (measured by the DPPH• assay). In contrast
to these inferences, the observed reduction in the radical-
scavenging activity in freeze-dried strawberry samples sub-
jected to MAP can also be attributed to high level of
oxidization that occur in fruits, followed by packaging.
However, it is worth to indicate again here that no reports
are available on the influence of employing MAP and

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refrigeration on antioxidant activities in freeze-dried fruit
samples, and hence as indicated earlier, we have attempted
to compare with the available data on fresh fruits.
Total Polyphenols
Polyphenolic compounds, a cluster of plant secondary
metabolites (such as phenolic acid, tannin, flavonoids,
flavonols), exhibit rich biological effects including those of
anti-aging, anticancer, anti-inflammatory and antioxidant
activities (Bhat et al. 2011; Bhat and Stamminger 2014). In
this study, control samples showed high levels of TPC
(1,919 g/100 g GAE), supporting the notion that strawber-
ries possess high antioxidant capacity.
Appealing results were recorded for TPC in strawberries
subjected to MAP and refrigeration/cold storage conditions.
With regard to samples maintained at room temperature
(25C), both unpacked (without MAP) and samples with
MAP showed a significant decrease in TPC on days 1 and 2,
while the contents gradually showed significant enhance-
ment on day 3. It has been opined that during cold storage,
antioxidant capacity in fruits can initially be augmented and
later significantly be reduced, which can be elucidated based
on various biochemical and physiological reactions (pheno-
lic metabolism) that persist to occur even after harvest.
These reactions further lead to the release of bioactive com-
pounds (antioxidant compounds) (Kalt et al. 1999;
Piljac-Žegarac and Šamec 2011). However, on day 4 (25C),
no activities or TPC could be measured as strawberries were
completely degraded. Concerning samples stored at 0C, a
significant increase in TPC was recorded in unpacked
samples on day 3, while for samples of 0C + MAP, a signifi-
cant and linear trend decrease was recorded during storage
of up to 4 days. Further, with regard to samples stored at
5C, a significant increase was recorded on day 3, while on
day 4, TPC decreased. For 5C + MAP samples, a significant
decrease was recorded as days progressed. Earlier, Connor
et al. (2002), while working on different blueberry cultivars,
which were subjected to refrigeration temperature of 5C,
concluded that fruits harvested prematurely can be stored at
this temperature without affecting the antioxidant com-
pounds (total phenolic compounds) and thus is capable of
maintaining the commercial quality. However, in case of
strawberries, as appropriate maturity and harvesting stages
are not yet completely standardized worldwide, this tem-
perature can be explored.
Further, pertaining to samples stored at 10C, a significant
decrease was recorded in unpacked samples, while in case of
10C + MAP, a rapid and significant loss in the total TPC was
recorded during storage days. Previously, Srivastava et al.
(2007), while working on blueberry cultivars (Tifblue and
Powder blue), which were subjected to storage at different
temperatures (−20, 6, 23 and 35C and up to 60 days),
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reported that temperature of −20C did not exhibit any sig-
nificant loss in antioxidant capacity during the first 30 days,
whereas at 6C, significant loss was recorded untimely from
15 days of storage. Nevertheless, in grapefruit and in orange
cultivars, enhancement in TPC has been reported during
long-term cold storage (Rapisarda et al. 2008). An increase
in TPC and antioxidant activity in “Black Amber” fruits
during cold storage is also reported (Diaz-Mula et al. 2009).
Stability in TPC without much significant differences is also
reported in various fruits during storage at room as well as
at refrigeration temperatures (Kevers et al. 2007). The same
authors have also noticed enhancement in TPC in asparagus
and leek, which were stored at 4C.
Overall, the recorded increase in TPC in strawberries can
also be caused by cell wall modifications (facilitating the
release of conjugated phenolic compounds as well as activa-
tion of phenylalanine ammonia-lyase enzyme activity)
which might have occurred owing to exposure to gases
(used for MAP) and chilling effects. In addition, as cooling
(between −1 and 8C) can contribute to reduction in tem-
perature of a food, this, in turn, can decrease the enzymatic
activities (Fellows 2000). Reports are available wherein loss
in phenolic content in loquat fruit during low temperature
storage (0 and 5C) was observed (Cao et al. 2009, 2011),
thus supporting our observations. In fact, combination of
gas used (high N2, and low O2 and CO2) is also reported to
prevent phenol in stored shredded carrots (Amanatidou
et al. 2000), thus further supporting our data. In strawber-
ries, concentration of some of the phenolics, such as
catechin, quercetin, ellagic acid, and kaempferol derivatives,
is reported to be enhanced during storage but remained
unaffected when CO2 was used as the storage atmosphere
(Gil et al. 1997). As various mechanisms can be playing a
pivotal role when combination treatments of MAP and cold
storage are used for storing strawberries, further in-depth
research is warranted in this regard. It is worth noting here
that no information is available on the effects of using MAP
and refrigeration on the changes of phenolic compounds
and antioxidant capacity in strawberries, and hence, we
have tried to give the best possible explanations. Further
mechanisms involved are worthy to be investigated.
In addition to these, usually, antioxidant activity, which is
measured as percentage inhibition of DPPH radical, can be
correlated with TPC (Gorinstein et al. 2004; Bhat and
Stamminger 2014). However, in this study, a positive corre-
lation was recorded only in selected strawberry samples
maintained at room temperature (25C) without MAP, 0C
MAP, 5C (MAP and without MAP) and 10C MAP.
Total Flavonoid Content
Flavonoids are extensively disseminated plant phenolic
group of compounds that are proved to be an effective
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 R. BHAT and R. STAMMINGER
STAMMINGER PRESERVATION
PRESERVATION OF STRAWBERRIES

antioxidant and a free radical scavenger (Pokorny et al.
2001; Giampieri et al. 2012, 2013). In this study, with regard
to TFC in strawberry sample, a nonconsistent result was
obtained. There was no clear correlation that could be
drawn with that of TPC or with the antioxidant activities
for TFC. However, in general, significant reductions were
recorded in all the samples compared with control batch of
strawberries. In one of the investigations on stored tomatoes
(at 7C and 15C), results revealed nonsignificant changes in
TFC (Toor and Savage 2006). Nevertheless, strawberries,
being a biological sample (which is rather active even
during postharvest stages), undertaking experiments at
various treatments and drawing accurate comparisons on
the effects can be difficult especially for bioactive com-
pounds (as that of flavonoids).
Ascorbic Acid Content
The results obtained for ascorbic acid (vitamin C) are
shown in Fig. 1. The results showed rapid and significant
degradation in the vitamin C contents in samples stored at
room temperature (25C, both with and without MAP) as
well as for those maintained at 10C (with and without
MAP). Whereas in 0C, samples without MAP could retain
significant amount of vitamin C, those of 0C + MAP could
retain the levels up to a certain extent until day 3. Interest-
ing results were obtained with respect to samples stored at
5C without MAP, wherein a gradual enhancement was
recorded with progress in storage days, while 5C + MAP did
not exhibit any significant changes during storage period. It
is a well-accepted fact that compared to some of the
common antioxidant compounds, production of vitamin C
tends to cease after the berry is picked or harvested. Never-
theless, the probability is still high, wherein vitamin C can
be oxidized to its non-antioxidant state (Klopotek et al.
2005). Besides, Marques et al. (2010) indicated that time
duration between harvesting and consumption of strawber-
ries (as fresh or frozen) can result in significant loss of
vitamin C.
Strawberries stored using modified atmosphere (20%
CO2, 20 days at 1C) showed the content of ascorbic acid to
be 27.0 mg/100 g (fresh weight) (Agar et al. 1997). A swift

A 300 B 350

250 300

200 250
mg AA/100 g
mg AA/100 g

150 200

100 150

100
50
50
0
RTO RT RTO RT RTO RT RTO RT 0
MAP MAP MAP MAP 0 O 0 MAP 0 O 0 MAP 0 O 0 MAP 0 O 0 MAP
Control D1 D2 D3 D4 Control D1 D2 D3 D4

C 400 D 400
350 350
300 300
mg AA/100 g

250 250
mg AA/100 g

200 200
150 150
100 100
50 50
0 0
5O 5 5O 5 5O 5 5O 5 10 O 10 10 O 10 10 O 10 10 O 10
MAP MAP MAP MAP MAP MAP MAP MAP
Control D1 D2 D3 D4 Control D1 D2 D3 D4

FIG. 1. CHANGES IN ASCORBIC ACID (AA) CONTENT IN DIFFERENT SET OF STRAWBERRY SAMPLES SUBJECTS TO MODIFIED ATMOSPHERE
PACKAGING (MAP) AND REFRIGERATION STORAGE: (A) ROOM TEMPERATURE (RT); (B) 0C; (C) 5C; (D) 10C
D1–D4 = days.

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PRESERVATION OF STRAWBERRIES
PRESERVATION OF STRAWBERRIES
loss in the ascorbic acid levels on refrigerated storage of
fresh-cut kiwifruit slices (maintained at 0, 5 and 10C up to 6
days) is reported (Agar et al. 1999). Significant loss in
vitamin c in fresh-cut strawberries and persimmons (stored
for 8 days using controlled atmosphere consisting of 2 kPa
O2, air + 12 kPa CO2, or 2 kPa O2 + 12 kPa CO2 and at 0C) is
also reported (Wright and Kader 1997). Changes in ascorbic
acid content in strawberries subjected to wrapping treat-
ments (O2 and CO2) at 1 or 10C (storage period of 8 days)
are reported by Nunes et al. (1998).
Although freeze drying is carried out in the absence of
oxygen, it is expected not to cause any loss in vitamin C
levels. However, in this study, a loss was recorded. A
decrease in vitamin C content in fruit juice during storage is
reported and has been correlated with the mode of process-
ing, storage conditions and packing (Polydera et al. 2003). A
decrease in ascorbic acid levels during storage, particularly
at temperature higher than 0C, is reported (Ajibola et al.
2009). In addition, a highly significant and negative correla-
tion is reported to occur between ascorbic acid versus
storage time and exposure to air. It has been shown that
ascorbic acid is highly stable during storage at refrigeration
condition (4–5C) (Oyetade et al. 2012). Further, enhance-
ment in ascorbic acid levels in ozone-treated and refriger-
ated strawberries is also reported (Perez et al. 1999). It can
be opined that enhancement in ascorbic acid in treated
fruits is attributed to the inhibitory action on enzymatic
activities. In general, ascorbic acid level can be affected by
processing temperature, enzymatic activities as well concen-
tration of oxygen and exposure to light (Davey et al. 2000).
CONCLUSIONS
In this study, we report on the effects of using combination
treatments of MAP and refrigerated storage on the level of
antioxidant compounds and their activity in strawberries.
The results generated were encouraging, wherein enhance-
ment in the levels/activity of selected antioxidant
compounds/activity was recorded. Samples stored with
MAP + 5C showed significant level of percentage inhibition
of DPPH radical (enhancement) on day 4 (96.21%) com-
pared with control (95.96%). Also, a positive correlation
was recorded for TPC in strawberry samples maintained at
room temperature (25C) without MAP, 0C MAP, 5C (MAP
and without MAP) and 10C MAP. Interesting results were
obtained for ascorbic acid content in strawberry samples
stored at 5C without MAP, wherein a gradual enhancement
was recorded with the progress in storage days, while
5C + MAP did not exhibit any significant changes during
storage period.
Further research studies are warranted to standardize the
combination treatment conditions (gas combinations as
well as refrigeration temperature and storage) in order to
128
8 Journal of of
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R.
R. BHAT
BHAT and R. STAMMINGER
improve and understand the overall effects on extending the
shelf life of highly perishable fruits such as strawberry.
Developing bio-based packaging materials for MAP could
be explored in the near future for safe storage of strawber-
ries. Additional research work needs to be initiated to evalu-
ate fruits’ respiration rate, texture and sensory qualities on
using the aforementioned combination treatments. Indeed,
appropriate modeling of the respiration rate in strawberries
subjected to MAP and refrigeration will be of help to extend
the shelf life. In fact, temperature abuse incurred during
transportation, distribution, storage as well as marketing is
also of main concern in farming as well as in marketing
sector. Hence, precautionary measures (such as mathemati-
cal modeling to test gas concentrations during storage, res-
piration phenomenon) need to be taken to overcome the
hindrances. Application of engineering principles to iden-
tify defective packages prior to reaching the consumers and
developing novel strategies to continuously monitor and
detect gas leakage along the entire supply chain of strawber-
ries is a prerequisite. Designing new low-cost cooling
systems that can effectively sustain MAP and that can be
readily installed at the farm levels should be explored.
ACKNOWLEDGMENTS
The first author thanks the International Bureau of the
BMBF, Germany, for providing fund. Both authors
acknowledge the help extended by all the staff members and
students of Household and Appliance Technology Section,
Universität Bonn. Special thanks to Ms. Edith Lambert for
the help rendered in freeze drying of samples and to Ms.
Rajan N for all the technical help rendered. Appreciation is
also due to Dr. J. Kreyenschmidt and H. Ulrik, Cold-Chain
Management Group, University Bonn, for the help rendered
in packaging of samples.
CONFLICT OF INTEREST
There is no conflict of interest in this manuscript.
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