Scientia Horticulturae 188 (2015) 44–48

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Changes in fruit firmness, cell wall composition and cell wall

degrading enzymes in postharvest blueberries during storage
Hangjun Chen a , Shifeng Cao b,∗ , Xiangjun Fang a , Honglei Mu a , Hailong Yang c , Xiu Wang a ,
Qingqing Xu a , Haiyan Gao a,∗∗
a
Key Laboratory of Fruits and Vegetables Postharvest and Processing Technology Research of Zhejiang Province, Zhejiang Academy of Agricultural Sciences,
Hangzhou 310021, China
b
Nanjing Research Institute for Agricultural Mechanization, Ministry of Agriculture, Liuying 100, Nanjing 210014, China
c
College of Life and Environmental Science, Wenzhou University, Wenzhou 325027, China

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

Article history: Blueberries are now the second most economically important soft fruit. However, they are highly per-
Received 5 January 2015 ishable and susceptible to rapid spoilage. One of the main factors limiting postharvest life of blueberries
Received in revised form 11 March 2015 is softening. The changes of fruit firmness, cell wall degrading enzymes and cell wall composition of
Accepted 13 March 2015
‘Brilliant’ blueberry (Vaccinium ashei cv. Brilliant) were investigated in this study. The results showed
Available online 3 April 2015
fruit firmness declined concomitantly with the increase of the content of water soluble pectin (WSP)
during storage paralleled by a decreasing amount of sodium carbonate soluble pectin (SSP), cellulose
Keywords:
and hemicellulose. Blueberries stored at low temperature (5 ◦ C) maintained higher fruit firmness than
Blueberry
Firmness
those stored at 10 ◦ C, which was due to the lower WSP content and higher contents of SSP, cellulose and
Cell wall composition hemicellulose. Meanwhile, the lower activities of cell wall degrading enzymes such as polygalacturonase,
Cell wall degrading enzymes cellulase, ␤-galactosidase and ␣-mannosidase in blueberries at 5 ◦ C were associated with greater fruit
firmness and lower WSP content as compared to those in fruit stored at 10 ◦ C.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction perishable and susceptible to rapid spoilage (Cantína et al., 2012).

Blueberries, one of the most widely consumed fruit in the
world, contain high amounts of phenolic compounds, including
anthocyanins, flavonols, chlorogenic acid and procyanidins (Koca
and Karadeniz, 2009), and have been shown a wide diversity
of bioactivities such as antioxidant, antidiabetic, antimicrobial,
antiproliferative, apoptotic, liver protection, lifespan-prolonging,
anti-inflammatory, cancer preventive and cardioprotective activi-
ties (Smith et al., 2000; Faria et al., 2005; Torri et al., 2007; Bingül
et al., 2013; Bunea et al., 2013). Due to their various health benefits,
unique taste, and nutritional value, worldwide production and con-
sumption of blueberries have increased rapidly in recent years and
they have become the second most important soft fruit species after
strawberry (Giongo et al., 2013). However, blueberries are highly
∗ Corresponding author at: Nanjing Research Institute for Agricultural Mechaniza-
tion, Ministry of Agriculture, Liuying 100, Nanjing 210014, China.
Tel.: +86 2558619521.
∗∗ Corresponding author at: Zhejiang Academy of Agricultural Sciences, Hangzhou
310021, China. Tel.: +86 57186406661.
E-mail addresses: shifengcao1@gmail.com (S. Cao), spsghy@163.com (H. Gao).
It is reported that fresh blueberries have a shelf life of 1–8 weeks
depending on stage of fruit ripeness, method of harvest, presence
of fruit disease, and storage conditions (Duan et al., 2011). One of
the main factors limiting postharvest life of blueberries is softening
(Angeletti et al., 2010), which may influence not only the quality of
the fruit, but also its storage life, transportability and resistance of
postharvest diseases (Deng et al., 2005).
Softening in any fruit is primarily due to the change in cell-wall
carbohydrate metabolism, leading to a net decrease in certain struc-
tural components (Sethu et al., 1996). During fruit softening, the
loss of firmness is associated with the decrease in total water sol-
uble pectin and the disassembly of primary cell wall and middle
lamella structures (Giongo et al., 2013). Hemicellulose depoly-
merisation and arabinose loss are the main cell wall modifications
(Vicente et al., 2007a). It is well documented that the changes in cell
wall composition and structure results from the coordinated action
of hydrolytic enzymes in the fruit (Deng et al., 2005). Prominent
enzyme, polygalacturonase (PG), as well as a variety of glycanases
and glycosidases, plays important roles in cell wall degradation
(Sethu et al., 1996).
Several preservation technologies, including cold storage
(Connor et al., 2002), high oxygen atmospheres storage (Zheng

http://dx.doi.org/10.1016/j.scienta.2015.03.018
0304-4238/© 2015 Elsevier B.V. All rights reserved.
 H. Chen et al. / Scientia Horticulturae 188 (2015) 44–48
et al., 2003), allyl isothiocyanate (Wang et al., 2010) and edible
coating (Duan et al., 2011), have been used to maintain bioactive
compounds, reduce deterioration, and prolong shelf life of fresh
blueberries. However, the modification of cell wall components in
postharvest blueberries during storage is still not clear, and its pos-
sible mechanism during softening is not understood. Maintaining
textural quality during storage is of interest to the fruit growing
and distribution industries of blueberries, therefore, in the present
work, composition modifications in the cell wall and changes of the
activities of cell wall degrading enzymes such as PG, cellulase, ␤-
galactosidase and ␣-mannosidase of blueberries were measured to
explore the softening mechanism in this kind of fruit during storage.
2. Materials and methods
2.1. Fruit material and treatment
Blueberry (Vaccinium ashei cv. Brilliant) fruit were hand-
harvested from five-year-old blueberry plants in a commercial
orchard located in Anji county (119◦ 68 N, 30◦ 63 E) of Zhejiang
Province. All fruit were harvested at commercial maturity, as deter-
mined by complete blue skin colour, and transported within 2 h
to the laboratory. Fruit with uniform size and colour were placed
in plastic containers with snap-on lids and each contained two
hundred fruit (about 300 g). The containers were divided into two
groups randomly. One group was then stored at 5 ◦ C and the other
10 ◦ C. Samples were taken initially and at 7-day intervals during
storage of 49 days.
2.2. Firmness determination
Fruit firmness measurement was conducted by a TA-XT plus
texture analyzer (Stable Micro Systems Ltd., U.K.) with a 5 mm
diameter stainless probe. Firmness was measured on the equatorial
region of each fruit. Twenty fruit from each treatment were com-
pressed 5 mm at a rate of 1.0 mm/s and firmness was expressed in
kilogram per square centimeter (kg/cm2 ).
2.3. Cell wall preparation and fractionation
Cell wall polysaccharides were obtained as ethanol insoluble
residue using the methods described by Deng et al. (2005). Briefly,
10 g of flesh were ground, extracted by 80% (v/v) ethanol and main-
tained in boiling water to inactivate enzymes. Then the sample was
centrifuged after cooling and the residue was re-extracted twice
with 80% ethanol. The retained residue was incubated overnight
with 90% (v/v) dimethysulphoxide at 4 ◦ C to remove starch, and
then washed twice with water, chloroform–ethanol (2:1), and ace-
tone, respectively. The isolated cell wall materials (CWM) were
dried in a vacuum oven at 40 ◦ C and stored over silica gel in a
vacuum desiccator.
The CWM was fractionated according to the methods of Deng
et al. (2005) and Li et al. (2006). Briefly, water soluble pectin
(WSP) was obtained by suspending CWM in 50 mM sodium acetate
buffer (pH 6.5) for 6 h of shaking, and collecting supernatant by
centrifuging at 4 ◦ C. The water-insoluble residue was re-suspended
in 50 mM sodium acetate buffer (pH 6.5) containing 50 mM EDTA,
shaken for 6 h, and centrifuged. The supernatant was collected
as chelator soluble pectin (CSP). Sodium carbonate soluble pectin
(SSP) was pooled by re-suspending the residue in 50 mM Na2 CO3
containing 2 mM EDTA, shaking, centrifuging and collecting the
supernatant. The remaining residue was re-suspended in 4 mM
NaOH containing 100 mM NaBH4 , shaken and centrifuged. The
supernatant was collected as hemicellulosic fraction and the final
residue was cellulosic fraction.
45
The pectin content in the fraction was measured by the
m-hydroxydiphenyl method (Paul and Jerome, 1982) using galac-
turonic acid as standard. The cellulose and hemicelluloses contents
were determined using the anthrone method (Vicente et al., 2005)
using glucose as standard.
2.4. Enzyme extraction and assay
Two grams of fruit tissues were treated with liquid nitrogen,
pulverized and extracted in 10 mL of 0.2 M sodium acetate buffer
(pH 5.0) containing 1 mM EDTA-Na, 5% polyvinylpyrrolidone (w/v).
The enzyme extract was obtained by centrifugation at 10,000×g for
20 min at 4 ◦ C.
PG activity was assayed by the method described by Deng et al.
(2005) and Pathak and Sanwal (1998) with slight modification. The
reaction mixture contained 2.0 ml of 0.2 M sodium acetate buffer
(pH 5.0), 1.0 ml of 1% (w/v) solution of citrus pectin, and 1.0 ml of
crude enzyme. The amount of reducing sugar released was deter-
mined using the dinitrosalicylate method after reaction 30 min at
50 ◦ C. One unit of enzyme was the amount which catalyses the for-
mation of 1 ␮g of reducing sugar per hour per g of original fresh
weight.
Cellulase activity was determined by measuring the reducing
sugar released from carboxymethyl cellulose (Deng et al., 2005).
The reaction mixture contained 2.0 ml of 1% (w/v) solution of car-
boxymethyl cellulose and 0.5 ml of crude enzyme. The amount of
reducing sugar released was determined using the dinitrosalicylate
method after reaction 30 min at 50 ◦ C. One unit of enzyme was the
amount which catalyses the formation of 1 mg of reducing sugar
per hour per g of original fresh weight.
␤-Galactosidase and ␣-mannosidase activity was determined
by measuring p-nitrophenol released from p-nitrophenyl-␤-
galactopyranoside and p-nitrophenyl-␣-mannopyranoside,
respectively (Sethu et al., 1996; Deng et al., 2005). The reaction
mixture contained 0.5 ml of sodium acetate buffer (0.2 M, pH 5.0),
0.18 ml of 16 mM p-nitrophenyl-␤-galactopyranoside and 0.12 ml
of crude enzyme. The amount of p-nitrophenol was measured
after reaction 90 min at 37 ◦ C. One unit of enzyme was the amount
which catalyses the formation of 1 ␮mol of p-nitrophenol per hour
per g of original fresh weight.
2.5. Statistical analysis
All the measurements were conducted in triplicate. Data pre-
sented were the means ± SD values. All statistical analyses were
performed with using SAS statistical software 8.01 (SAS institute,
Cary, NC).
3. Results and discussion
3.1. Changes in fruit firmness
Fruit firmness is an important quality attribute in blueberry, and
excessive softening is one of the main factors reducing quality and
limiting commercialization for fresh consumption (Angeletti et al.,
2010). As shown in Fig. 1, irrespective of the storage temperatures,
firmness of blueberries increased during the first 7 days of storage,
and declined gradually afterwards. Fruit stored at 5 ◦ C maintained
greater firmness than those at 10 ◦ C after 7 days of storage.
3.2. Changes in cell wall composition
Previous work analyzing cell wall changes in blueberry fruit
during development showed that pectin solubilization increased
during ripening (Vicente et al., 2007b). Less attention has been
46 H. Chen et al. / Scientia Horticulturae 188 (2015) 44–48

2.5 three pectin-rich fractions as WSP, CSP, and SSP and hemicellulose
and cellulose. Among them, WSP was thought to represent wall
o
5 C polymers solubilized in vivo but remaining in the apoplast which
o
10 C could be obtained by water extraction of the purified cell wall,
2.0
whereas CSP and SSP are generally considered to be enriched for
* * * ionically and covalently bound pectins, respectively. Typically, dur-
ing fruit softening, depolymerisation of cell wall polysaccharides
Firmness (N)

1.5
1.0
0.5
0.0
0 7
Fig. 1. Changes on firmness in blueberry fruit during storage at 10 ◦ C and 5 ◦ C. Val-
ues are the means ± SE. Vertical bars represent the standard errors of the means.
Asterisks indicate significant differences between the fruit stored at 10 ◦ C and 5 ◦ C
(Duncan’s multiple range test, * P < 0.05).
paid to the changes in pectin, hemicellulose and cellulose con-
tent of blueberries after harvest. In this study, we examined the
possible role that cell wall modification might play in response
to fruit softening during storage. Cell walls were extracted to give
*
* was accompanied by increases in the levels of WSP, whilst the lev-
els of CSP, SSP, hemicellulose and cellulose decreased (Carrington
* et al., 1993; Vicente et al., 2007a). In our present study, the pectin
fractions changed significantly in blueberries during storage at 5 ◦ C
and 10 ◦ C. The amount of WSP increased gradually with storage
time paralleled by a decreasing amount of SSP (Fig. 2A and B). Mean-
while, the amount of CSP increased during the first 21 days, and
then decreased afterwards (Fig. 2C). These changes suggested that,
similar to the other fruit, pectins, as indicated by uronic acid con-
tent, were solubilized from the wall during softening in blueberries,
indicating the possible alterations in the cross-linking of carbohy-
14 21 28 35 42 49 drates in the cell walls (Manning, 1993). Moreover, it should be
noted that the increase in fruit firmness in blueberries during first
Storage time (day) 7 days of storage observed in our present study might be resulted
from the CSP accumulation (Fig. 2C) in this fruit regardless of the
storage temperatures.
Additionally, in our present study, we also observed that blue-
berries stored at 5 ◦ C experienced higher levels of SSP and CSP
but lower WSP content as compared with those at 10 ◦ C, which
were 31.2% and 116.7% higher in levels of SSP and CSP, respec-
tively, and 14.6% lower in WSP content than those in fruit stored
at 10 ◦ C at the end of storage. Thus, results here suggested that low
temperature storage somehow prevented solubilization of these

0.6 2.8

Na2CO3 soluble pectin (%)

* * * B
Chelator soluble pectin (%) Water soluble pectin (%)

A * *
* *
* *
2.1
0.4 *
1.4
o
0.2 o
5 C 5 C
o
o 10 C 0.7
10 C

0.0 0.0
1.8 1.5
D
Hemicellulose (%)

C
* *
1.2 * * 1.0
* * * *
0.6 0.5

0.0 0.0

3.5 E
3.0
*
*
Cellulose (%)

2.5 * *
2.0

1.5

1.0
0 7 14 21 28 35 42 49
Storage time (day)

Fig. 2. Changes in water-soluble pectin (A), sodium carbonate-soluble pectin (B), chelator soluble pectin (C), hemicellulose (D) and cellulose (E) contents of blueberry fruit
during storage at 10 ◦ C and 5 ◦ C. Values are the means ± SE. Vertical bars represent the standard errors of the means. Asterisks indicate significant differences between the
fruit stored at 10 ◦ C and 5 ◦ C (Duncan’s multiple range test, * P < 0.05).
 H. Chen et al. / Scientia Horticulturae 188 (2015) 44–48
10000
A *
8000 * 12000
Polygalacturonase
* * *
(μg h g FW)
6000
*
-1 -1
4000
2000 o
5 C
o o
10 C
0 0
800
C
* *
600
β-Galactosidase
(μmol h g FW)
* * *
-1 -1
400
200
0 0
Storage time (day)
Fig. 3. Changes in activities of polygalacturonase (A), cellulase (B), ␤-galactosidase (C) and ␣-mannosidase (D) of blueberry fruit during storage at 10 ◦ C and 5 ◦ C. Values
are the means ± SE. Vertical bars represent the standard errors of the means. Asterisks indicate significant differences between the fruit stored at 10 ◦ C and 5 ◦ C (Duncan’s
multiple range test, * P < 0.05).
polymers. It was reported that the architectural strength of cell
wall can be increased by the cross-linking of pectin and the associ-
ation between pectin and extensin proteins (Vicente et al., 2007a).
As higher amount of ionically and covalently bound pectins (CSP
and SSP) was maintained in blueberries stored at 5 ◦ C, it probably
cross-linked to other cell wall polymers and strengthened the cell
wall, which was associated with the greater firmness.
Increasing evidence highlighted solubilization and depolymer-
ization of hemicellulose and cellulose also played important roles
in fruit softening (Wakabayashi et al., 2000). Vicente et al. (2007b)
demonstrated hemicellulose degradation was among the main
modifications of cell wall disassembly in blueberry fruit dur-
ing development. Results found in the present work show that
contents of hemicellulose and cellulose decreased gradually in
blueberries during storage regardless of the storage tempera-
tures, lower temperatures maintained higher levels of these two
components in blueberries, which were 50.0% and 66.7% higher,
respectively, than those in fruit at 10 ◦ C after 49 days of storage
(Fig. 2D and E). It was documented that the rigidity of cellular
walls was probably attributed to the cellulose-hemicellulose net-
work structure, formed by hydrogen-bond and crosslinks between
cellulose microfibrils (Wakabayashi et al., 2000). The breakdown
of hemicellulose was revealed to result in the disassembly of the
cellulose-hemicellulose network and fruit softening (Cheng et al.,
2009). On the other hand, due to the crystalline nature of cellulose,
its change was related to the resistance of cell wall to enzymatic
degradation (Zhou et al., 2011). Taken together, our results suggest
that the continuous degradation of hemicellulose and cellulose is of
importance to fruit softening of blueberries during storage as well
as an array of other fruit (Wakabayashi et al., 2000; Zhou et al., 2011;
Wang et al., 2015). Cold storage suppressed the depolymerization
and degradation of cellulose and hemicellulose, thus delaying fruit
softening.
3.3. Changes in cell wall degrading enzymes
It was increasingly apparent that softening and textural changes
were brought about by the actions of a multitude of cell
47
15000
*
B
(mg h g FW)
* * * *
Cellulase
9000
-1 -1
* *
6000
o
5 C 3000
10 C
1000
* D
*
800
(μmol h g FW)
*
α-Mannosidase
*
* 600
-1 -1
400
200
wall-localized enzymes acting on specific, potentially highly local-
ized substrates (Brummell et al., 2004). PG hydrolyzes pectin acid
along with the main chain of polygalacturonic acid, causing pectin
degradation, cell wall dissolution, and ultimately, fruit softening
(Wei et al., 2010). Cellulase degrades both cellulose and ␤-1,4-
glucan backbone of xyloglucan, a hemicellulosic polysaccharide
abundant in cell walls of dicotyledons (Vicente et al., 2007a; Bu
et al., 2013). ␤-Galactosidase has been characterized in association
with the removal of galactosyl residues from cell wall polymers
during fruit softening (Wei et al., 2010). The removal of pec-
tic galactan side-chains was an important factor in the cell wall
changes leading to ripening-related firmness loss (Payasi et al.,
2009). Glycosidases such as ␣-mannosidase and ␣-galactosidase
are a class of carbohydrate hydrolyses that act on short chain
oligosaccharides, which may be present in glycoproteins, and on
carbohydrate heteromers or homomers (Vicente et al., 2007a). In
order to reveal the mechanism involved in fruit softening of blue-
berries during storage, the activities of four cell wall degrading
enzymes such as PG, cellulase, ␤-galactosidase and ␣-mannosidase
were measured. Regardless of storage temperatures, activities of
all these four enzymes in blueberries showed similar changes dur-
ing storage, which increased and peaked firstly and then declined
afterwards (Fig. 3). Low temperature at 5 ◦ C remarkably suppressed
activities of the four enzymes and maintained lower WSP con-
tent and higher contents of hemicellulose and cellulose compared
with the fruit stored at 10 ◦ C, which suggested that cold stor-
age inhibited cell wall degradation by inhibiting its degrading
enzymes. At the end of the storage, activities of cellulase, ␤-
galactosidase and ␣-mannosidase in fruit at 5 ◦ C were 3.2-, 1.5-
and 1.2-fold, higher than those at 10 ◦ C, respectively. Vicente et al.
(2005) also found that softening process was delayed via the
inhibition of cell wall degrading enzymes including PG and ␤-
galactosidase in heat treated strawberries, which confirmed that
cell wall degrading enzymes play an important role in fruit soften-
ing. Therefore, our results indicated that the effect of cold storage
on delaying softening in blueberries fruit may be partially due to
relatively lower activities of PG, cellulase, ␤-galactosidase and ␣-
mannosidase.
48 H. Chen et al. / Scientia Horticulturae 188 (2015) 44–48
4. Conclusion
In summary, the present work showed that during fruit soften-
ing in blueberries, the decline of fruit firmness was associated with
increased WSP content and decreased levels of SSP, hemicellulose
and cellulose. Compared with those at 10 ◦ C, fruit stored at 5 ◦ C
showed greater firmness and the process of softening was delayed
due to lower WSP content and higher levels of SSP, hemicellulose
and cellulose. Meanwhile, cold storage also inhibited activities of
cell wall degrading enzymes such as PG, cellulase, ␤-galactosidase
and ␣-mannosidase. The combined effect of cold storage on these
enzymes could slow down pectin solubilization and depolymeriza-
tion which was related to the firmer structure in blueberries stored
at 5 ◦ C.
Acknowledgements
This study was supported by the National Natural Science Foun-
dation of China (31471635) and Special Fund for Agro-scientific
Research in the Public Interest (201303073).
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