Lebensm.-Wiss. u.-Technol.

, 33, 188 } 201 (2000)

The E!ect of Di!erent Prefreezing Treatments on the Structure of Strawberries Before and After Jam Making
J. Suutarinen*, K. Honkapa Ka K , R-L. Heinio K , K. Autio and M. Mokkila

VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT (Finland) (Received September 9, 1999; accepted January 7, 2000)

The structural changes in strawberry tissues during prefreezing treatments, freezing, thawing and jam making were studied by means of instrumental textural measurements and by bright-xeld as well as by Fourier transform infrared microscopical studies and sensory evaluation. Calcium chloride, pectin methylesterase (PME) or crystallized sucrose were used as pretreatment agents before freezing. Calcium chloride and PME treatments were used either at normal air pressure or in a vacuum. In addition, strawberries were dipped in calcium chloride solution after which they were sprinkled with crystallized sucrose. Strawberries were also just sprinkled with crystallized sucrose. Jams made from strawberries treated with CaCl2 and PME in a vacuum or with CaCl2 and crystallized sucrose, respectively, had the highest xrmness values (about twice as great as the reference sample). Firmness of jam berries correlated negatively with xrmness of jam media, i.e. jams with xrmer strawberries had less xrm medium. According to microscopical studies, both CaCl2 and PME in a vacuum and CaCl2 and sucrose pretreatments, respectively, awected the microstructure of strawberry tissues. These pretreatments seemed to stabilize the vascular tissue and to awect pectin, protein and structural carbohydrate. The use of a vacuum seemed to awect the pretreatment solutions, awording more ewective absorption to the cortex and pith and providing stabilization there, especially for pectin and structural carbohydrate. According to sensory evaluation of the jams, diwerent prefreezing treatments were shown to have a signixcant inyuence on the sensory attributes evaluated. The textural attributes in particular were statistically signixcantly diwerent among the strawberry jams: wholeness of the berries in the jam ( P(0.001), xrmness ( P(0.001) and clarity ( P"0.001) of the jam medium as well as redness of the jam colour ( P(0.05) were diwerent among the strawberry jams analysed.

2000 Academic Press Keywords: strawberries; prefreezing treatments; structure; jam

Introduction The initial condition of the fruit, pre- and postharvest treatments, and method of cooking in#uence fruit textural changes. The "rmness of fresh strawberries is of considerable importance when harvesting and handling the fruits prior to processing. The response to postharvest treatment varies with the cultivar and apparently depends on the Ca> content of the fruit at the time of treatment and on the ability of the plant to accumulate and distribute Ca> (1}3). Chung and Youn (4) found that by spraying the strawberry leaves at the #owering stage with a 0.5 % calcium chloride solution, a new membrane protein of the treated fruit was formed. In addition, the cell wall of the treated fruits had a distinct middle lamella that was absent from the control. According to Derbedeneva (5), changes in histological structure of strawberry depend not only on speed of freezing and size of ice crystals but also on the structure of the individual types of tissues. Tissues consisting of small cells close to each other were more resistant. The cell wall structure of strawberries varies in the di!erent
* Author to whom correspondence should be addressed.

tissues (Fig. 1). A strawberry is composed of "ve distinct tissue zones. Epidermal cells form the outer layer. The second layer is composed of hypodermal cells and the third layer of cortical cells. Vascular bundles beginning from the achenes and connecting to the pith play a very important role in the texture of strawberries. Jewell et al. (6) found, in microscopic studies, that the vascular strands and achenes of strawberries formed the majority

Fig. 1 Schematic presentation of a strawberry showing location of epidermal and cortical cells, achenes, hypodermis and vascular tissue (adapted from Jewell et al. [6])

0023-6438/00/030188 # 14 $35.00/0 2000 Academic Press

doi:10.1006/fstl.2000.0638 All articles available online at http://www.idealibrary.com on

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of the structures that maintained their structural integrity during the jam-making process, although in many instances the epidermal cells were also intact. Most of the remaining cells, that is, parenchymal and hypodermal cells, exhibited severe plasmolysis, cellular collapse and in many instances the cell walls were ruptured. Strawberries show increased levels of soluble polyuronides during softening, yet the nature of this solubilization is unknown. It might be that strawberry softening results not from polyuronide hydrolysis but rather from the inability of the tissue to maintain its structural integrity as a result of the addition to the wall of less tightly bound, more freely soluble polyuronides. The increased proportions of arabinose, galactose and rhamnose might all be contributing factors (7,8). A key factor in the maintenance of fruit "rmness in strawberries is the chelation of calcium ions as cross-links between carboxyl groups of adjacent polyuronide chains. The formation of cation cross-bridges between uronic acids may make the cell wall less accessible to enzymes in the fruit that cause softening or to cell wall-degrading enzymes produced by fungal pathogens. Calcium is more e!ective in "rming thermally processed fruit than frozen fruit. This increased "rming action is probably due to heat breaking the chemical bonds on the polyuronide chains, thus rendering more sites available for the formation of calcium pectate crosslinkages in the middle lamella region (9 } 12). During vacuum impregnation treatment, the pore structure of the fruit is immersed in a liquid phase. Fito (13) described a fast mass transfer phenomenon which occurs when porous structures are immersed in a liquid phase. This involves the in-#ow of the external liquid throughout the capillary pores. Reduced pressure is imposed in a solid}liquid system followed by restoration of atmospheric pressure. During the vacuum step the internal gas in the fruit pores is expanded and partially #ows out. In the atmospheric step, the residual gas is compressed and the external liquid #ows into the pores as a function of the compression ratio. Pressure changes can also promote deformities of the fruit because of the viscoelastic properties of its solid matrix. The use of a vacuum reduces the overall appearance of the fruit somewhat, which may not be noticeable in a jam-type product (14,15). In jam making, high mechanical forces are used because the level of dissolved solids is raised from less than 10% to almost 70% in a matter of minutes. Sugar syrup is di!used into the fruit during jam making, whilst at the same time osmotic e!ects tend to remove water and cause collapse of the fruit. Intercellular air is also replaced by syrup. In addition, the surrounding matrix is raised to boiling point of the syrup at which temperature the cell contents are super-heated, resulting in elevated internal pressure (3). In fruit heated during processing, endogenous Ca> is used to form calcium pectate by the heat-enhanced activity of pectinesterase (PE). If the activity of PE has been enhanced during heating, the exogenously supplied Ca> is used to form calciumpectate, an action that increases decay resistance and maintains "rmness (11). Examination of the cell wall of the strawberry fruit has indicated very low levels of PE.

However, vacuum infusion of commercially available fungal PE, under optimized preprocessing conditions of pH and calcium levels, has resulted in great improvement in the texture of heat-processed strawberry halves (16). In our earlier studies, we developed methods based on bright-"eld and Fourier transform infrared (FT-IR) microscopy (17,18). In this investigation we have used these methods together with instrumental texture and sensory evaluation to study the structure of the pretreated strawberries. Di!erent calcium chloride, pectin methylesterase and sucrose prefreezing treatments were used, and strawberries were studied after freezing and jam making.

Materials and Methods Strawberry Fresh Spanish strawberries (Fragaria ananassa, cv. Camarosa) grown in the spring of 1999 were used for the studies.

Enzyme Pectin methylesterase (EC 3.1.1.11) from orange peel was purchased from Sigma (P 5400, Sigma, Deisenhofen, Germany). The PME activity was assayed by titration of the liberated carboxyl groups of 1% citrus pectin with 50 mM NaOH solution using an automatic titrator at room temperature. One unit of PME activity (nkat) was de"ned as the amount of enzyme, that liberated 1 nmol of carboxyl groups per second under the assay conditions.

Strawberry treatments The fresh, whole fruits were randomly distributed among the experiments. Fresh nonpretreated strawberries were used as a reference (Test 1). In the enzyme test, strawberries were dipped in PME solution at 37 3C for 15 min. The enzyme dosage used was 4200 nkat/kg fresh strawberries (Test 2). In the calcium chloride tests strawberries were dipped in a 1% (w/v) CaCl solution (a solution made from  CaCl ;2H O, Riedel-de Hae K n AG, Seelze, Germany   and tap water, 90 mmol/L) with PME at 37 3C either at normal air pressure (760 mmHg) for 15 min (Test 3a) or in a vacuum (125 mmHg) for 10 min (Test 3b). The enzyme dosage used was 4200 nkat/kg fresh strawberries. Two kinds of sucrose treatments were used. Strawberries were either sprinkled with crystallized sucrose (Test 4) or dipped at 37 3C in a 1% CaCl -solution for 15 min  and then sprinkled with crystallized sucrose (crystal size approximately 0.53 mm, Suomen Sokeri Oy, Kantvik, Finland) (Test 5). The amount of sucrose added, 150 g/kg strawberries, was selected as being the typical amount used in household strawberry freezing. The strawberries were put in 1-L PS cases (l 120 mm, w 85 mm and h 105 mm, respectively). Crystallized sucrose (75 g) was sprinkled on each 0.5 kg layer of strawberries (18).

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Experimental procedure In Tests 2 and 5, the dipping bowl was kept in a temperature-controlled bath at 37 3C. In Tests 3a and 3b, the dipping bowl was kept in a temperature-controlled vacuum chamber (W.C. Heraeus GmbH, Hanau, Germany) at 37 3C either at normal air pressure or in a vacuum, respectively. In Test 3b, the desired vacuum level, 125 mmHg, was achieved in 2.5 min. The dipping bowl was kept at the desired vacuum level for 10 min after which the vacuum was shut o! and the bowl was taken out of the chamber. As in the other dipping tests, the total dipping time of Test 3b was 15 min. In all tests 1.25 kg strawberries were dipped in a 1.5 L dipping solution. A light weight was put on the strawberries to ensure that all the berries were under the dipping solution. The total amount of strawberries per test was 2.5 kg. 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. About 1 h after each dipping the strawberries were frozen at !20 3C. The temperature of the strawberries stayed at !2 3C for 5 h. After 10 h the temperature of the strawberries reached !20 3C. The strawberries were stored at !20 3C for 2 d before jam making and for 2 wk before thawing and analysis, respectively.

Calcium analysis Frozen untreated reference and pretreated strawberries were used for calcium analyses. In addition, the amounts of calcium in the jams (strawberries and medium) were determined. Calcium analysis was carried out 1 wk after either freezing or jam cooking, respectively. Frozen strawberries as well as berries in jams and mediums were homogenized before analysis. The strawberries of each jam sample (230 g) were separated from the medium with a fork. The separated strawberries were weighed and homogenized. The medium was mixed with a spoon. Calcium was analysed after dry ashing by atomic absorption spectrometer (AAS) using the #ame technique (method VTT-4289-91, accredited by Finnish Accreditation Service). The uncertainty of the measurements was $10%. The total calcium content of the jam was calculated from the calcium contents of the jam strawberries and the medium assuming that the jam consisted of 43% strawberries and 57% medium.

Jam making The jam recipe is shown in Table 1. Frozen strawberries, crystallized sucrose and water were mixed together. The mixture was heated until it boiled. The mixture was allowed to boil for 10 min after which citric acid (Riedede Hae K n AG, Seelze, Germany) and pectin (Grinsted TM Pectin LA 410, Danisco Ingredients, Brabrand, Denmark) solution were added. The jam was allowed to cool at room temperature for 60 min before "lling 0.5 L glass jars. After "lling the jars were closed and stored at 5 3C for 1 wk before analysis. In Tests 4 and 5 (sucrose prefreezing treatments) the amount of sucrose added in jam cooking was calculated so that all the test jams contained an equal amount of sucrose. The content of soluble solids in the jam was determined with an Opton 74016 (West Germany) refractometer, pH was measured with an Orion Research Digital Ionanalyzer 501 (Orion Research Inc., Cambridge, MA, U.S.A. and electrode Orion 8155SC, Orion Research Inc., Boston, MA, U.S.A.).

Table 1 Jam recipe

Ingredients Pectin solution: Grinsted TM Pectin LA 410 Sucrose Water g/ca. 3.5 kg jam 21 84 420 1575 1309 * 248.5 20 g/100 g 0.57 2.28 11.42 42.82 35.59 6.77 0.54

Texture Analyser measurements After 2 wk of freezer storage, the reference and pretreated strawberries were thawed for analysis by keeping them in their storage cases at 5 3C for 24 h. Thawed fruits were equilibrated to 17 3C by storing them at 20 3C for 4 h. Thawed strawberries were then dried with tissue paper and weighed into 120 g portions for "rmness measurements. The compression force of strawberries was measured with a Texture Analyser (model TA-HDi, Stable Micro Systems, Godalming, U.K.) with a 250 kg load cell using an Ottawa Cell (A/OTC) with a Holed Extrusion Plate (A/HOL). The starting position of the plunger was 50 mm from the base and the "nal position was 1 mm above the base plate. The plunger speed was 1.5 mm/s. The compression force at a plunger position of 5 mm from the base and the area of deformation curve were recorded as the result. The result was taken as an average of three replicates. After cooking, the jams were stored at 5 3C for 1 wk and equilibrated to 20 3C by keeping them at 20 3C for 2 h. The strawberries and the medium were separated with a spoon on a plate. For "rmness measurements, strawberries or the medium were weighed into 100 g portions. The compression force of strawberries or the medium was measured using an A/BE/45 Back Extrusion Rig. The starting position of the plunger was 50 mm from the base and the "nal position 1 mm above the base plate. The plunger speed was 1.5 mm/s. The area of deformation curve was recorded as the result. In both cases the result was an average of three replicates. All "rmness results were analysed with analysis of variance (ANOVA) and Tukey's HSD test (P(0.05) with SPSS software (SPSS version 8.0, SPSS Inc., 1997).

Strawberries Sucrose Water Citric acid solution (50% w/v)

* In the experiments 4 and 5, sugar added in jam making: 1073 g/ca. 3.5 kg jam

Microstructural studies After 1 wk of freezer storage, the untreated reference and pretreated strawberries were freeze-dried for 20 h (Dr Morand freeze-drier, Germany) (17,18). The freezedried strawberries were cut into smaller pieces, in"ltrated

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with a Historesin Embedding Kit (Jung, Germany) for 5 d, polymerized and cut into thin sections (4 m) with a Microm HM355 (Microm Laborgera K te GmbH, Walldorf, Germany) microtome. Sections were stained with the following staining solutions: 0.2 g/L Ruthenium Red in water for 2 h followed by washing with water; and 0.1 g/L Light Green for 1 min and thereafter with iodine. Ruthenium Red stains pectin pink; Light Green and iodine stains protein green or yellow (19,20). Samples were examined and photographed with an Olympus BX50 microscope (Olympus, Optical Co. Ltd, Japan). The images were viewed with a Soft Imaging System analySIS 3.0 software (Soft Imaging System GmbH, Germany) and printed.

Fourier transform infrared microscopical 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 1 wk of freezer storage the untreated reference as well as the pretreated strawberries were randomly chosen and thawed at room temperature (20 3C). The strawberries were thawed in an upright position, hulls on the table, so as to ascertain what e!ect thawing would have on the structure of pretreated strawberries. Thawed strawberries were frozen with liquid nitrogen and 7- m sections were cut using a cryostat (Kryostat 1720 digital, Wild Leitz GmbH, Wetzlar, Germany). Before freezing the thawed strawberries were bound to a sample table of the cryostat using Tissue-Tek compound (O.C.T. 4583, Sakura Finetechnical Co., Japan). The solution was not used to surround or cover the tissue specimen but only for "xing the sample to the sample. Ethanol-washed 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, i.e. 32 m with a 36;objective) was adjusted to frame the desired portion for scanning and to exclude unwanted tissue. Strawberry sections were manually mapped systematically across the achene, vascular tissue and pith, or across the epidermis, hypodermis and cortical cells. The transmission mode was used and 50 scans were accumulated to produce a spectrum over the 4000 } 700 cm\ range at a resolution of 4 cm\. A reference was scanned using the aluminium foil free of any tissue and 100 scans were accumulated to produce the transmission spectrum in the 4000 } 700 cm\ range. The absorbance spectra were stored after logarithmic transformation and baseline correction (17).

scale corresponded to the lowest or opposite intensity (value 0) and the right side to the highest intensity (value 10) of the attribute. The sensory attributes evaluated were redness of colour (brown}red), wholeness of berries (broken}whole), clarity of medium (opaque}clear, transparent), evenness of medium (uneven}even), "rmness of medium ("rm}#uid), softness of berries (hard}soft), leatheriness of berries (not leathery}leathery), sweetness of odour and #avour (not sweet}sweet), sourness of odour and #avour (not sour}sour), balance of odour and #avour (unbalanced}balanced) and faultlessness of odour and #avour (defective}faultless). The samples were presented to the panellists coded and in random order, and water and crackers were provided for cleansing the palate between the jam samples. The samples were analysed in two sensory replicates on two sequential days. Analysis of variance (ANOVA) and Tukey's HSD test (P(0.05) were executed with the SPSS software (SPSS version 8.0, SPSS Inc., 1997) for the sensory results. ANOVA was used to test statistical di!erences in sensory attributes between the jam samples, and the statistical di!erence between the two sessions (P(0.05). When the di!erence in the analysis of variance was statistically signi"cant, pairwise comparisons of the attributes between the jam samples were analysed by Tukey's test.

Results and Discussion Soluble solids and pH The total soluble solids and pH-values of the jams are presented in Table 2. All soluble solids values were quite low (between 48 and 56 3Brix). The pectin used for the study was intended for jams, with a Brix value around 45. Furthermore, in these investigations it was more important to follow the e!ect of calcium on the strawberries than to optimize the jam making. Therefore, the amount of soluble solids were not adjusted to a certain level but the jams were cooked using similar total amounts of each ingredient including the sucrose used for pretreatments. Jams 3b and 5 had the highest Brix values and jam 4 had the lowest value. pH values of the jams were around 3.4. After jam cooking, on the surface of the jams that contained added calcium chloride (Tests 3a, 3b and 5), no froth was observed, whereas on the other jams some froth was noticed. Protein denaturation during cooking was probability caused by CaCl . 

Table 2 Total soluble solids (3Brix) and pH-values of the jams

Test 3Brix 50 52 54 56 48 55 pH 3.4 3.4 3.3 3.1 3.4 3.4

Sensory evaluation The sensory quality of the strawberry jams was evaluated by a trained panel with proven skills (n"11) using descriptive analysis (21). Attribute intensities were rated on a 10-unit graphical intensity scales. The scales were verbally anchored at each end, and the left side of the

1. 2. 3a. 3b. 4. 5.

Reference PME CaCl #PME  CaCl #PME in a vacuum  Sucrose CaCl #sucrose 

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Firmness The "rmness results of all the tests are given in Table 3. The prefreezing treatments had a statistically signi"cant in#uence on the "rmness of frozen strawberries when measuring the compression force (P(0.05) and on the "rmness of jam berries (P(0.01) and media (P(0.001) when measuring the area of deformation curve. In pairwise comparisons (Tukey's test, P(0.05) the "rmness values of pretreated or untreated reference frozen strawberries did not di!er from each other. Frozen strawberries treated with only PME (Test 2) or with sucrose (Test 4) were less "rm than strawberries treated with CaCl  and PME in a vacuum (Test 3b). The deformation force and curve area values of the strawberries treated with CaCl and PME in a vacuum (Test 3b) were greater than  those of other strawberries though there was no statistically signi"cant di!erence between them. One reason for this is that the height of the thawed strawberries surface at the used base plate varied quite a lot from test to test. Consequentially, the force and curve area values varied to such a degree between replicates that more measurements would have been required to decrease the deviation and to attain more reliable results. The studies of Main et al. (15), who used strawberries treated with a 1% calcium lactate solution in a vacuum, and those of GarcmH a et al. (10), who used strawberries treated with a 1% calcium chloride solution at 45 3C, showed that the pretreated strawberries were signi"cantly "rmer than the untreated reference strawberries. In addition, Main et al. (15) reported that in their studies whole fruit maintained its integrity better than sliced fruit after heat treatment in a 35 3C warm bath. Jams made from strawberries treated with CaCl and  PME in a vacuum (Test 3b) or with CaCl and crystal lized sucrose (Test 5), had signi"cantly higher "rmness values than the reference sample (approximately twice as great). The "rmness values of jams made from strawberries given other pretreatments did not di!er from those of the reference strawberries. Jams made from the reference strawberries had a harder medium than other jams except for the jam made from sucrose-treated strawberries (Test 4). Jams made from fruit pretreated with CaCl in 

a vacuum (Test 3b), had a softer medium than other jams except for the jam made from CaCl - and PME-treated  strawberries at normal air pressure (Test 3a). The "rmness of jam berries correlated negatively with the "rmness of jam media, i.e. the jams with "rmer strawberries had softer media. Jam berries were more homogenous in size and shape than the thawed ones. Therefore, in jam "rmness analysis, three replicates seemed to be su$cient to obtain reliable results.

Calcium analysis Calcium chloride treatments at normal air pressure as well as together with the sucrose-treatment (Tests 3a and 5, respectively) clearly increased the Ca content of strawberries relative to the reference or other treatments in which Ca had not been added (Tests 1, 2 and 4) (Table 4). The amount of Ca in the strawberries that had been dipped in the calcium chloride solution in a vacuum (Test 3b) was about three times greater than the amount of Ca in the strawberries that were not dipped in the calcium chloride solution (Tests 1, 2 and 4). The same e!ect can be seen in the amounts of Ca in jam strawberries and media. According to GarcmH a et al. (10) the strawberries treated with 1% calcium chloride solution at 25 3C had a signi"cantly higher calcium content than the fruits of the Table 4 Amounts of calcium in pretreated frozen strawberries, jam strawberries and media. Calcium content of tap water used for analysis was 18 mg/L
Ca, mg/kg Test 1. 2. 3a. 3b. 4. 5. Reference PME CaCl #PME  CaCl #  PME in a vacuum Sucrose CaCl #sucrose  Frozen Jam Medium Jam, berries berries total 130 140 250 450 150 230 100 110 180 290 110 180 67 57 130 190 58 110 80 79 150 240 81 140

Table 3 Firmness* of pretreated frozen and thawed strawberries as well as of strawberries in jams and jam mediums
Firmness Frozen berries Compression force (kg) Test 1. 2. 3a. 3b. 4. 5. Reference PME CaCl #PME  CaCl #PME in a vacuum  Sucrose CaCl #sucrose  x N 32.9?@ 26.3? 32.2?@ 41.6@ 27.1? 32.9?@ s  3.5 1.6 2.0 2.9 3.4 7.0 Frozen berries Deformation curve area (kgs) x N 313.7 258.3 315.5 335.1 239.4 288.8 s  53.6 11.0 21.2 31.0 30.1 62.3 Jam berries Deformation curve area (kgs) x N 49.0? 75.9?@ 35.7?@ 101.0@ 70.7?@ 104.5@ s  3.5 4.3 6.2 15.1 3.0 32.9 Jam medium Deformation curve area (gs) x N 10.6B 7.1@A 4.9?@ 3.4? 8.2AB 6.8@A s  0.5 1.2 0.8 0.1 1.9 1.3

* Mean (x N ) and standard deviation (s ) of three measurements.  ?}B Means in each column followed by the same letter signify that the pretreated strawberries, jam berries or media are not statistically signi"cantly di!erent in respect of "rmness (Tukey's HSD test; P(0.05)

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di!erent controls after 1 d of storage at 1 3C or 8 3C. Heating at 45 3C enhanced the penetration of the calcium from the 1% calcium chloride solution into the fruits. This is in agreement with our results.

Microstructural studies Microstructural studies were carried out after storage in a freezer for 1 wk using the reference fruit, PME-treated fruit, or CaCl - and PME-treated strawberries in a vac uum (Tests 1, 2 and 3b, respectively). The pectin and protein in cortical cells and vascular tissue were stained with speci"c staining systems, and their locations were studied. In the untreated reference samples, pectin in the middle lamella of the cortical cells was poorly stained compared to the CaCl - and PME-treated strawberries  in a vacuum (Fig. 2a+c). Moreover, the cell walls seemed to be broken and pectin appeared outside the middle lamella. In the PME-treated strawberries, pectin appeared outside the middle lamella as in the reference sample. In the CaCl - and PME-treated strawberries in  a vacuum, pectin was well stained and quite homogeneously spread in the middle lamella. In some parts the middle lamella had swollen and was "lled with pectin. Swelling was not observed in the reference sample. In the cortical cells, protein was stained very similar to that in the reference sample as well as in the PME-treated or CaCl - and PME-treated strawberries in a vacuum (Fig.  3a+c). In the vascular tissue of the reference strawberries, protein was poorly stained and lignin spirals seemed to be badly broken (not shown). The vascular tissues of the PME-treated strawberries were totally broken and it was impossible to carry out the microstructural studies. In the vascular tissue of the CaCl - and PME-treated straw berries in a vacuum, protein was well stained and homogeneously spread (Fig. 4). Furthermore, the lignin-rich, spiral-type structures seemed to be quite intact and coherent.

Fourier transform infrared microscopical studies These were carried out on fruit treated in Tests 1}5. The analyses of the spectra was mainly based on our earlier studies of strawberries (17,18). Fourier transform infrared microspectroscopy spectra are shown only for the reference, PME-, and CaCl - and PME-pretreated strawber ries in a vacuum. The measured spectra were scattered to some extent. 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 the spectra, and some peaks, i.e. peaks typical of protein (amide I), acidi"ed pectin and lignin, tended to overlap somewhat in 1700 } 1500 cm\ spectral region.

Achene, vascular tissue and path Figures 5 and 6 show typical spectra of the vascular tissue and pith (taken from approximately the centre of the tissues) of the reference, PME-treated and CaCl - and  PME-treated strawberries in a vacuum in the

4000 } 700 cm\ region. Figure 7 shows a series of spectra in the 4000 } 700 cm\ region of sections of strawberry tissue obtained from the outer edge of the achene through the vascular tissue into the centre of the pith. In all these spectra, the OH and CH stretching vibration bands were present in the 3500 } 2900 cm\ region. At approximately 1660 and 1550 cm\, amide I and small amide II bands due to the CO and CN stretching bands, respectively, were present. In each spectrum, a group of peaks situated approximately in the 1460 } 1330 cm\ region represented structural carbohydrate bands. Also, in each spectrum, the CO stretching vibration band at approximately 1240 cm\ and strong carbohydrate bands in the 1200 } 1000 cm\ region were present. The "rst two spectra * three or four in the cases of the PME (Fig. 7b) * or the Ca- and PME-treated strawberries in a vacuum (Fig. 7c) * represent the achene of the reference sample (Fig. 7a). The following twelve spectra from 3 } 14 represent the vascular tissue of the reference sample * 4 } 13, and 5}19, in the cases of Tests 2 (Fig. 7b) and 3b (Fig. 7c), respectively. The next eight spectra from 15}22 or 14 }29 and 20 } 29 in the cases of Tests 2 (Fig. 7b) and 3b (Fig. 7c), respectively, of the reference sample represent the pith in Fig. 7a. An overview of a series of spectra of the reference (Fig. 7a) and pretreated sections of strawberry tissues shows that peak absorbances of the CaCl  and PME-treated strawberries in a vacuum were higher and peak areas larger than the spectra of other pretreated or reference strawberries. In each of the achene spectra (Fig. 7a+c), a strong CO stretching vibration band was present at approximately 1740 cm\ due to esteri"ed group vibration of the pectin. In addition, quite a strong CO stretching vibration band was present in each of the baseline-corrected (before smoothing) achene spectra at approximately 1640 cm\ due to acidi"ed group vibration of the pectin. This peak was di$cult to recognize after smoothing because of the overlapping e!ects. At approximately 1605 and 1510 cm\, peaks typical of lignin were present in the achene spectrum of the reference, calcium chloride-, and PME- or sucrose-treated strawberries. It was di$cult to recognize the lignin peaks in the achene spectra of the reference and the calcium chloride- and PME-treated samples in a vacuum as well as of calcium chloride- and sucrose-treated strawberries due to destruction of the composition during or after treatments. In the vascular tissues of the reference and pretreated strawberries (Figs 5, 7), there is a carbonyl band at approximately 1725 cm\, representing pectin. The acidi"ed group of pectin at approximately 1640 cm\ was just recognizable from the baseline-corrected spectra of each of the measured strawberries. In particular, in the calcium chloride- and PME-treated strawberries in a vacuum, the amide I and II bands were higher than in the spectra of the reference or other pretreated strawberries. Also, the pectin and protein bands as well as the structural carbohydrate bands in the 1460 } 1330 cm\ region were higher and their areas larger in the spectra of the CaCl - and PME-treated strawberries in a vacuum  compared to the spectra of the reference or other pretreated strawberries. The pectin band of the spectra of the

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Fig. 2 Micrographs of (a) untreated reference; (b) PME-treated; and (c) CaCl - and PME-treated (in a vacuum) strawberry cortical  tissue (two micrographs per treatment). Pectin (marked with an arrow) was stained and appeared pink

other CaCl -treated strawberries was higher relative to  the nonpretreated reference strawberries. In the PMEtreated strawberries, the pectin and protein as well as the structural carbohydrate and carbohydrate peaks had lost

their spectral intensities relative to the reference spectra. This indicated, on the one hand, that PME had just cut carboxyl units from the methylesteri"ed pectin groups based on the smaller pectin peaks at approximately

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Fig. 3 Micrographs of (a) untreated reference; (b) PME-treated; and (c) CaCl - and PME-treated (in a vacuum) strawberry cortical  tissue (two micrographs per treatment). Proteins (marked with an arrow) were stained and appeared green or yellow

1725 cm\ and, on the other hand, that the commercial enzyme preparation had also cut protein and structural carbohydrate (cellulose) and for that reason had to have some protease and cellulase activities too. The vascular

tissue spectra of the sucrose-treated strawberries were very similar to the reference strawberries. This indicated that the surface of the fresh strawberries themselves were quite hard in the pretreatment stage and thus did not

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allow crystallized sugar to penetrate much. In the spectra of the vascular tissue of the reference and pretreated strawberries, the typical lignin peaks cannot clearly be detected. The reason for this is presumably the overlapping e!ects together with the small amount of lignin in strawberry. From the edge of the vascular tissue to the centre of the pith, no dramatic changes can be detected in the pectin, protein or structural carbohydrate peaks of the other pretreated strawberries except the CaCl - and PME-treated strawberries in a vacuum.  The absorbances of these compounds were higher and

peak areas larger in the vacuum pretreatments compared to the nonpretreated reference or other pretreated strawberries.

Fig. 4 Micrograph of CaCl - and PME-treated (in a vacuum)  strawberry vascular tissue. Protein (marked with an arrow) was stained and appeared green or yellow

Epidermis, hypodermis, cortex Figure 8 shows typical spectra of the cortex of the reference, PME- and CaCl and PME-treated strawberries in  a vacuum in the 4000 } 700 cm\ region (about from the centre of the tissues). Figure 9 shows transitions from the edge of the epidermis through the hypodermis to the end of the cortex in the 4000 } 700 cm\ region. The "rst two spectra represent the epidermis and hypodermis. The rest of the spectra represent the cortex. There was quite strong carbonyl band at approximately 1725 cm\ representing esteri"ed pectin. At approximately 1660 and 1550 cm\, there were the CO and CN stretching vibration bands representing amide I and II. The amide I and II peaks were better recognized from the baseline-corrected spectra. The structural carbohydrate bands were present in the 1460 } 1360 cm\ region as well as the CO stretching vibration band at approximately 1240 cm\ and carbohydrate bands in the 1200 } 1000 cm\ region. There were no considerable changes in pectin, protein, structural carbohydrate or carbohydrate peaks of the spectra of the epidermis and hypodermis of the pretreated strawberries relative to the reference. Instead, pectin and structural carbohydrate peaks of the cortex of the Ca- and PME-treated strawberries in a vacuum (Fig. 9c) were higher and their areas larger than in the reference sample. An overview of the spectra of the cortex of the other pretreated strawberries indicated that their structures in some points of the cortex were badly broken during or after these pretreatments.

Fig. 5 Spectra of the vascular tissue of (a) untreated reference; (b) PME-treated; and (c) CaCl - and PME-treated (in a vacuum)  strawberry

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Fig. 6 Spectra of the pith of (a) untreated reference; (b) PME-treated; and (c) CaCl - PME-treated (in a vacuum) strawberry 

According to FT-IR microscopy studies, both the CaCl  and PME-pretreatments (Test 3b) as well as the CaCl  and sucrose-pretreatments (Test 5), before freezing, stabilized the sections of strawberry tissues. These pretreatments seemed to stabilize "rst the vascular tissue and to a!ect pectin, protein and structural carbohydrate peaks compared to the untreated reference. The use of a vacuum seemed to a!ect the pretreatment solutions, a!ording more e!ective absorption to the cortex and pith and providing stabilization there, especially for pectin and structural carbohydrate. Commercial PME showed, in addition to pectinase, also protease and cellulase activities. Neither of the pretreatments seemed to a!ect the achene, epidermis or hypodermis. On the other hand, because of the partial destruction of the compounds during or after the pretreatments, the analysis of the achene, epidermis or hypodermis tissues were quite di$cult. Furthermore, both the epidermis and hypodermis consist of quite a few cellular layers, which were di$cult to recognize after thawing and refreezing. Generally, the overlapping e!ects of acidi"ed pectin, amide I and lignin peaks in the 1700 } 1500 cm\ region, together with their small natural amounts in strawberry, decreased the spectral resolution. Therefore, it was not possible to recognize these compounds from this area of spectra clearly. In future, it would be interesting to study the di!erent pretreatment e!ects on the strawberry tissues with Raman- or UV-Raman microscopes. Thus, extra sample preparation for the measurements might be avoided and the overlapping 1700 } 1500 cm\ spectral region would probably be easier to handle and it would be easier to identify the interesting peaks. According to the microscopical studies of Szczesniak and Smith (19), frozen strawberry halves in syrup showed

plasmolysed cells, disorganized and clumped cytoplasm, pale pigmentation and folding of cell walls and cell surfaces. Furthermore, staining indicated some de"nite changes in cellulose, pectin and lignin structures. Cellulose and pectin stained intermittently * some walls stained heavily, others stained lightly, while some did not stain at all. According to their studies, degradation of cellulose and pectin may have accounted for thinning of the cell wall. Armbruster's (22) microscopical observations showed that epidermis and xylem (a part of a bundle zone) were not changed by freezing regardless of the variety or pretreatment (dry sucrose or sucrose syrup) applied. Parenchyma (cortical) cell walls were ruptured without cell separation on freezing, the disorganization being greater in varieties with large cells. These studies are mostly parallel to our earlier microscopical observations (18).

Sensory proxles of strawberry jams Di!erent prefreezing treatments were shown to have a signi"cant in#uence on the sensory attributes evaluated. The textural attributes in particular were statistically di!erent among the strawberry jams: redness of the jam colour (P(0.05), wholeness of the berries in the jam (P(0.001), "rmness (P(0.001) as well as clarity (P"0.001) of the jam medium were di!erent among the strawberry jams analysed. The sensory results of the jams were also analysed in pairwise comparisons (Tukey's test, P(0.05). The jam made from berries treated with CaCl and sucrose (Test 5) was found to be redder in  colour than the reference jam (Test 1) whereas the latter jam was evaluated to be browner than the former jam. The reference jam also consisted more broken berries

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Fig. 7 Transition from the strawberry achene through the vascular tissue to the centre of the pith in the 4000 } 700 cm\ region baseline-corrected stack map. (a) Untreated reference; (b) PME-treated; and (c) CaCl - and PME-treated (in a vacuum) strawberry 

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Fig. 8 Spectra of the cortical tissue of (a) untreated reference; (b) PME-treated; and (c) CaCl - and PME-treated (in a vacuum)  strawberry

than all the jams treated with CaCl (Tests 3a, 3b and 5)  (Table 5). It may be concluded that after dipping in the CaCl -solution followed by sugar sprinkling or dipping  in CaCl - and PME-solutions, the strawberries retained  their shape better than without these treatments. The medium was more #uid in the jam containing strawberries treated with CaCl and sucrose in a vacuum (Test 3b)  than in the others. The medium was "rmer in the reference jam (Test 1) than in the jams made from berries treated with CaCl (Tests 3a, 3b and 5). The medium of  the reference jam (Test 1) was more turbid than that of jams made from strawberries treated with CaCl and  sucrose in a vacuum or with CaCl and crystallized  sucrose (Tests 3b and 5). There seems to be a negative correlation between clarity and "rmness of the medium. The medium was clear in all the jams treated with the CaCl -solution, and they were also more #uid than the  others. Also, juice was somewhat separated from the jams in the CaCl -treatments. Other sensory attributes evalu ated did not di!er statistically signi"cantly from each other. The results were somewhat di!erent depending on the replicate number, which might result from di!erences between the two jam replicates presented to the panellists. In fact, because of the large size of the strawberries and the rather small amount of jam samples, the amount of strawberries for one assessor was rather small. From the evaluated attributes, only leatheriness of the berries was assessed di!erently in the two replicates (P(0.005). According to the sensory studies of Main et al. (15), the strawberries treated with 1% or 2% calcium lactate solutions in a vacuum, frozen with sucrose and thawed in a 35 3C water bath for 30 min, showed no signi"cant di!erences in #avour or texture with reference to the

untreated control. Instead, colour attribute, when determined as acceptability, tended to be lower on all fruit that had been exposed to a vacuum and was rated lower than the control on all soaks except that in the 2% calcium lactate solution. This may be due to bleeding of pigment that occurred due to cell rupture. According to GarcmH a et al. (11), strawberry pretreatments in calcium chloride solutions either at temperatures of 25 or 45 3C did not a!ect the sensory quality (appearance and o!-#avour) of fruits.

Conclusion Instrumental texture- and microscopy studies as well as sensory analysis showed that the CaCl and PME in  a vacuum as well as calcium chloride and sucrose prefreezing treatments clearly improved "rmness of strawberry tissues. Fourier transform infrared microscopy and bright-"eld microscopy studies showed that the pectin, protein and structural carbohydrate components of the above pretreated strawberries were more stable than those of the untreated reference samples. The calcium chloride and pectin methylesterase pretreatments in a vacuum seemed to stabilize "rst the structure of the vascular tissue and then the cortex and pith. The di!erent prefreezing treatments for the strawberries gave promising results on the sensory quality of the jams. The textural properties of the strawberry jams, in particular, were in#uenced signi"cantly. Treatment with the CaCl  solution seemed to result in a clear and #uid medium in the jam, and these sensory attributes had negative correlations with each other. Treatments with the CaCl  solution followed by sprinkled sugar or with the

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Fig. 9 Transition from the strawberry epidermis through the hypodermis and cortex to the end of the vascular tissue in the 4000 } 700 cm\ region baseline-corrected stack map. (a) Untreated reference; (b) PME-treated; and (c) CaCl - and PME-treated  (in a vacuum) strawberry

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Table 5 Sensory evaluation by descriptive analysis of strawberry jams produced using di!erent prefreezing treatments (n"11). Means representing results combined from two independent replicates
Attribute Redness of colour Wholeness of berries Clarity of medium Evenness of medium Firmness of medium Softness of berries Leatheriness of berries Sweetness of odour and #avour Sourness of odour and #avour Balance of odour and #avour Faultlessness of odour and #avour Reference 7.2 ? 4.8 ? 5.6 ? 4.6 7.6 B 7.2 6.3 7.2 5.0 6.8 7.8 PME 7.4 ?@ 5.9 ?@A 6.2 ?@ 4.8 6.9 AB 7.1 5.3 7.3 5.0 7.1 8.0 CaCl #PME  7.9 ?@ 6.9 @AB 7.1 ?@A 5.4 5.7 @A 6.6 6.2 7.6 4.9 6.7 8.1 CaCl #PME  in a vacuum 8.3 ?@ 7.8 AB 8.2 A 6.4 2.8 ? 6.4 6.0 7.9 5.3 6.7 7.6 Sucrose 7.9 ?@ 5.3 ?@B 6.8 ?@A 4.9 7.4 AB 6.1 6.1 7.3 5.6 6.8 7.6 CaCl #sucrose  8.5 @ 8.2 B 8.0 @A 5.4 4.9 @ 5.4 5.9 7.2 5.4 6.5 7.1

?\B Means in each row followed by the same letter signify that the strawberry jams are not statistically signi"cantly di!erent in respect of that attribute (Tukey's HSD test; P(0.05)

CaCl - and PME-solutions in a vacuum were found to  retain the shape of the strawberries.

Acknowledgements We wish to thank Annikka Mustranta from VTT Biotechnology for her expert knowledge in enzyme applications. Furthermore, we wish to thank Anne Ala-Kahrakuusi, Helena Liukkonen-Lilja and Liisa A G na K ka K inen and the sensory group from VTT Biotechnology for their skilful technical assistance in carrying out the pretreatments as well as the calcium and microscopical measurements and sensory analysis.

References
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