The Effect of Prefreezing Treatments on the Structure of Strawberry Jam Before and After Jam Maki

8/12/2019 The Effect of Prefreezing Treatments on the Structure of Strawberry Jam Before and After Jam Maki

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* Author to whom correspondence should be addressed.

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

Lebensm.-Wiss. u.-Technol., 33, 188 }201 (2000)

The E ! ect of Di ! erent Prefreezing Treatments on theStructure of Strawberries Before and After Jam Making

J. Suutarinen *, K. Honkapa K Ka K K, R-L. Heinio K, K. Autio and M. Mokkila

VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT (Finland)(Recei ved 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 sensoryevaluation. 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 incalcium chloride solution after which they were sprinkled with crystallized sucrose. Strawberries were also just sprinkled withcrystallized sucrose. Jams made from strawberries treated with CaCl 2 and PME in a vacuum or with CaCl 2 and crystallized sucrose,respecti vely, had the highest xrmness values (about twice as great as the reference sample). Firmness of jam berries correlated negati vely with xrmness of jam media, i.e. jams with xrmer strawberries had less xrm medium. According to microscopical studies, bothCaCl 2 and PME in a vacuum and CaCl 2 and sucrose pretreatments, respecti vely, a w ected the microstructure of strawberry tissues. These pretreatments seemed to stabilize the vascular tissue and to a w ect pectin, protein and structural carbohydrate. The use of a vacuum seemed to a w ect the pretreatment solutions, a w ording more e w ecti ve absorption to the cortex and pith and pro viding stabilization there,especially for pectin and structural carbohydrate. According to sensory e valuation of the jams, di w erent prefreezing treatments were shown to ha ve a signi xcant in yuence on the sensory attributes e valuated. The textural attributes in particular were statistically signi xcantly di w erent 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 di w erent among the strawberry jams analysed.

2000 Academic Press

Keywords: strawberries; prefreezing treatments; structure; jam

Introduction

The initial condition of the fruit, pre- and postharvesttreatments, and method of cooking in #uence fruit tex-tural changes. The "rmness of fresh strawberries is of considerable importance when harvesting and handlingthe fruits prior to processing. The response to posthar-vest treatment varies with the cultivar and apparentlydepends on the Ca content of the fruit at the time of treatment and on the ability of the plant to accumulateand distribute Ca (1}3). Chung and Youn (4) foundthat by spraying the strawberry leaves at the #oweringstage with a 0.5 % calcium chloride solution, a newmembrane protein of the treated fruit was formed. Inaddition, the cell wall of the treated fruits had a distinct

middle lamella that was absent from the control.According to Derbedeneva (5), changes in histologicalstructure of strawberry depend not only on speed of freezing and size of ice crystals but also on the structureof the individual types of tissues. Tissues consisting of small cells close to each other were more resistant. Thecell wall structure of strawberries varies in the di ! erent

tissues (Fig. 1 ). A strawberry is composed of "ve distincttissue zones. Epidermal cells form the outer layer. Thesecond layer is composed of hypodermal cells and thethird layer of cortical cells. Vascular bundles beginning

from the achenes and connecting to the pith play a veryimportant role in the texture of strawberries. Jewell et al .(6) found, in microscopic studies, that the vascularstrands and achenes of strawberries formed the majority

0023-6438/00/030188 # 14 $35.00/0 doi:10.1006/fstl.2000.06382000 Academic Press All articles available online at http://www.idealibrary.com on

188BALA H. RAMESH VVC LWT 20000638 Date 21-2-2000.......................... DISC USED: NOYES


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Table 1 Jam recipe

Ingredients g/ca. 3.5 kg jam g/100 g

Pectin solution: Grinsted TMPectin LA 410

21 0.57

Sucrose 84 2.28Water 420 11.42

Strawberries 1575 42.82Sucrose 1309 * 35.59Water 248.5 6.77Citric acid solution (50% w/v) 20 0.54

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

Experimental procedureIn Tests 2 and 5, the dipping bowl was kept in a temper-ature-controlled bath at 37 3C. In Tests 3a and 3b, thedipping bowl was kept in a temperature-controlled vac-uum 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 bowlwas kept at the desired vacuum level for 10 min afterwhich the vacuum was shut o ! and the bowl was takenout of the chamber. As in the other dipping tests, the totaldipping time of Test 3b was 15 min. In all tests 1.25 kgstrawberries were dipped in a 1.5 L dipping solution.A light weight was put on the strawberries to ensure thatall the berries were under the dipping solution. The totalamount of strawberries per test was 2.5 kg. After dipping,the berries were drained for 5 min in a plastic strainerand packaged in 0.5-kg portions in 1-L PET cases. About1 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 straw-berries reached ! 20 3C. The strawberries were stored at! 20 3C for 2 d before jam making and for 2 wk beforethawing and analysis, respectively.

Jam making The jam recipe is shown in Table 1 . Frozen strawberries,crystallized sucrose and water were mixed together. Themixture was heated until it boiled. The mixture wasallowed to boil for 10 min after which citric acid (Riede-de Hae Kn AG, Seelze, Germany) and pectin (GrinstedTM Pectin LA 410, Danisco Ingredients, Brabrand,Denmark) solution were added. The jam was allowed tocool at room temperature for 60 min before " lling 0.5 Lglass jars. After " lling the jars were closed and stored at5 3C for 1 wk before analysis. In Tests 4 and 5 (sucroseprefreezing 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 Opton74016 (West Germany) refractometer, pH was measuredwith an Orion Research Digital Ionanalyzer 501 (OrionResearch Inc., Cambridge, MA, U.S.A. and electrodeOrion 8155SC, Orion Research Inc., Boston, MA,U.S.A.).

Calcium analysisFrozen untreated reference and pretreated strawberrieswere used for calcium analyses. In addition, the amountsof calcium in the jams (strawberries and medium) weredetermined. Calcium analysis was carried out 1 wk after

either freezing or jam cooking, respectively. Frozenstrawberries as well as berries in jams and mediums werehomogenized before analysis. The strawberries of each jam sample (230 g) were separated from the medium witha fork. The separated strawberries were weighed andhomogenized. The medium was mixed with a spoon.Calcium was analysed after dry ashing by atomic absorp-tion spectrometer (AAS) using the #ame technique(method VTT-4289-91, accredited by Finnish Accredita-tion Service). The uncertainty of the measurementswas $ 10%. The total calcium content of the jam was calculated from the calcium contents of the jamstrawberries and the medium assuming that the jamconsisted of 43% strawberries and 57% medium.

Texture Analyser measurementsAfter 2 wk of freezer storage, the reference and pretreatedstrawberries were thawed for analysis by keeping them intheir storage cases at 5 3C for 24 h. Thawed fruits wereequilibrated to 17 3C by storing them at 20 3C for 4 h.Thawed strawberries were then dried with tissue paperand weighed into 120 g portions for "rmness measure-ments. The compression force of strawberries was mea-sured with a Texture Analyser (model TA-HDi, StableMicro Systems, Godalming, U.K.) with a 250 kg load cellusing an Ottawa Cell (A/OTC) with a Holed ExtrusionPlate (A/HOL). The starting position of the plunger was50 mm from the base and the "nal position was 1 mmabove the base plate. The plunger speed was 1.5 mm/s.The compression force at a plunger position of 5 mmfrom the base and the area of deformation curve wererecorded as the result. The result was taken as an averageof three replicates.

After cooking, the jams were stored at 5 3C for 1 wk andequilibrated to 20 3C by keeping them at 20 3C for 2 h.The strawberries and the medium were separated witha spoon on a plate. For "rmness measurements, straw-berries or the medium were weighed into 100 g portions.The compression force of strawberries or the mediumwas measured using an A/BE/45 Back Extrusion Rig.The starting position of the plunger was 50 mm from thebase and the "nal position 1 mm above the base plate.The plunger speed was 1.5 mm/s. The area of deforma-tion curve was recorded as the result. In both cases the

result was an average of three replicates. All "rmnessresults 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).

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

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

Test 3Brix pH

1. Reference 50 3.42. PME 52 3.43a. CaCl # PME 54 3.33b. CaCl # PME in a vacuum 56 3.14. Sucrose 48 3.45. CaCl # sucrose 55 3.4

with a Historesin Embedding Kit (Jung, Germany) for5 d, polymerized and cut into thin sections (4 m) witha Microm HM355 (Microm Laborgera Kte GmbH, Wal-ldorf, Germany) microtome. Sections were stained withthe following staining solutions: 0.2 g/L Ruthenium Red

in water for 2 h followed by washing with water; and0.1 g/L Light Green for 1 min and thereafter with iodine.Ruthenium Red stains pectin pink; Light Green andiodine stains protein green or yellow ( 19,20). Sampleswere examined and photographed with an Olympus BX-50 microscope (Olympus, Optical Co. Ltd, Japan). Theimages were viewed with a Soft Imaging System analySIS3.0 software (Soft Imaging System GmbH, Germany) andprinted.

Fourier transform infrared microscopical studiesAll FT-IR spectra were recorded using a Bruker IFS 66(Bruker Optik GmbH, Germany) FT-IR spectrometer.The spectrometer was equipped with a microscope acces-sory. A liquid nitrogen cooled mercury cadmium tellu-ride (MCT) detector was attached to the microscope. Thespectrometer system was purged with predried nitrogengas. After 1 wk of freezer storage the untreated referenceas well as the pretreated strawberries were randomlychosen and thawed at room temperature (20 3C). Thestrawberries were thawed in an upright position, hulls onthe table, so as to ascertain what e ! ect thawing wouldhave on the structure of pretreated strawberries. Thawedstrawberries were frozen with liquid nitrogen and 7- msections were cut using a cryostat (Kryostat 1720 digital,Wild Leitz GmbH, Wetzlar, Germany). Before freezingthe 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 notused to surround or cover the tissue specimen but onlyfor "xing the sample to the sample. Ethanol-washedaluminium foil, "xed with tape to the object lens, wasused to surround the specimens. After focusing on the

subject to be scanned, the aperture (size 1.2, i.e. 32 mwith a 36 objective) was adjusted to frame the desiredportion for scanning and to exclude unwanted tissue.Strawberry sections were manually mapped systemati-cally across the achene, vascular tissue and pith, or acrossthe epidermis, hypodermis and cortical cells. The trans-mission mode was used and 50 scans were accumulatedto produce a spectrum over the 4000 }700 cm range ata resolution of 4 cm . A reference was scanned using thealuminium foil free of any tissue and 100 scans wereaccumulated to produce the transmission spectrum in the

4000 }700 cm range. The absorbance spectra werestored after logarithmic transformation and baselinecorrection ( 17).

Sensory e valuationThe sensory quality of the strawberry jams was evaluatedby a trained panel with proven skills ( n" 11) usingdescriptive analysis (21). Attribute intensities were ratedon a 10-unit graphical intensity scales. The scales wereverbally anchored at each end, and the left side of the

scale corresponded to the lowest or opposite intensity(value 0) and the right side to the highest intensity (value10) of the attribute. The sensory attributes evaluatedwere redness of colour (brown }red), wholeness of berries(broken }whole), clarity of medium (opaque }clear, trans-

parent), evenness of medium (uneven }even), "rmnessof medium ( " rm }#uid), softness of berries (hard }soft),leatheriness of berries (not leathery }leathery), sweetnessof 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 werepresented to the panellists coded and in random order,and water and crackers were provided for cleansing thepalate between the jam samples. The samples were ana-lysed 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 (SPSSversion 8.0, SPSS Inc., 1997) for the sensory results.ANOVA was used to test statistical di ! erences in sensoryattributes between the jam samples, and the statisticaldi! erence between the two sessions ( P ( 0.05). When thedi! erence in the analysis of variance was statisticallysigni "cant, pairwise comparisons of the attributes be-tween the jam samples were analysed by Tukey 's test.

Results and DiscussionSoluble solids and pH The total soluble solids and pH-values of the jams arepresented in Table 2 . All soluble solids values were quitelow (between 48 and 56 3Brix). The pectin used for thestudy was intended for jams, with a Brix value around 45.Furthermore, in these investigations it was more impor-tant to follow the e ! ect of calcium on the strawberriesthan to optimize the jam making. Therefore, the amountof soluble solids were not adjusted to a certain level butthe 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 4had the lowest value. pH values of the jams were around3.4. After jam cooking, on the surface of the jams thatcontained added calcium chloride (Tests 3a, 3b and 5), nofroth was observed, whereas on the other jams some frothwas noticed. Protein denaturation during cooking wasprobability caused by CaCl .

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Table 3 Firmness * of pretreated frozen and thawed strawberries as well as of strawberries in jams and jam mediums

Firmness

Frozen berries Frozen berries Jam berries Jam mediumCompression Deformation Deformation Deformation

force (kg) curve area (kgs) curve area (kgs) curve area (gs)

Test x N s x N s x N s x N s

1. Reference 32.9 3.5 313.7 53.6 49.0 3.5 10.6 0.52. PME 26.3 1.6 258.3 11.0 75.9 4.3 7.1 1.23a. CaCl # PME 32.2 2.0 315.5 21.2 35.7 6.2 4.9 0.83b. CaCl # PME in a vacuum 41.6 2.9 335.1 31.0 101.0 15.1 3.4 0.14. Sucrose 27.1 3.4 239.4 30.1 70.7 3.0 8.2 1.95. CaCl # sucrose 32.9 7.0 288.8 62.3 104.5 32.9 6.8 1.3

* Mean ( x N) and standard deviation (s ) of three measurements.} 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)

Table 4 Amounts of calcium in pretreated frozen straw-berries, jam strawberries and media. Calcium content of tap water used for analysis was 18 mg/L

Ca, mg/kg

Frozen Jam Medium Jam,Test berries berries total

1. Reference 130 100 67 802. PME 140 110 57 793a. CaCl # PME 250 180 130 1503b. CaCl #

PME in a vacuum

450 290 190 240

4. Sucrose 150 110 58 815. CaCl # sucrose 230 180 110 140

FirmnessThe "rmness results of all the tests are given in Table 3.The prefreezing treatments had a statistically signi "cantin#uence on the "rmness of frozen strawberries whenmeasuring 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 pair-wise comparisons (Tukey 's test, P ( 0.05) the "rmnessvalues of pretreated or untreated reference frozen straw-berries did not di ! er from each other. Frozen strawber-ries treated with only PME (Test 2) or with sucrose (Test4) were less "rm than strawberries treated with CaCland PME in a vacuum (Test 3b). The deformation forceand curve area values of the strawberries treated withCaCl and PME in a vacuum (Test 3b) were greater thanthose of other strawberries though there was no statist-ically signi "cant di ! erence between them. One reason forthis is that the height of the thawed strawberries surfaceat the used base plate varied quite a lot from test to test.Consequentially, the force and curve area values variedto such a degree between replicates that more measure-ments would have been required to decrease the devi-ation 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 Garc m Haet al . (10), who used strawberries treated with a 1%calcium chloride solution at 45 3C, showed that the pre-treated strawberries were signi "cantly "rmer than theuntreated reference strawberries. In addition, Main et al .(15) reported that in their studies whole fruit maintainedits integrity better than sliced fruit after heat treatmentin a 35 3C warm bath.

Jams made from strawberries treated with CaCl andPME in a vacuum (Test 3b) or with CaCl and crystal-lized sucrose (Test 5), had signi "cantly higher "rmnessvalues than the reference sample (approximately twice asgreat). The "rmness values of jams made from strawber-ries 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 ex-cept 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 jamsexcept for the jam made from CaCl - and PME-treatedstrawberries at normal air pressure (Test 3a). The " rm-ness of jam berries correlated negatively with the "rm-ness of jam media, i.e. the jams with " rmer strawberries

had softer media. Jam berries were more homogenous insize and shape than the thawed ones. Therefore, in jam" rmness analysis, three replicates seemed to be su $ cientto obtain reliable results.

Calcium analysisCalcium chloride treatments at normal air pressure aswell as together with the sucrose-treatment (Tests 3a and5, respectively) clearly increased the Ca content of straw-berries relative to the reference or other treatments inwhich Ca had not been added (Tests 1, 2 and 4) ( Table 4 ).The amount of Ca in the strawberries that had beendipped in the calcium chloride solution in a vacuum (Test3b) was about three times greater than the amount of Cain the strawberries that were not dipped in the calciumchloride solution (Tests 1, 2 and 4). The same e ! ect can beseen in the amounts of Ca in jam strawberries and media.According to Garc m Ha et al . (10) the strawberries treatedwith 1% calcium chloride solution at 25 3C had a signi-"cantly higher calcium content than the fruits of the

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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 calciumfrom the 1% calcium chloride solution into the fruits.This is in agreement with our results.

Microstructural studiesMicrostructural studies were carried out after storage ina freezer for 1 wk using the reference fruit, PME-treatedfruit, or CaCl - and PME-treated strawberries in a vac-uum (Tests 1, 2 and 3b, respectively). The pectin andprotein in cortical cells and vascular tissue were stainedwith speci "c staining systems, and their locations werestudied. In the untreated reference samples, pectin in themiddle lamella of the cortical cells was poorly stainedcompared to the CaCl - and PME-treated strawberriesin a vacuum (Fig. 2a +c). Moreover, the cell walls seemedto be broken and pectin appeared outside the middlelamella. In the PME-treated strawberries, pectin ap-peared outside the middle lamella as in the referencesample. In the CaCl - and PME-treated strawberries ina vacuum, pectin was well stained and quite homogene-ously spread in the middle lamella. In some parts themiddle lamella had swollen and was "lled with pectin.Swelling was not observed in the reference sample. In thecortical cells, protein was stained very similar to that inthe reference sample as well as in the PME-treated orCaCl - 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 tobe badly broken (not shown). The vascular tissues of thePME-treated strawberries were totally broken and it wasimpossible to carry out the microstructural studies. Inthe vascular tissue of the CaCl - and PME-treated straw-berries in a vacuum, protein was well stained and homo-geneously spread ( Fig. 4 ). Furthermore, the lignin-rich,spiral-type structures seemed to be quite intact andcoherent.

Fourier transform infrared microscopical studiesThese were carried out on fruit treated in Tests 1 }5. Theanalyses of the spectra was mainly based on our earlierstudies of strawberries ( 17,18). Fourier transform infraredmicrospectroscopy spectra are shown only for the refer-ence, PME-, and CaCl - and PME-pretreated strawber-ries in a vacuum. The measured spectra were scattered tosome extent. Before analysis, the spectra were manipu-lated by smoothing to compensate for these e ! ects. Thesmoothing was done using the Savitzky }Golay algo-

rithm, 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 overlapsomewhat in 1700 }1500 cm spectral region.

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

4000 }700 cm region. Figure 7 shows a series of spectrain the 4000 }700 cm region of sections of strawberrytissue obtained from the outer edge of the achenethrough the vascular tissue into the centre of the pith. Inall these spectra, the OH and CH stretching vibration

bands were present in the 3500 }2900 cm region. Atapproximately 1660 and 1550 cm , amide I and smallamide 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 cmregion represented structural carbohydrate bands. Also,in each spectrum, the CO stretching vibration band atapproximately 1240 cm and strong carbohydratebands 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 ina vacuum ( Fig. 7c ) * represent the achene of the refer-ence sample (Fig. 7a ). The following twelve spectra from3}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 }22or 14 }29 and 20 }29 in the cases of Tests 2 (Fig. 7b ) and3b (Fig. 7c ), respectively, of the reference sample repres-ent the pith in Fig. 7a . An overview of a series of spectraof the reference (Fig. 7a ) and pretreated sections of straw-berry tissues shows that peak absorbances of the CaCl -and PME-treated strawberries in a vacuum were higherand peak areas larger than the spectra of other pretreatedor reference strawberries.In each of the achene spectra ( Fig. 7a +c), a strong COstretching vibration band was present at approximately1740 cm due to esteri "ed group vibration of the pectin.In addition, quite a strong CO stretching vibration bandwas present in each of the baseline-corrected (beforesmoothing) achene spectra at approximately 1640 cmdue to acidi "ed group vibration of the pectin. This peakwas di $ cult to recognize after smoothing because of the overlapping e ! ects. At approximately 1605 and1510 cm , peaks typical of lignin were present in the

achene spectrum of the reference, calcium chloride-, andPME- or sucrose-treated strawberries. It was di $ cult torecognize the lignin peaks in the achene spectra of thereference and the calcium chloride- and PME-treatedsamples in a vacuum as well as of calcium chloride- andsucrose-treated strawberries due to destruction of thecomposition during or after treatments.In the vascular tissues of the reference and pretreatedstrawberries (Figs 5 , 7), there is a carbonyl band atapproximately 1725 cm , representing pectin. Theacidi "ed group of pectin at approximately 1640 cm

was just recognizable from the baseline-corrected spectraof each of the measured strawberries. In particular, in thecalcium chloride- and PME-treated strawberries ina vacuum, the amide I and II bands were higher than inthe spectra of the reference or other pretreated strawber-ries. Also, the pectin and protein bands as well as thestructural carbohydrate bands in the 1460 }1330 cmregion were higher and their areas larger in the spectra of the CaCl - and PME-treated strawberries in a vacuumcompared to the spectra of the reference or other pre-treated 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 corticaltissue (two micrographs per treatment). Pectin (marked with an arrow) was stained and appeared pink

other CaCl -treated strawberries was higher relative tothe nonpretreated reference strawberries. In the PME-treated strawberries, the pectin and protein as well as thestructural carbohydrate and carbohydrate peaks had lost

their spectral intensities relative to the reference spectra.This indicated, on the one hand, that PME had just cutcarboxyl units from the methylesteri "ed pectin groupsbased 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 corticaltissue (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 commercialenzyme preparation had also cut protein and structuralcarbohydrate (cellulose) and for that reason had to havesome protease and cellulase activities too. The vascular

tissue spectra of the sucrose-treated strawberries werevery similar to the reference strawberries. This indicatedthat the surface of the fresh strawberries themselves werequite hard in the pretreatment stage and thus did not

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

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

allow crystallized sugar to penetrate much. In the spectraof the vascular tissue of the reference and pretreatedstrawberries, the typical lignin peaks cannot clearly bedetected. The reason for this is presumably the overlap-ping e ! ects together with the small amount of lignin in

strawberry. From the edge of the vascular tissue to thecentre of the pith, no dramatic changes can be de-tected in the pectin, protein or structural carbohydratepeaks of the other pretreated strawberries except theCaCl - and PME-treated strawberries in a vacuum.The absorbances of these compounds were higher and

peak areas larger in the vacuum pretreatments comparedto the nonpretreated reference or other pretreated straw-berries.

Epidermis, hypodermis, cortexFigure 8 shows typical spectra of the cortex of the refer-ence, PME- and CaCl and PME-treated strawberries ina vacuum in the 4000 }700 cm region (about from thecentre of the tissues). Figure 9 shows transitions from theedge of the epidermis through the hypodermis to the endof the cortex in the 4000 }700 cm region. The "rst twospectra represent the epidermis and hypodermis. The restof the spectra represent the cortex. There was quitestrong carbonyl band at approximately 1725 cm rep-resenting esteri "ed pectin. At approximately 1660 and1550 cm , there were the CO and CN stretching vibra-tion bands representing amide I and II. The amide I andII peaks were better recognized from the baseline-correc-ted spectra. The structural carbohydrate bands werepresent in the 1460 }1360 cm region as well as the COstretching vibration band at approximately 1240 cmand carbohydrate bands in the 1200 }1000 cm region.There were no considerable changes in pectin, protein,structural carbohydrate or carbohydrate peaks of thespectra of the epidermis and hypodermis of the pret-reated strawberries relative to the reference. Instead, pec-tin and structural carbohydrate peaks of the cortex of theCa- and PME-treated strawberries in a vacuum (Fig. 9c )were higher and their areas larger than in the referencesample. An overview of the spectra of the cortex of theother pretreated strawberries indicated that their struc-tures in some points of the cortex were badly brokenduring or after these pretreatments.

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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 regionbaseline-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 theCaCl -solution followed by sugar sprinkling or dippingin CaCl - and PME-solutions, the strawberries retainedtheir shape better than without these treatments. Themedium was more #uid in the jam containing strawber-ries treated with CaCl and sucrose in a vacuum (Test 3b)than in the others. The medium was " rmer in the refer-ence jam (Test 1) than in the jams made from berriestreated 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 andsucrose in a vacuum or with CaCl and crystallized

sucrose (Tests 3b and 5). There seems to be a negativecorrelation between clarity and "rmness of the medium.The medium was clear in all the jams treated with theCaCl -solution, and they were also more #uid than theothers. Also, juice was somewhat separated from the jamsin the CaCl -treatments. Other sensory attributes evalu-ated did not di ! er statistically signi "cantly from eachother.The results were somewhat di ! erent depending on thereplicate number, which might result from di ! erencesbetween the two jam replicates presented to the panel-

lists. In fact, because of the large size of the strawberriesand the rather small amount of jam samples, the amountof strawberries for one assessor was rather small.From the evaluated attributes, only leatheriness of theberries was assessed di ! erently in the two replicates(P ( 0.005).According to the sensory studies of Main et al . (15), thestrawberries treated with 1% or 2% calcium lactatesolutions in a vacuum, frozen with sucrose and thawed ina 35 3C water bath for 30 min, showed no signi "cantdi! erences in #avour or texture with reference to the

untreated control. Instead, colour attribute, when deter-mined as acceptability, tended to be lower on all fruit thathad been exposed to a vacuum and was rated lower thanthe control on all soaks except that in the 2% calciumlactate solution. This may be due to bleeding of pigmentthat occurred due to cell rupture. According to Garc m Ha etal . (11), strawberry pretreatments in calcium chloridesolutions either at temperatures of 25 or 45 3C did nota! ect the sensory quality (appearance and o ! -#avour) of fruits.

Conclusion

Instrumental texture- and microscopy studies as well assensory analysis showed that the CaCl and PME ina vacuum as well as calcium chloride and sucrose pref-reezing treatments clearly improved "rmness of straw-berry tissues. Fourier transform infrared microscopy andbright- "eld microscopy studies showed that the pectin,protein and structural carbohydrate components of theabove pretreated strawberries were more stable thanthose of the untreated reference samples. The calcium

chloride and pectin methylesterase pretreatments ina vacuum seemed to stabilize "rst the structure of thevascular tissue and then the cortex and pith. The di ! erentprefreezing treatments for the strawberries gave promis-ing results on the sensory quality of the jams. The tex-tural 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 inthe jam, and these sensory attributes had negativecorrelations 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 the4000 }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 treat-ments ( n" 11). Means representing results combined from two independent replicates

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

Redness of colour 7.2 7.4 7.9 8.3 7.9 8.5Wholeness of berries 4.8 5.9 6.9 7.8 5.3 8.2Clarity of medium 5.6 6.2 7.1 8.2 6.8 8.0Evenness of medium 4.6 4.8 5.4 6.4 4.9 5.4Firmness of medium 7.6 6.9 5.7 2.8 7.4 4.9Softness of berries 7.2 7.1 6.6 6.4 6.1 5.4Leatheriness of berries 6.3 5.3 6.2 6.0 6.1 5.9Sweetness of odour and #avour 7.2 7.3 7.6 7.9 7.3 7.2Sourness of odour and #avour 5.0 5.0 4.9 5.3 5.6 5.4Balance of odour and #avour 6.8 7.1 6.7 6.7 6.8 6.5Faultlessness of odour and #avour 7.8 8.0 8.1 7.6 7.6 7.1

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

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

Acknowledgements

We wish to thank Annikka Mustranta from VTTBiotechnology for her expert knowledge in enzyme ap-plications. Furthermore, we wish to thank Anne

Ala-Kahrakuusi, Helena Liukkonen-Lilja and LiisaA Gna Kka Kinen and the sensory group from VTT Biotechnol-ogy for their skilful technical assistance in carrying outthe pretreatments as well as the calcium and microscop-ical measurements and sensory analysis.

References

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The Effect of Prefreezing Treatments on the Structure of Strawberry Jam Before and After Jam Maki

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