Lebensm.-Wiss. u.-Technol., 33, 89}102 (2000)
The E!ects of Calcium Chloride and Sucrose Prefreezing
Treatments on the Structure of Strawberry Tissues
J. Suutarinen, K. Heiska, P. Moss and K. Autio
J. Suutarinen, K. Heiska, K. Autio: VTT, Biotechnology, P.O. Box 1500, Fin-02044 VTT (Finland)
P. Moss: Department of Forest Products Technology, Technical University of Helsinki, FIN-02151 Espoo
(Finland)
(Received April 28, 1999; accepted September 30, 1999)
The structural changes in strawberry tissues during prefreezing treatments, freezing and thawing were studied by means of textural and
drip loss measurements as well as by bright-xeld and confocal laser scanning microscopy (CLSM), and by Fourier transform infrared
(FT-IR) microscopy. Calcium chloride or sucrose were used as dipping pretreatment agents before freezing. In the calcium chloride test
series a full factorial screening design was used. The three-level factors included were the CaCl2 concentration of the dipping solution,
the dipping time and the temperature of the solution. Two kinds of sucrose treatments were used. Strawberries were either dipped in
water}sucrose solutions or were sprinkled with crystallized sucrose. The CaCl2 or sucrose pretreatments did not signixcantly awect the
drip loss of thawed strawberries compared to water dips or untreated reference samples. The pretreated strawberries were xrmer than
the untreated control samples but some of the CaCl2- and sucrose-treated strawberries were less xrm than the strawberries dipped in
water. According to microscopical studies, both CaCl2 and crystallized sucrose pretreatments awected the microstructure of strawberry
tissues. These pretreatments especially awected pectin, protein, lignin and structural carbohydrates in the vascular tissue and cortex
compared to the untreated reference samples. CaCl2-treatment had an even stronger ewect than sucrose on the above compounds except
lignin. The pretreatments did not seem to awect the epidermis, hypodermis or pith.
( 2000 Academic Press
Keywords: strawberries; prefreezing treatments; structure
Introduction
Consumers demand high quality jams with more natural
#avour, colour and whole fruit content. The changes in
the texture of berries appear as tissue softening and loss
of cohesiveness as well as a decrease in the extent of
intermolecular bonding between cell wall polymers (1). In
Nordic countries, the season for fresh strawberries is
short and very few industrially processed jams are made
from fresh berries during the harvesting season; instead,
frozen berries are commonly used (2). In order to maintain the original shape of the fruit it is sometimes necessary to pretreat it to modify its structure. One way of
achieving this is to use calcium, which forti"es the fruit by
changing the pectin structure (3). Calcium ions are important intracellular messengers in plants. Studies on leaf
senescence and fruit ripening have indicated that the rate
of senescence often depends on the calcium status of the
tissue and that by increasing calcium levels, various parameters of senescence such as respiration, protein and
chlorophyll content, and membrane #uidity are altered.
Calcium maintains the cell wall structure in fruits by
interacting with the pectic acid in the cell walls to form
0023-6438/00/020089#14 $35.00/0
( 2000 Academic Press
calcium pectate. Thus, fruits treated with calcium are
generally "rmer than controls (4,5,6). Another pretreatment of fruit is the addition of crystallized sucrose. The
added sucrose acts as a dehydrating agent in order to
decrease the water content of strawberries. Water is
transferred out of the fruit into the syrup medium and
sucrose di!uses into the berries (7). A reduction in free
water causes less cell wall rupture and rearrangement of
the cell contents during freezing. Decreasing drip losses
ensure better fruit appearance, #avour, aroma and
rheological behaviour in cooking.
The strong susceptibility of strawberries to textural
damage is not surprising when one considers their extremely low solid content. About 10% of solid matter in
the strawberry must be arranged into a very intricate
polymeric structure capable of supporting a large
amount of liquid (8). Tissue structural features play an
essential role in determining texture. Such factors as the
size and shape of cells, the volume of intercellular spaces
and the thickness of the cell wall are important. Thin
areas of the cell walls may present sites of easier wall
breakage (9). The microscopic structure and molecular
architecture of the cell wall has an important bearing on
doi:10.1006/ fstl.1999.0616
All articles available online at http://www.idealibrary.com on
89
lwt/vol. 33 (2000) No. 2
its function. The wall of most interest is the primary cell
wall. Secondary cell walls are virtually absent from mature fruits (10). The wall has been presented as being
composed of cellulose "brils embedded in a matrix consisting largely of pectic substances, hemicelluloses, proteins, lignins, lower molecular weight solutes and water
(11). The relative composition of the wall varies as one
goes from the plasmalemma, separating the wall from the
cytoplasm, to the middle lamella. In a very simpli"ed
sense the cellulose gives rigidity and resistance to tearing,
while the pectic substances and hemicelluloses confer
plasticity and the ability to stretch. Upon strawberry
maturation, the most observable changes take place in
the parenchymal cells situated in the cortex. These cells
undergo considerable enlargement and become separated from each other. The cortex grows most rapidly and
the pith most slowly (12). The structure of the ripe fruit
depends largely on maintenance of turgor in the cortical
parenchymal cells (13). During freezing, no changes were
found in epidermal and xylem cells but signi"cant rupture of parenchymal (cortex) cells was found to occur (14).
Varieties with large cells su!er more damage than those
with small cells (14,15). Microscopical examination of
fruit pieces from strawberry jams has shown that only the
epidermal and vascular tissues retain their structural
integrity during the boiling stage of jam production and
that the bulk of the cells rupture (16).
Bridging the gap between the molecular nature (e.g. data
from the chemical structure and interaction) and the
perceived functionality (texture, #avour, colour, etc.) is an
absolute requirement in reaching a true understanding of
the chemical, physical and biological nature of a food at
all levels. One of the most powerful techniques for studying microstructure is confocal laser scanning microscopy
(CLSM), a form of light microscopy employing laser light
which enables nondestructive optical sectioning of samples in both the normal plane and z-dimension. It can be
used in #uorescence mode and a wavelength is selected to
excite a speci"c substance. This method not only provides an image with better resolution than conventional
light microscopy or #uorescence microscopy, but also
provides an opportunity to observe a three-dimensional
image without the need for mechanical sectioning. In
addition, it can distinguish the spatial location of di!erent components by detecting #uorescence from dyes speci"c to di!erent chemical species (17).
In our earlier studies we developed methods based on
Fourier transform infrared (FT-IR) and light microscopy
(18). In this investigation we have used these methods
and CLSM to study how the calcium chloride and sucrose prefreezing treatments a!ect the di!erent strawberry
tissues and their cell wall components during freezing
and thawing.
harvested and hulled manually and stored in 1-L PET
(polyethylene) cases at 5 3C before being treated. The
treatments were administered to the whole strawberries
within a few hours of harvesting. Strawberries (2.5 kg)
were randomly chosen for each treatment. The average
weight of a Finnish strawberry was 8.4 g with an average
cross-sectional area of 490$140 mm2. Fresh Egyptian
strawberries (cv. unknown) grown in the spring of 1999
were used for the confocal laser scanning microscopy
studies.
Experimental procedure
In the calcium chloride test series, a full factorial screening design containing 11 tests was used. The three-level
factors included were CaCl concentration of the dipping
2
solution, 1, 5.5 or 10 g/L, respectively, dipping time, 0.25,
7.625 or 15 min, respectively, and temperature of the
solution, 25, 37.5 or 50 3C, respectively (Table 1). Treatment in 1% CaCl solution has been shown to be e!ec2
tive in maintaining the "rmness of strawberries (5). In the
preliminary test the treatment of fresh strawberries in
solutions with a CaCl concentration greater than 1%
2
was found to cause an o!-taste in fresh strawberries.
Therefore CaCl concentrations over 1% were omitted
2
from the investigation. Di!erent temperatures of dipping
solutions were used to study the e!ect of CaCl on
2
strawberry tissues. The experimental design was created
using Modde for Windows 3.0 software (Umetri Ab,
Umeas , Sweden).
Two kinds of sucrose treatments were used. Strawberries
were either dipped in water}sucrose solutions (350 and
700 g sucrose/1 L water) or they were sprinkled with
crystallized sucrose (Table 2) The amount of sucrose
(crystal size approximately 0.53 mm), 150 g/kg strawberries, was selected as being the normal amount used in
household strawberry freezing. Crystallized sucrose
(75 g) was sprinkled on each 0.5 kg layer of strawberries.
The strawberries were put in 1-L PET cases (length
130 mm, width 105 mm and height 72 mm, respectively)
and stored for further analysis.
Table 1 Experimental design used in CaCl pretreat2
ments
Materials and Methods
Strawberries
Fresh Finnish strawberries (Fragaria ananassa, cv.
Jonsok) grown in central Finland in the summer of 1998
were used for the studies. The mature strawberries were
90
Experiment
CaCl
Dipping time Temperature of
2
number concentration
(min)
dipping solution
(g/L)
(3C)
1
2
3
4
5
6
7
8
9
10
11
12
13
1
0.25
10
0.25
1
15
10
15
1
0.25
10
0.25
1
15
10
15
5.5
7.625
5.5
7.625
5.5
7.625
0 (Water)
15
Untreated control sample
25
25
25
25
50
50
50
50
37.5
37.5
37.5
25
lwt/vol. 33 (2000) No. 2
Table 2 Experimental design used in sucrose pretreatments. The temperature of the dipping solutions was 5 3C
Experiment
number
Dipping/
sprinkling
1
2
3
4
5
Dipping
Dipping
Dipping
Dipping
Sprinkling
6
7
Sucrose
concentration
700 g/1 L water
700 g/1 L water
350 g/1 L water
350 g/1 L water
150 g/1 kg
Strawberries
Dipping in water
0
Untreated control sample
Dipping
time (min)
1
15
1
15
1
In the CaCl and sucrose dipping experiments, 1.25 kg
2
strawberries were dipped in 1.5 L dipping solution. In the
CaCl experiments where the temperature of the dipping
2
solutions was 37.7 or 50 3C, the dipping bowl was kept in
a temperature-controlled bath of 37.5 or 50 3C, respectively. A light weight was put on the strawberries to
ensure that all the berries were submerged in the dipping
solution. After dipping, the berries were drained for 5 min
in a plastic strainer and packaged in 0.5 kg portions in
1-L PET cases. The strawberry cases were put into 2-L
plastic bags, which were left open until the strawberries
had been frozen. About 1 h after either dipping in CaCl
2
solutions or sucrose solutions or adding crystallized sucrose, the strawberries were frozen in an industrial freezer
at !20}25 3C without mechanical ventilation. The temperature of the strawberries stayed at !2 3C for 10 h.
After 36 h the temperature of the strawberries reached
!20 3C. The strawberries were stored at !20 3C for
approximately 2 mo before thawing and analysis.
Texture analyser measurements
After approximately 2 mo of freezer storage, the strawberries were thawed for analysis by keeping them in their
storage cases in a refrigerator at 5$1 3C for 24 h.
Thawed fruits were equilibrated to 17 3C by keeping
them at 20$1 3C on a counter for 4 h. Thawed strawberries were then dried with paper tissue and weighed into
80 g portions for "rmness measurements. The compression force of strawberries was measured with a Texture
Analyser (model TA-XT2, Stable Micro Systems, U.K.)
with a 25 kg load cell using an Ottawa Cell (A/OTC) with
a Holed Extrusion Plate (A/HOL) and large plunger. The
starting position of the plunger was 50 mm from the base
and the "nal position 10 mm above the base plate. The
plunger speed was 1.5 mm/s. Maximum force was calculated by averaging four replicates.
Drip loss
The strawberries were weighed before and after thawing.
The after-thaw weight was taken after 3.5 h equilibration
at 20$1 3C. For separating the drip from the berries,
0.5 kg strawberries were put onto a plastic screen and
allowed to drip for 30 s. Drip loss was calculated by
averaging the percentages of the original weights of the
strawberries of three replicates.
Microstructural studies
After approximately 2 mo of freezer storage, the untreated control and pretreated samples were freeze-dried
for 20 h (Dr Morand freeze-drier, Germany). The freezedried strawberries were cut into smaller pieces, mixed
gently (Vari-Mix Aliquot Mixer, USA) in an in"ltration
solution for 5 d, polymerized in a Historesin Embedding
Kit (Jung, Germany) and cut into thin sections (4 km)
with a Microm HM355 (Microm LaborgeraK te GmbH,
Germany) microtome. Sections were stained with the
following staining solutions: 0.2 g/L Ruthenium Red in
water for 2 h, followed by rinsing with water; 0.1 g/L
Light Green for 1 min, rinsing with iodine; and 2 g/L
phloroglucinol in 95 g/L ethanol and 50 g/L sulphur acid
(2 : 1). Ruthenium Red stains pectin pink; Light Green
and iodine stain protein green or yellow, and phloroglucinol and lignin light pink. For the #uorescence microscopic examinations, the sections were stained with
0.1 g/L Calco#uor White M2R New and measured at
400}410 nm (8). Samples were examined and photographed with an Olympus BX-50 microscope (Olympus,
Japan). The images were viewed with a Soft Imaging
System Analysis (Soft Imaging System GmbH, Germany)
and printed.
Confocal laser scanning microscopical studies
A Leica CLSM was used to examine cryo-sections of
pretreated frozen strawberries and untreated fresh strawberries. The CLSM was used in #uorescence mode with
an excitation wavelength of 488 nm. Samples were
stained with acridine orange (Merck) which is a good
general stain, although it appears to stain lignin much
more intensely than other cell-wall components. This
was demonstrated by staining commercial preparations
of cellulose, lignin, pectin and protein with acridine
orange and comparing the image intensities. A surgery scalpel was used to cut the strawberry in half.
Samples were mounted in distilled water and examined with oil immersion objectives. Series of optical
sections were collected and then added together to
make an extended focus image of the sample area. The
depth to which a sample can be optically sectioned depends on the density and transparency of the material;
with the fresh strawberry material, the limit was about
160 km.
FT-IR microspectroscopical studies
All FT-IR spectra were recorded using a Bruker IFS 66
(Bruker Optik GmbH, Germany) FT-IR spectrometer.
The spectrometer was equipped with a microscope accessory. A liquid nitrogen cooled mercury cadmium telluride (MCT) detector was attached to the microscope. The
spectrometer system was purged with predried nitrogen
gas. After approximately 2 mo storage in a freezer, pretreated calcium and sucrose as well as untreated reference
91
lwt/vol. 33 (2000) No. 2
strawberries were randomly chosen and thawed at room
temperature (20$1 3C). The strawberries were thawed
in an upright position with the hulls on the table so as to
ascertain what e!ect thawing would have on the structure of pretreated strawberries. Thawed strawberries
were frozen at !40 3C for 15 min and 7-km sections
were cut using a cryostat (Leitz, Germany). Ethanolwashed aluminium foil, "xed with tape to the object lens,
was used to surround the specimens. After focusing on
the subject to be scanned, the aperture (size 1.2 with
a 36]objective) was adjusted to frame the desired portion for scanning and to exclude unwanted tissue. Strawberry sections were manually mapped step by step either
across the achene, vascular tissue and pith, or across the
epidermis, hypodermis, cortical cells and pith. The transmission mode was used and 100 scans were accumulated
to produce a spectrum over the 4000}700 cm~1 range
at a resolution of 4 cm~1. A reference was scanned
using the clean aluminium foil and 100 scans were accumulated to produce the transmission spectrum in the
4000}700 cm~1 range. After logarithmic transformation
and baseline correction, the absorbance spectra were
stored.
Results and Discussion
Firmness and drip loss
The CaCl or sucrose pretreatments did not a!ect the
2
drip loss of thawed strawberries signi"cantly when compared to water-dipped or untreated control samples. The
pretreated strawberries were "rmer than the untreated
control samples, but some of the CaCl -treated strawber2
ries were less "rm than the strawberries dipped in
water. Sample number 11 had the highest "rmness value
and was selected together with crystallized sucrosesprinkled sample number 5 for the microscopical studies
(Tables 3, 4).
Table 3 Drip loss and "rmness of the strawberries in
CaCl test seriesa
2
Experiment
number
1
2
3
4
5
6
7
8
9
10
11
12
13
Drip loss (g/100 g)
Firmness (kg)
xN
s
9
xN
s
9
24.5
26.5
25.6
28.7
25.3
27.2
25.3
25.7
26.3
26.4
24.6
24.9
24.5
0.6
0.3
1.1
0.2
1.2
1.6
0.7
0.6
0.4
0.3
1.5
1.2
1.1
10.6
11.3
10.2
9.7
10.1
10.3
9.6
10.3
9.9
10.1
12.5
10.3
9.2
0.9
1.4
0.9
0.6
0.3
0.5
1.0
1.1
1.1
1.5
0.5
1.1
0.4
a Mean (xN ) and standard deviation (s ) of three measurements of
9
drip loss and four of "rmness
Table 4 Drip loss and "rmness of the strawberries pretreated with sucrosea
Experiment
number
1
2
3
4
5
6
7
Drip loss (g/100 g)
Firmness (kg)
xN
s
9
xN
s
9
25.0
26.7
27.5
26.9
25.0
25.7
23.0
0.2
0.9
1.3
0.5
0.9
0.7
0.7
11.6
10.5
10.2
11.6
12.0
10.9
9.2
1.0
0.4
1.2
0.9
0.6
0.8
0.4
a Mean (xN ) and standard deviation (s ) of three measurements of
9
drip loss and four of "rmness
Microstructural studies
The di!erent chemical components in di!erent parts of
the strawberry cell walls, cortical cells and vascular tissue
were stained with di!erent staining systems, and the
locations of pectin, protein and lignin were studied. In the
untreated reference samples, the pectin in the middle
lamella was poorly stained compared to the pretreated
samples (Fig. 1). In calcium-treated strawberries, pectin
was more strongly stained than in the reference or sucrose-treated samples. Furthermore, the protein was
poorly stained especially in the reference and sucrosetreated samples compared to the Calcium-treated sample
(Fig. 2). The lignin in the vascular tissue was less changed
in the sucrose-treated sample (Fig. 3). In particular, the
lignin of the reference strawberry vascular tissue was
seriously damaged. The cell walls in the cortex of the
calcium-treated strawberries appeared to have more
bright blue staining than the sucrose-treated or reference
samples (Fig. 4).
CLS microscopical studies
Fresh and calcium-treated strawberry sections of cortex
stained with acridine orange are seen in Fig. 5. The fresh
strawberry sample was scanned at di!erent depths and
16 sections (taken at 5-km incremental steps with a 10]
objective) were reconstructed by computer (Fig. 5a). The
cell content was quite homogeneously spread inside
the fresh strawberry cell walls whereas in the calciumtreated strawberry cryo-section (single section taken with
a 40]objective) there was shrinkage of the cytoplasm
due to osmotic water loss, although the cell walls themselves looked quite undamaged (Fig. 5b). The same e!ect
was found in the sucrose-treated strawberry cryo-sections (not shown). In the untreated reference strawberry
section the cell walls were mostly broken (not shown).
Figure 6 shows cells around the vascular tissue in a fresh
strawberry (single section taken with a 25]objective,
Fig. 6a) and vascular tissue of a sucrose-treated cryosection (taken at 1-km incremental steps with a 40]objective, Fig. 6b). In the fresh strawberry, the cell walls
around the vascular tissue were undamaged. The ligni"ed
annular and helical thickenings of the primary xylem
92
lwt/vol. 33 (2000) No. 2
Fig. 1 Micrographs of strawberry vascular (a) and cortical (b) tissue. Pectin (marked with arrow) was stained and appeared pink
93
lwt/vol. 33 (2000) No. 2
Fig. 2 Micrographs of strawberry vascular (a) and cortical (b) tissue. Proteins (marked with arrow) were stained and appeared
green or yellow
94
lwt/vol. 33 (2000) No. 2
Fig. 3 Micrographs of strawberry vascular tissue. Lignin (marked with arrow) was stained and appeared pink
95
lwt/vol. 33 (2000) No. 2
and especially in the reference strawberry sections, the
lignin spirals were also broken to some extent and their
#uorescence was rather low (not shown).
FT-IR microspectroscopical studies
The surfaces of the strawberry sections were rough, probably due to compression of structural compounds after
thawing and refreezing. Therefore, the measured spectra
were scattered. Before analysis, the spectra were manipulated by smoothing to compensate for these e!ects. The
smoothing was done using the Savitzky}Golay algorithm, which decreases the signal to noise ratio (S/N) of
spectra and some peaks, for example, peaks typical of
protein, lignin and pectin, tended to overlap to some
degree. Some of the strawberry tissues, e.g. the vascular
tissue of each sample, were broken at some points, so
measurements were recorded only from the points where
tissue existed. Trying to keep the direction from the edge
of the epidermis into the centre of the pith at each stage
while at the same time trying to avoid the tissue-free
areas therefore meant that the number of spectra of each
tissue was quite low.
Fig. 4 Micrographs of strawberry cell walls of cortical tissue
were also coherent and intact. In the sucrose-treated
strawberry, the lignin spirals were broken to some extent but were mostly coherent. In the calcium-treated,
Achene, vascular tissue and pith
Figure 7 shows a series of spectra in the 4000}700 cm~1
region of sections of strawberry tissue obtained from the
outer edge of the achene through the vascular tissue into
the centre of the pith of the untreated reference and
CaCl -treated as well as the sucrose-treated strawberries.
2
The "rst two spectra represent the surface of the achene.
The next three spectra represent the achene. The following eight spectra, 5 to 13, represent the vascular tissue.
The spectra from 14 to 17 (15 or 16 in the cases of the
sucrose- or calcium-treated strawberries) of the reference
represent the pith in Fig. 7. In all these spectra, the OH
and CH stretching vibration bands were present in the
3500}2900 cm~1 region. In each spectrum, a group of
peaks situated approximately in the 1460}1330 cm~1
region represent structural carbohydrate bands. Also, in
each spectrum, the CO stretching vibration band at approximately 1240 cm~1 and strong carbohydrate bands
in the 1200}1000 cm~1 region were present. An overview
of series of spectra of reference and pretreated sections of
strawberry tissues showed that peak absorbances of
spectra of the Calcium-treated strawberries were higher
and their areas larger, especially in vascular tissue, compared to the sucrose-treated or reference strawberries.
Resolution of spectra of the achene and its surface, especially in pretreated sections, was quite low. This was
probably due to compression of structural compounds
after destruction of composition during or after treatments. In consequence, very concentrated material did
not allow light to penetrate.
In each of the achene spectra (Fig. 7), a strong CO
stretching vibration band was present at approximately
1740 cm~1 due to an ester stretching vibration. This
is characteristic of pectin. At approximately 1680
and 1550 cm~1, small amide I and amide II bands
were present in the spectrum of the calcium- as well as
96
lwt/vol. 33 (2000) No. 2
Fig. 5 Fresh (a) and calcium-treated (b) strawberry sections of cortical tissue
97
lwt/vol. 33 (2000) No. 2
Fig. 6 Fresh (a) and sucrose-treated (b) strawberry sections of vascular tissue
98
lwt/vol. 33 (2000) No. 2
Fig. 7 Transition from the strawberry achene through vascular tissue to the centre of the pith in the 4000}700 cm~1 region
baseline-corrected stack map. (a) untreated reference; (b) calcium-treated strawberry; (c) sucrose-treated strawberry
99
lwt/vol. 33 (2000) No. 2
Fig. 8 Transition from the strawberry epidermis through the hypodermis, cortex and the vascular tissue to the centre of the pith in
the 4000}700 cm~1 region baseline-corrected stack map. (a) untreated reference; (b) calcium-treated strawberry; (c) sucrose-treated
strawberry
100
lwt/vol. 33 (2000) No. 2
the sucrose-treated strawberries. At approximately 1605
and 1510 cm~1, peaks typical of lignin were present in
the achene spectrum of the reference. One cannot recognize very clearly the peak at 1605 cm~1 in every measured spectrum of pretreated sections of the strawberry
achene. This means that either lignin has been broken in
the pretreated strawberry from the measured points or
&more likely' the lignin peak was not seen due to overlapping e!ects. In the achene spectra of pretreated strawberries, especially in the sucrose-treated strawberries, structural carbohydrate bands in the 1460}1330 cm~1 region
as well as carbohydrate bands in the 1200}1000 cm~1
region were weaker than in the reference. This was probably due to the overall destruction of the structural
compounds in the achenes of these strawberries.
In the vascular tissues of the reference and pretreated
strawberries there was a carbonyl band at approximately
1725 cm~1, representing pectin (Fig. 7). Also, at approximately 1650 and 1550 cm~1, amide I and amide II bands
were present in each spectra. In particular, in the spectra
of the vascular tissue of the calcium-treated strawberries,
the pectin and protein peak absorbances were higher and
peak areas larger than in the reference or sucrose-treated
strawberries. Furthermore, the pectin and protein peak
areas were also slightly larger in the spectra of the vascular tissue of the sucrose-treated strawberry compared to
the control. The structural carbohydrate peaks in the
1460}1330 cm~1 region as well as carbohydrate peaks
absorbances in the 1240}1000 cm~1 region were higher
and peak areas larger in the spectra of the calciumtreated strawberries than in the spectra of the reference
or the sucrose-treated strawberries. In the spectra of
the vascular tissue of the reference and pretreated strawberries, the typical lignin peaks could not be detected
even if the lignin was quite evidently present in the
vascular tissue of Fig. 3. The reason for this is presumably the overlapping e!ects followed by smoothing.
From the edge of the vascular tissue to the centre of the
pith, no dramatic changes can be seen in the pectin,
protein or carbohydrate peaks of the spectra of the calcium- and sucrose-treated strawberries compared to the
reference.
Epidermis, hypodermis, cortex and pith
Figure 8 shows transitions from the edge of the epidermis
through the hypodermis and cortex to the centre of the
pith in the 4000}700 cm~1 region. The "rst three
spectra represent the epidermis and hypodermis. The
next three spectra (six in the case of calcium-treated
strawberries) from the fourth to the sixth represent the
cortex. The last four spectra in Fig. 8 represent the pith.
In all of the spectra in Fig. 8 there were quite strong
carbonyl bands at approximately 1725 and 1640 cm~1,
representing esteri"ed and acidi"ed groups of pectin. At
approximately 1550 cm~1 there was the strong CN
stretching vibration band representing the amide II.
Structural carbohydrate bands were present in the
1435}1360 cm~1 region as well as carbohydrate bands in
the 1240}1000 cm~1 region. There were no considerable
changes in pectin, protein or carbohydrate peaks of the
spectra of the epidermis and hypodermis of the calcium- or sucrose-treated strawberries compared to the
untreated reference. Instead the pectin, protein and
carbohydrate peak absorbances of the cortex of the pretreated strawberries were slightly higher and peak areas
larger than in the reference. From the edge of the cortex
to the centre of the pith, no dramatic changes could be
seen in the pectin, protein or carbohydrate peaks of
the pretreated strawberries spectra compared to the
reference.
According to FT-IR microscopy studies, both CaCl
2
and sucrose pretreatments before freezing seemed to stabilize the sections of strawberry tissues. These pretreatments especially seemed to a!ect pectin, protein and
structural carbohydrate peaks in the vascular tissue and
cortex compared to the untreated reference. Calcium
chloride treatment had an even stronger e!ect on the
above compounds than sucrose. Neither of the pretreatments seemed to a!ect the epidermis, hypodermis or pith.
On the other hand, because of the partial destruction of
the compounds during or after the pretreatments, analysis of the achene, epidermis or hypodermis tissues was
quite di$cult. Furthermore, the epidermis and hypodermis comprised quite a few cellular layers which were
di$cult to recognize after thawing and refreezing. Generally, overlapping e!ects followed by smoothing decreased
the resolution of spectra and lignin peaks, for example, in
the pretreated strawberries which probably remain under
these e!ects. Therefore, lignin could not be recognized
clearly from the spectra of the vascular tissue or in some
achene spectra.
Conclusion
Instrumental texture as well as microscopical studies
showed that in most cases CaCl and sucrose prefreezing
2
treatments stabilized the structure of strawberry tissues.
The drip loss of the thawed strawberries did not seem
to act as a useful indicator of cohesiveness. Fourier
transform infrared microspectroscopical studies, together
with bright "eld microscopical studies, showed that
the pectin, protein, lignin and structural carbohydrate
components of pretreated strawberries remained more
stable after freezing and thawing than untreated reference samples. The CLSM studies showed that the
cell walls of pretreated strawberries remained quite undamaged after treatments. In the untreated reference
strawberries, the cell walls were mostly broken and the
cytoplasm was shrunk due to osmotic water loss. All
methods except drip loss measurements were found to
be useful tools for analysing the pretreatment e!ects
in studying the di!erent chemical components and
their changes in strawberry tissues during and after
treatments.
Acknowledgements
We wish to thank Anne Ala-Kahrakuusi and Liisa
AG naK kaK inen from VTT/Biotechnology for their skillful
101
lwt/vol. 33 (2000) No. 2
technical assistance in carrying out the pretreatments
and the microstructural as well as FT-IR microspectroscopic measurements.
References
1 BUREN, J. P. The chemistry of texture in fruits and vegetables. Journal of ¹exture Studies, 10, 1}23 (1979)
2 GORMLEY, T. R. Freezing strawberries for jam. Farm and
Food Research, 1, 106}107 (1970)
3 BROOMFIELD, R. W. The manufacture of preserves, #avourings and dried fruits. In: ARTHEY, D. AND ASHURST, P. R.
(Eds), Fruit Processing. London: Chapman & Hall, pp.
165}195 (1996)
4 POOVAIAH, B. W. Role of calcium in prolonging storage
life of fruits and vegetables. Food ¹echnology, 40, 88}89
(1986)
5 GARCID A, J. M., HERRERA, S. AND MORILLA, A. E!ects of
postharvest dips in calcium chloride on strawberry. Journal
of Agriculture and Food Chemistry, 44, 30}33 (1996)
6 MAIN, G. L., MORRIS, J. R. AND WEHUNT, E. J. E!ect of
preprocessing treatments on the "rmness and quality characteristics of whole and sliced strawberries after freezing and
thermal processing. Journal of Food Science, 51, 391}394
(1986)
7 PONTING, J. D., WATTERS, G. G., FORREY, R. R., JACKSON,
R. AND STANLEY, W. L. Osmotic dehydration of fruits. Food
¹echnology, 20, 1365}1368 (1966)
8 SZCZESNIAK, A. S. AND SMITH, B. J. Observations on strawberry texture a three-pronged approach. Journal of ¹exture
Studies, 1, 65}89 (1969)
9 REEVE, R. G. Relationships of histological structure to texture of fresh and processed fruits and vegetables. Journal of
¹exture Studies, 1, 247}284 (1970)
10 NELMES, B. J. AND PRESTON, R. D. Wall development in
apple fruits: a study of the life history of a parenchyma cell.
Journal of Experimental Botany, 19, 496}518 (1968)
11 MUG HLETHALER, K. Ultrastructure and formation of plant
cell walls. Annual Review of Plant Physiology, 18, 1}24
(1967)
12 HAVIS, A. L. A developmental analysis of the strawberry
fruit. American Journal of Botany, 30, 311}314 (1947)
13 NEAL, G. E. Changes occurring in the cell walls of strawberries during ripening. Journal of the Science of Food and
Agriculture, 16, 604}611 (1965)
14 ARMBRUSTER, G. Cellular and textural changes in three
varieties of strawberries as a result of prefreezing treatments.
American Society for Horticultural Science, 91, 876}880
(1967)
15 SCHOLEY, J. Suitability of fruit for jam manufacture. Food
Manufacture, 48, 19}20 (1973)
16 CROSS, D. E. Changes during processing. In: GOODENOUGH, P. W. AND ATKIN, R. K. (Eds), Quality in Stored
and Processed <egetables and Fruit. London: Academic
Press, pp. 383}387 (1981)
17 VODOVOTZ, Y., VITTADINI, E., COUPLAND, J., MCCLEMENTS, D. J. AND CHINACHOTI, P. Bridging the gap: use of
confocal microscopy in food research. Food ¹echnology, 50,
75}82 (1996)
18 SUUTARINEN, J., AG NAG KAG INEN, L. AND AUTIO, K. Comparison of light microscopy and spatially resolved Fourier
transform infrared (FT-IR) microscopy in the examination
of cell wall components of strawberries. ¸ebensmittel =issenschaft und ¹echnologie, 31, 595}601 (1999)
102