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The Effects of Calcium Chloride and Sucrose Prefreezing Treatments on the Structure of Strawberry Tissues
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
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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
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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
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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
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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
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Fig. 1 Micrographs of strawberry vascular (a) and cortical (b) tissue. Pectin (marked with arrow) was stained and appeared pink
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Fig. 2 Micrographs of strawberry vascular (a) and cortical (b) tissue. Proteins (marked with arrow) were stained and appeared green or yellow
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Fig. 3 Micrographs of strawberry vascular tissue. Lignin (marked with arrow) was stained and appeared pink
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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
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Fig. 5 Fresh (a) and calcium-treated (b) strawberry sections of cortical tissue
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Fig. 6 Fresh (a) and sucrose-treated (b) strawberry sections of vascular tissue
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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
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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
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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
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technical assistance in carrying out the pretreatments and the microstructural as well as FT-IR microspectroscopic measurements.
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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
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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
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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
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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
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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
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Fig. 1 Micrographs of strawberry vascular (a) and cortical (b) tissue. Pectin (marked with arrow) was stained and appeared pink
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Fig. 2 Micrographs of strawberry vascular (a) and cortical (b) tissue. Proteins (marked with arrow) were stained and appeared green or yellow
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Fig. 3 Micrographs of strawberry vascular tissue. Lignin (marked with arrow) was stained and appeared pink
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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
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Fig. 5 Fresh (a) and calcium-treated (b) strawberry sections of cortical tissue
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Fig. 6 Fresh (a) and sucrose-treated (b) strawberry sections of vascular tissue
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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
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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
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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
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technical assistance in carrying out the pretreatments and the microstructural as well as FT-IR microspectroscopic measurements.
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