Journal of the Science of Food and Agriculture
J Sci Food Agric 84:1765–1770 (online: 2004)
DOI: 10.1002/jsfa.1884
The effect of calcium and cellular
permeabilization on the structure of the
parenchyma of osmotic dehydrated
‘Granny Smith’ apple
Amparo Quiles, Isabel Hernando, Isabel Pérez-Munuera, Empar Llorca,
Virginia Larrea and M Ángeles Lluch∗
Departamento de Tecnologı́a de Alimentos, Universidad Politécnica de Valencia, PO Box 22012, 46071 Valencia, Spain
Abstract: Calcium plays a very important role in the maintenance of quality in vegetable foods and it
is used as a texturing agent to preserve the structure of minimally processed vegetables. The osmotic
dehydration (OD) of ‘Granny Smith’ apple parenchyma by immersion in a sucrose solution allows us to
obtain products with sensorial characteristics similar to those of fresh products, but it produces a loss
of cellular integrity. Here we study the use of calcium as a possible preservative for the microstructural
integrity of the product during OD, as well as to test whether cellular permeabilization improves the
impact of calcium on the edible parenchyma of the ‘Granny Smith’ apple. Sodium dodecyl sulfate (SDS)
has been used for convenience as a permeating agent although we recognize this may not be used in
practice. The microstructure of the OD apple treated with calcium chloride and with SDS has been
compared with fresh apple by means of light microscopy, transmission electron microscopy and low
temperature scanning electron microscopy. The results show that calcium has a protective effect on the
structural integrity during OD: it strengthens the cellular walls cements, avoids cellular collapse and
protects protoplasts from plasmolysis. Although SDS favours calcium penetration, its use is not advisable
since it encourages the destructuring effect of the OD and counteracts the consolidating effect of calcium.
 2004 Society of Chemical Industry
Keywords: Granny Smith apple; osmotic dehydration; microstructure; cellular wall; calcium chloride;
permeabilization; sodium dodecyl sulfate
INTRODUCTION
There is an increasingly high demand for dehydrated
products that can be preserved easily due to
continuous changes in lifestyle.1 In recent years
osmotic dehydration (OD) has proved to be an
important preservation technique that allows the
partial exit of water by the direct contact of vegetable
or animal tissue with a solution of high osmotic
pressure and low water concentration (aw ). OD
allows us to obtain products with a longer shelflife with sensorial characteristics similar to those of
fresh products and a high nutritive value.2,3 Although
the ‘Granny Smith’ apple variety is not the most
produced cultivar in EU and Spain, it is one of
the varieties most appreciated by the consumers. For
instance, children’s attitudes towards eating apples
have been investigated4 and results show that the
‘Granny Smith’ apple is their favourite of six different
varieties. The ‘Granny Smith’ apple can be therefore
considered a typical fruit used in OD conservation
studies.
The OD of apple parenchyma by immersion
into a sucrose solution nevertheless affects its
microstructure, producing collapsed and deformed
cells, as well as intercellular spaces that are lengthened
and longitudinally contracted.5,6 Quantification by
image analysis (IA) shows that the contraction of a
macroscopic portion of the apple is due to microscopic
contraction of the cells as well as to the microscopic
contraction of the intercellular spaces.7
Calcium is a fundamental element in the structure,
function and stability of the cellular membrane and
cellular wall.8 The quantity of calcium surrounding
the cell affects its degree of firmness and its fragility
against breaking and deterioration. Calcium could
therefore be used as a structuring agent to minimize
the damage produced by OD on the structural
integrity. Microstructural9 studies carried out with
∗
Correspondence to: M Ángeles Lluch, Departamento de Tecnologı́a de Alimentos, Universidad Politécnica de Valencia, PO Box 22012,
46071 Valencia, Spain
E-mail: [email protected]
Contract/grant sponsor: Comisión Interministerial de Ciencia y Tecnologı́a (CICYT), Spain; contract/grant number: ALI 96-1126
(Received 22 October 2003; revised version received 6 April 2004; accepted 16 April 2004)
Published online 4 August 2004
 2004 Society of Chemical Industry. J Sci Food Agric 0022–5142/2004/$30.00
1765
A Quiles et al
IS
a
b
IS
c
PT
d
DCW
PT
g
f
PL
ML
ML
TN
Figure 1. a. Cryo-SEM: fresh apple. b. Cryo-SEM: OD apple. c. LM: fresh apple. d. LM: OD apple. f. TEM: fresh apple. Plasmalemma contiguous to
the cellular wall. g. TEM: OD apple. Degraded middle lamella. No plasmalemma is observed near the cellular wall. DCW: neighbouring cell walls
detached. ML: middle lamella. PL: plasmalemma. PT: protoplast. IS: intercellular space. TN: tonoplast.
‘Golden Delicious’ apples treated with calcium
showed that the middle lamella and the cellular walls
did not suffer deformation during storage and their
cell-to-cell contacts remained intact. However, on
untreated apples, the middle lamella and walls suffered
degradation and the cells became progressively
separated. The degradation of the cellular wall starts
with the deterioration and destruction of the middle
lamella.9,10 These changes result in the loss of cellular
cohesion, and in the loss of firmness and textural
quality of the fruit.
Although there are microstructural studies on the
behaviour of the edible parenchyma of the ‘Granny
Smith’ apple during OD,5,6,11 there are no data on the
influence that calcium chloride (CaCl2 ) treatment has
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on the parenchyma of this apple variety, nor are there
any data available on the possible preserving effect that
calcium may exert on the structural integrity during
OD. This paper studies the microstructural effect of
a CaCl2 treatment in order to maintain the structural
integrity of apple during OD.
Calcium penetration on the fruit is favoured
by permeabilization of the cellular wall. Adding
surfactants to the CaCl2 solution could improve the
final calcium content in some fresh apple varieties,
such as ‘McIntosh’ and ‘Delicious’, while for other
varieties, such as ‘Golden Delicious’ and ‘Spartan’, no
difference has been found.12,13 This work also studies
the combined effect of sodium dodecyl sulfate (SDS),
used as a permeating agent, with the CaCl2 , in order
J Sci Food Agric 84:1765–1770 (online: 2004)
Calcium and cellular permeabilization of parenchyma of apple
Figure 2. Cryo-SEM.a. Fresh apple treated with CaCl2 . b. Detail of fresh apple treated with CaCl2 . c. Cell-to-cell contact in fresh apple treated with
CaCl2 . d. OD apple treated with CaCl2 . f. Detail of OD apple treated with CaCl2 . g. Reinforced cements in OD apple treated with CaCl2 . CC:
cellular cements.
to observe a possible improvement of the penetration
of calcium in ‘Granny Smith’ apple during OD.
syrup:kg of fruit was higher than 50:1, so it could
be assumed that the concentration of the solution
remained constant during dehydration.
MATERIALS AND METHODS
Materials
‘Granny Smith’ apples were purchased from a local
market and stored at 4 ◦ C until their treatment. For
each treatment, four apple rings (15 mm thick, 75 mm
external diameter and 23 mm internal diameter,
without skin and core) were obtained from four
different apples. The rings were cut into cubes (15 mm
side, four cubes from each ring).
In order to carry out the CaCl2 treatment, the apple
cubes were submerged in a 40 g l−1 CaCl2 solution
for 30 min at room temperature and with constant
stirring. For the treatment with SDS, the apple cubes
were submerged in a 2 g l−1 SDS solution for 30 s at
room temperature and with constant stirring. For OD,
the untreated or treated CaCl2 and/or SDS cubes were
submerged in a sucrose solution (65◦ Brix, 25 ◦ C) and
placed within a shaker during 8 h. The ratio kg of
Methods
Cryo-SEM (low-temperature scanning electron
microscopy)
A Jeol JSM-5410 SEM instrument (Izasa, Barcelona,
Spain) was used with a Cryo CT-1500C unit (Izasa,
Barcelona, Spain). The sample was placed in the
holder, fixed with slush nitrogen (T ≤ −210 ◦ C),
transferred frozen to the Cryo unit, etched, and
gold-coated (2 mbar, 2 mA). The sample was then
transferred onto the microscope and examined at
15 kV and −130 ◦ C.
J Sci Food Agric 84:1765–1770 (online: 2004)
LM (light microscopy) and TEM (transmission electron
microscopy)
Samples were fixed with a 25 g l−1 glutaraldehyde
solution (0.025 M phosphate buffer, pH 6.8, at
4 ◦ C, 24 h), postfixed with a 20 g l−1 OsO4 solution
(1.5 h), dehydrated using a graded ethanol series
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A Quiles et al
a
b
PT
PT
c
d
PT
ML
PT
Figure 3. a. LM: Fresh apple treated with CaCl2 . b. LM: OD apple treated with CaCl2 . c. TEM: Fresh apple treated with CaCl2 . d. TEM: OD apple
treated with CaCl2 . ML: middle lamella. PT: protoplast.
(300, 500, 700, 960 and 1000 g kg−1 ), contrasted
in 40 g l−1 uranyl acetate and embedded in epoxy
resin (ANAME, Madrid, Spain). The samples were
cut using a Reichter Jung ultramicrotome (Leila,
Barcelona, Spain). Ultrathin sections (10 nm) were
stained with 40 g l−1 lead citrate and observed in a
Philips EM 400 (Eindhoven, Holland) transmission
electronic microscope at 80 kV. The thick sections
(0.5 µm) were stained with 10 g l−1 toluidine blue and
examined in a Nikkon Eclipse E800 light microscope
(Izasa, Barcelona, Spain).
RESULTS AND DISCUSSION
The tissue of fresh ‘Granny Smith’ apple is composed
of numerous cells and intercellular spaces; intercellular
spaces are the result of the joining of three,
four, or even eight, ten or more cells (Fig 1a).
This structure has also been described for ‘Granny
Smith’ and ‘Red Delicious’ tissues by SEM14
and for ‘Golden Delicious’ by cryo-SEM.15 In
fresh apples, cells are swollen (Figs 1a and c) and
closely bonded to each other (Fig 1c) by means
of a well-delimited medium lamella (Fig 1f). Inside
the cells, a large vacuole occupies most of the
protoplast (Fig 1c), and both the plasmalemma and
the tonoplast are kept close to the cellular wall
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(Fig 1c, f). In fresh apples, cellular walls are formed
by cellulose fibrils closely packed together with no
discontinuities (Fig 1f) and consequently, with a dense
appearance.
Osmotic dehydration produces plasmolysis on the
cells, which become deformed, contracted and collapsed (Fig 1b); the intercellular spaces are contracted
and deformed (Fig 1b). Those spaces closest to the
interphase food-osmotic solution are dumped by the
osmotic solution that acts as a physical barrier against
enzymatic browning.16 The close cell-to-cell contacts that characterize fresh apples are not present in
dehydrated apples, where neighbouring cell walls are
detached (Fig 1d) and the middle lamella is destructed
(Fig 1g). The protoplasts and cellular content residues
appear in plasmolysis and are retracted to the centre
of the cell (Fig 1d, g). OD produces thicker and edgetwisted cellular walls, as well as detached cellulose
fibrils, producing less density on some areas of the
wall (Fig 1g).
Fresh apple parenchyma treated with CaCl2 shows
well preserved and non-fractured cells (Fig 2a).
Cellular walls are swollen and compact (Fig 2b),
but thicker (Fig 3a) than in non-treated fresh apples.
The calcium treatment reinforces the cellular cements
and maintains the strength of the cell-to-cell contacts
(Fig 2c). The middle lamella is strengthened and more
J Sci Food Agric 84:1765–1770 (online: 2004)
Calcium and cellular permeabilization of parenchyma of apple
a
PCW
b
CC
d
c
PT
PT
PCW
Figure 4. a. OD apple treated with SDS and CaCl2 . a and b. Cryo-SEM. c. LM: d. TEM. CC: cellular cements. PCW: perforated cellular wall. PT:
protoplast.
intensely stained (Fig 3c) than in CaCl2 non-treated
fresh apple (Fig 1f). The protoplast (plasmalemma,
tonoplast and the cytoplasmic lumen) is located
near the cellular wall (Fig 3a and c) as in fresh
apples. Byung et al 17 studied the effect of calcium
application on cell structure of stored apple (cv ‘Fuji’
and ‘Tsugaru’). Electron microscopy analysis of cell
structure in calcium-treated and untreated fruit stored
for 120 days showed that untreated fruit had severe
degradation of cell middle lamella whereas calciumtreated fruit maintained lamella integrity. Seok and
Lee18 investigated the effects of postharvest calcium
infiltration on cell wall structure of ‘Fuji’ apple cultivar
stored for 6 months. The cell wall of calcium treated
fruit maintained the middle lamella region but in
untreated fruits the cell wall was separated due to
its dissolution.
The images obtained for OD apples treated with
CaCl2 show that calcium has a preserving effect on
the microstructure (Fig 2d). CaCl2 -treated OD cells
are not as collapsed (Fig 2d and f) as in OD apples
(Fig 1b); calcium has a strengthening effect on the
cellular walls (Fig 3b) and protects protoplast against
plasmolysis (Figs 3b and d). However, OD produces
striations and wrinkles on the surface of the cells
(Fig 2g), as well as distortion and deformation of the
edges of the cellular walls (Fig 3d). Calcium keeps
neighbouring cells bonded (Fig 3b) and reinforces
the middle lamella, minimizing cellular detachment
and cellulose fibrils separation (Fig 3d). The cellular
walls are more intensely stained and are more
reinforced (Fig 3d) than the cells of the non-treated
J Sci Food Agric 84:1765–1770 (online: 2004)
OD apple parenchyma. Glenn and Poovaiah9 also
described well-preserved cell-to-cell contacts and a
strong stained middle lamella in calcium-treated
‘Golden Delicious’ apples. Our results show that the
structuring and consolidating effect of calcium may
partly counteract and minimize the destabilizing effect
of the OD on the cellular integrity of ‘Granny Smith’
apple.
Osmotic dehydrated apples treated with SDS and
CaCl2 show opposing effects. On the one hand, the
typical effects of the OD are present: cellular collapse,
cellular plasmolysis (Fig 4a), contraction of the
protoplast towards the centre of the cell (Fig 4c) and
distortion of the edges of the cellular walls (Fig 4d).
On the other hand, because of the treatment with
CaCl2 , many areas show reinforced cell-to-cell bonds
(Figs 4a) and intensely stained cellular walls with
well-packed cellulose fibrils (Fig 4d). Nevertheless,
SDS produces alteration and perforation of the
cellular walls (Fig 4b and c). Therefore, although the
permeabilization effects of the SDS could probably
help calcium penetration, its use cannot be advised,
since it increases the destructuring effect of the
OD and it counteracts the consolidating effect of
calcium.
ACKNOWLEDGEMENT
The authors would like to thank the Comisión
Interministerial de Ciencia y Tecnologı́a for its
financial support (Project ALI 96-1126, CICYT,
SPAIN).
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