Effects of Phenolic Compounds on Gelation Behavior ofGelatin Gels

JIN WU, SHIH-CHIEN CHIU, ELI M. PEARCE, T. K. KWEI

Department of Chemical Engineering and Chemistry, Herman F. Mark Polymer Research Institute, Polytechnic University,Six MetroTech Center, Brooklyn, New York 11201

Received 23 June 2000; accepted 12 October 2000Published online 00 Month 2000

ABSTRACT: The effects of phenolic additives on the gelation behavior of gelatin gelswere investigated using thermomechanical analysis (TMA) for study of gel-meltingtemperature, dynamic mechanical analysis (DMA) for study of gel-storage modulus andgel-aging stability, viscometry for study of gelation time, and texture analyzer for studyof gel strength and gel melting. Thermodynamically, the addition of 1,3-benzenediol,1,4-benzenediol or 1,3,5-benzenetriol favored the gelation process of gelatin solutions(increases in Tm and aging stability) due to the introduction of extra physical crosslinksamong gelatin chains through hydrogen bonding, while the addition of 1,2-benzenediolhad a negative effect (decreases in Tm and aging stability) possibly due to intra-hydrogen bonding of the additive molecule itself. All the phenolic compounds had littleeffect on gel moduli. Kinetically, the introduction of 1,2-benzenediol or 1,4-benzenediolslowed the gelation process, while introduction of catechin, a polyphenol, acceleratedthe first stage of the gelation process. © 2000 John Wiley & Sons, Inc. J Polym Sci A: PolymChem 39: 224–231, 2001Keywords: gelatin gel; phenolic compounds; thermomechanical analysis; dynamicmechanic analysis; physical crosslinks; hydrogen bonding

INTRODUCTION

Gelatin gels are widely used in photographic,medical, pharmaceutical, and food industries.The gelation behavior of gelatin gels is influencedby temperature, pH,1 ash content, method ofmanufacture, thermal history, and concentra-tion.2 Fine tuning of the gelation behavior of gel-atin gels, such as gel-melting temperature, gela-tion rate, and gel modulus, are of past as well asfuture practical importance. For instance, thethermal and mechanical stability of gelatin gelswere reported to be improved by chemicalcrosslinking with carbodiimide.3,4 The increased

water-sorption behavior of several glycol-crosslinked gelatin gels was observed by Iannaceet al.5 Further, chemical crosslinking was alsoconducted on gelatin itself.6, 7 Mechanical proper-ties were improved for chemically crosslinked ori-ented gelatin.8,9,10 In addition, thermal crosslink-ing of gelatin was studied and higher micro-hard-ness values were reported on these gelatinfilms.11

Besides chemical crosslinking, the addition ofcertain reagents such as sugar and polyols hasstabilized higher order structures of polypeptidechains by mediation of these reagents with thewater structure.12–15 However, some compoundssuch as poly(ethylene glycol) and 2-methyl-2,4-pentanediol acted as destabilizers for someglobular proteins.16,17 Fujitsu et al.18 systemati-cally studied the effects of hydroxyl compounds onthe formation of gelatin gels, and concluded that

Correspondence to: E. M. Pearce (E-mail: epearce@duke.poly.edu)Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 39, 224–231 (2001)© 2000 John Wiley & Sons, Inc.

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the network structures of gelatin gels were influ-enced by the number and position of the hydroxylgroups as well as the carbon atoms of these coex-isting compounds. The rationale of adding hy-droxyl compounds was to increase the number ofcrosslinking junctions in the gel through hydro-gen bonding. It is known that aliphatic hydroxylcompounds are not as good hydrogen-bonding do-nors as phenolic hydroxyl groups, therefore inthis study we decided to incorporate phenoliccompounds, which are excellent hydrogen-bond-ing donors, into gelatin gels to investigate theeffects of these compounds on the gelation behav-ior of these gels.

The rationale of this study was to introduceadditional physical crosslinks from the phenoliccompounds with the gelatin chains through hy-drogen bonding. The number and relative posi-tion of hydroxyl groups on aromatic rings werebelieved to affect this. In this study, we startedwith simple phenolic compounds, then extendedthe study to polyphenols19,20 to investigatechanges in the gelation behavior of gelatin gelsincluding gel-melting temperature, gelation rate,gel modulus, and gel-aging stability. The informa-tion from this investigation might be useful forfuture applications.

EXPERIMENTAL

Materials

Type B gelatin (225 bloom, from calf skin) waspurchased from Aldrich. Phenolic compoundsused in this study were purchased from Aldrichincluding phenol, 1,2-benzenediol, 1,3-benzene-diol, 1,4-benzenediol, 1,2,3-benzenetriol, 1,2,4-benzenetriol, 1,3,5-benzenetriol, and (6) catechinhydrate (98%).

Preparation of Gelatin Gels

All gelatin gels used in this study were based on10 wt % gelatin or gelatin plus additive in water.Gelatin was dissolved in deionized water at 50 °C.A given amount of phenolic compound (weight vsgelatin itself) was added and mixed with gelatinsolution for 1 h. The sample solutions were storedat 4 °C for 24 h to complete the formation of gelnetworks.

Determination of Gel-Melting Temperature

Thermomechanical analysis (TMA) was con-ducted on a PerkinElmer TMA-7 Thermome-

chanical Analyzer, which was computerized witha Pyris controller for this study. The gelatin gel(10 wt %) was formed in a PerkinElmer samplepan at 4 °C for 24 h. The following conditions wereoptimized and utilized in this study: penetrationmode, 50-mN static force, and 1 °C/min heatingrate. The quartz probe tip was placed down totouch the gel surface before the penetration modewas started. The temperature scan was per-formed with heating from 4 °C to 70 °C at the rateof 1 °C/min. The probe position was plotted versustemperature, and the onset temperature of thesudden penetration was used as the gel meltingtemperature.

Determination of Gel Modulus and Gel-AgingStability

Dynamic mechanical analysis (DMA) was con-ducted on a PerkinElmer DMA-7e dynamic me-chanical analyzer, which was computerized witha Pyris controller for this study. The followingconditions were optimized and utilized in thisstudy: the parallel plate-disc measuring system,6-mN static force, 5-mN dynamic force, 5-Hz fre-quency, and 2 °C/min heating rate. All DMA ex-periments were performed in the compressionmode in the linear viscoelastic range with cylin-drical gel samples (10 wt %, diameter 6 mm andthickness 2 mm). The preset gelatin gel wasplaced between the parallel plates before com-pression mode was started. The gel was held at 4°C for 10 min, heated to 14 °C at 2 °C/min andheld at 14 °C for aging as programmed. The stor-age modulus (E’, compression) was determined at4 °C and 14 °C, and the gel stability was charac-terized with the time during which the storagemodulus dropped to 0 at 14 °C.

Determination of Gelation Rate

The gelation rate was determined using a Brook-field DV-II calculating digital viscometer. The 10wt % gelatin solution (with or without additive)was preheated at 60 °C for 2 h to erase any ther-mal history, and then immersed into a 22 °Cwater-bath for gelation. The cylindrical Spindle#28 with a spinning rate of 0.5 rpm was chosen forthe experiment. The viscosity (centpoise, cps) wasplotted versus time, and the experiment endedwhen viscosity reached 2.0 3 106 cps (off instru-ment limit).

GELATION BEHAVIOR OF GELATIN GELS 225

Determination of Gel-Melting Rate and GelStrength

The gel-melting rate was determined by theStevens L.F.R.A. texture analyzer (model TA-100)with a 1-inch diameter AOAC plummet. The pre-set gel sample (as prepared previously at 4 °C)was put into a 22 °C-water bath for 10 min toattain temperature equilibrium, and then the gelstrength was measured as a function of time at 22°C. The gel strength was characterized by mea-suring the resistant force (gram) when the plum-met penetrated into the gel set at 4 mm with theplummet speed set at 1.0 mm/s.

RESULTS AND DISCUSSION

The effects of phenolic compounds on the gelationbehavior of gelatin solutions were generalized inthe following aspects: (1) gel-melting tempera-ture; (2) gel modulus and gel-aging stability; (3)gelation rate. The pH values of the samples usedin this study were determined to be about 5.2,which were very close to that of the pure gelatinsolution. Therefore, phenolic additives did notchange the pH values of the samples. Type Bgelatin was chosen for this study because of itsweakly acidic isoelectric point because the phe-nolic compounds used in this study are weaklyacidic. It is interesting to compare the effects ofphenolic compounds on the gelation behavior ofboth Type B and Type A gelatin (weakly basicisoelectric point) in the future.

Gel-Melting Temperature (Tm)

Previous literature reported the determination ofgel-melting temperature (Tm) using the falling

ball method18 or DSC21 based on a slow temper-ature increase. Another method was to determinethe gel-induction period of gelatin as a function oftemperature and extrapolate this period to infi-nite induction time (Tgel). However, Tgel cannot beidentified with Tm as, in general, the temperatureat which gelation took place was lower than themelting temperature of an already formed gel. Inthis study, thermomechanical analysis (TMA)was used for the first time to determine gel-melt-ing temperature, which was proven to be accurateand reproducible. The probe position was plottedversus temperature for each formulation in Fig-ure 1, and the onset point of the transition wastaken as gel-melting temperature. Each formula-tion consisted of gelatin plus 6 wt % additive(weight vs gelatin) in a 10 wt % aqueous solution.Gelatin without any additive in a 10 wt % aque-ous solution was used as comparison. The effectson gel-melting temperature were summarized inTable I. As can be seen, these simple phenolicadditives did change the gel-melting tempera-tures of gelatin gels and the extent of changedepended on the positions of hydroxyl groups onthe benzene ring when the additive was kept at acertain constant amount. Phenol itself had littleeffect on the Tm; for the diols, 1,3-benzenediol and1,4-benzenediol increased the Tm’s by about 15' 20 °C, while 1,2-benzenediol had a negativeeffect; for the triols, 1,2,4-benzenetriol and 1,3,5-benzenetriol increased the Tm’s, while 1,2,3-ben-zenetriol had little effect. It was interesting thatgel-melting temperatures may be finely tuned forgelatin gels by minor changes in the chemical

Figure 1. TMA diagrams of 10 wt % gelatin hydro-gels with different additives.

Table I. Gel-Melting Temperatures of DifferentGelatin Hydrogels Determined by ThermomechanicalAnalysis (TMA)

10 wt % Gelatin Hydrogel

Gel-MeltingTemperature

(°C)

Gelatin 30.1 6 1.7Gelatin 1 6 wt % 1,2-benzenediol 23.9 6 1.6Gelatin 1 6 wt % 1,3-benzenediol 49.6 6 1.5Gelatin 1 6 wt % 1,4-benzenediol 45.7 6 2.8Gelatin 1 6 wt % 1,2,3-benzenetriol 33.2 6 1.3Gelatin 1 6 wt % 1,2,4-benzenetriol 38.8 6 2.3Gelatin 1 6 wt % 1,3,5-benzenetriol 43.1 6 0.9Gelatin 1 6 wt % phenol 32.2 6 1.9

Note: Three measurements were performed for each hydro-gel, and the average gel melting temperature and its standarddeviation were indicated.

226 WU ET AL.

structure of the additives. The phenolic com-pounds that raised the Tm’s were believed to helpthe formation of physical crosslinks among gela-tin chains through hydrogen bonding.

During the gelation process, the Gibbs free en-ergy change (DGgel) from the random coil into thetriple-helix configuration is negative. Because ofthe negative entropy contribution DSgel (fromboth the decreased entropy of the gelatin chainitself and the decreased entropy of water mole-cules that were immobilized and oriented in theaxis direction), the contribution of the transitionenthalpy DHgel has to be considerably negative.The addition of phenolic compounds was thoughtto promote the formation of hydrogen bondsamong the three helices, thereby contributing tothe negative enthalpy change. The clue can befound in the detailed structures of the phenoliccompounds that differed in their capabilities toengage in inter-hydrogen bonding versus intra-hydrogen bonding as shown in Figure 2. Both1,4-benzenediol and 1,3-benzenediol acted asphysical crosslinking agents through inter-hydro-gen bonds with gelatin chains, while 1,2-ben-zenediol was more readily intra-hydrogen bondedand thus contributed little to interchain cross-linking. On the other hand, all the phenolic com-pounds might have had a deleterious effect on theformation of physical crosslinks among gelatinchains due to steric hindrance. The net effect wasdifficult to predict from the experimental results.We found that 1,4- and 1,3-benzenediol increasedthe gel-melting temperatures with 1,3-ben-zenediol having the larger effect, while 1,2-ben-zenediol decreased the Tm. Similar results wereobtained for the triols. 1,2,3-benzenetriol had lit-tle effect, 1,2,4-benzenetriol had a positive effect,and 1,3,5-benzenetriol provided the largest Tmincrease. Thus, the meta- and para-hydroxylgroups on the benzene ring seem to help hydrogen

bonding among gelatin chains, while the ortho-hydroxyl groups had no or negative effects due tointra-hydrogen bonding of the additive itself. Insummary, the positions and numbers of hydroxylgroups on benzene ring of these phenolic com-pounds affected gelatin gels differently.

The effect of the amount of the additive ongel-melting temperature was also investigated.The results for 1,4-benzenediol were shown inFigure 3. The gel-melting temperature increasedwith the amount of the additive up to ' 20 wt %beyond which the additive was insoluble.

Gel Modulus and Gel-Aging Stability

For this series of experiments, all the gel sampleswere set at 4 °C for 24 h for complete gelation,which was confirmed by the DMA measurementsthat indicated that the storage moduli did notchange with aging time at 4 °C (data not shown).The DMA diagrams were shown in Figure 4. Allthe curves can generally be divided into threeportions. The first portion represented aging at 4°C for 8 min, which showed that the modulus didnot change with time. The second portion repre-sented the heating process, which showed themodulus decreasing with time. The last portionrepresented aging at 14 °C, which showed thatthe modulus remained constant at first, thendropped gradually to zero. The aging temperatureof 14 °C was selected because the aging time(during which modulus dropped to zero) was lo-cated within suitable experimental ranges. Ascan be seen here, the moduli of all the gel samplesat 4 °C were around 5.5 3 104 Pa, while those at

Figure 2. Schematic representation of a phenolic-compound-enhanced physical gelatin network.

Figure 3. Effects of additive amounts (vs gelatinweight) on gel-melting temperatures of 10 wt % gelatingels.

GELATION BEHAVIOR OF GELATIN GELS 227

14 °C were around 2.0 3 104 Pa. The introductionof physical crosslinks by phenolic additives hadlittle effect on gel modulus both at 4 °C and 14 °Cwithin experimental error, unlike the chemicalcrosslinked gelatin gels, which were reported tohave increased moduli.4 However, the introduc-tion of either chemical crosslinks3,4 or physicalcrosslinks in this study (increased Tm) improvedthe thermal stability of gelatin gels.

The other important aspect of the gel was itsaging stability. For example, gelatin gels losttheir strength when aged at room temperaturebecause of its thermal reversibility. From the pre-vious Tm data, we reported improved thermal sta-bility of the gelatin gels having the addition ofsome phenolic compounds. Encouraged by thisresult, we found that these phenolic compoundsalso stabilized the gelatin gel aging at 14 °C (Fig.

4). The aging stability was characterized by thetime period during which the storage modulus ofthe gel dropped from a certain value to zero at theaging temperature of 14 °C. The results of gelmodulus and gel-aging stability were summa-rized in Table II. As can be seen, compared to thepure gelatin gel, 1,3-benzenediol, 1,4-benzenedioland 1,3,5-benzenetriol stabilized the gelatin gels.Phenol, 1,2,3-benzenetriol, and 1,2,4-benzenetriolhad little effect on aging stability. 1,2-benzenedioldestabilized the gelatin gel with decreased agingtime. This result was consistent with that of thegel melting temperature in that the phenolic ad-ditives that increased the Tm of the gelatin gelalso increased the aging stability of the gel, andthose that decreased the Tm also destabilized thegel. A graphic comparison was shown in Figure 5,which demonstrated the effects of various phe-nolic additives on gel-melting temperature, gel-aging stability, and gel modulus of the gelatingels. The gel-melting temperature and gel-agingstability at 14 °C increased with the introductionof 1,3-benzenediol, 1,4-benzenediol, and 1,3,5-benzenetriol, which we believed to provide extraphysical crosslinks among gelatin chains. In con-trast, the Tm and aging stability at 14 °C de-creased with the introduction of 1,2-benzenediolfor which intra-hydrogen bonding of the moleculeitself decreased its ability to crosslink. In addi-tion, 1,2,3-benzenetriol, like phenol, had little ef-fect on both gel-melting temperature and gel-ag-ing stability, while 1,2,4-benzenetriol increasedthe Tm but had little effect on aging stability. Wehypothesized that the 1,2,3-benzenetriol mole-cule, in which the three neighboring hydroxyl

Figure 4. DMA diagrams of 10 wt % gelatin hydro-gels with different additives.

Table II. Gel-Storage Modulus (E9) and Gel Stability of Different Gelatin Hydrogels Studied by DynamicMechanical Analysis (DMA)

10 wt % Gelatin Hydrogel

Storage Modulus (3 1024 Pa)Gel-Aging Stability

at 14 °C (min)4 °C 14 °C

Gelatin 6.1 6 0.2 2.1 6 0.3 39.9 6 1.4Gelatin 1 6 wt % phenol 5.1 6 0.4 2.1 6 0.2 38.0 6 0.9Gelatin 1 6 wt % 1,2-benzenediol 5.4 6 0.3 1.9 6 0.4 31.2 6 2.0Gelatin 1 6 wt % 1,3-benzenediol 5.9 6 0.2 2.0 6 0.1 56.7 6 2.2Gelatin 1 6 wt % 1,4-benzenediol 5.3 6 0.4 2.3 6 0.2 50.2 6 1.9Gelatin 1 6 wt % 1,2,3-benzenetriol 5.3 6 0.5 1.6 6 0.4 38.9 6 1.3Gelatin 1 6 wt % 1,2,4-benzenetriol 5.5 6 0.3 2.5 6 0.3 38.6 6 1.9Gelatin 1 6 wt % 1,3,5-benzenetriol 4.7 6 0.8 1.9 6 0.2 48.1 6 2.5

Note: Three measurements were performed for each hydrogel, and the average modulus and aging stability and theircorresponding standard deviations were indicated.

228 WU ET AL.

groups might be intra-hydrogen bonded betweeneach other, appeared to be like a phenol molecule(as shown in Fig. 6). The 1,2,4-benzenetriol mol-ecule, in which only two neighboring hydroxylgroups might be intra-hydrogen bonded, ap-peared to be the hybrid of a phenol molecule and1,4-benzenediol (also as shown in Fig. 6).

Gelation Rate

To date, there was no generally recognized stan-dard method of measurement for gelation ratebecause of the nature of the sol–gel transitionalthough the gel point was statistically well-de-fined. Some gelation-time methods22 measuredthe time when a gelatin solution of a standardconcentration attained a certain high viscosityvalue or certain gel-modulus value. These timeswere not necessarily close to the actual gelationtime, but could nevertheless serve to determine auseful ranking of gelatin gels with respect to ac-tual setting. In this investigation, the gelatin so-lutions were maintained at 22 °C and the viscos-ity-time relation was plotted for each sample

shown in Figure 7. As can be seen, the 1,4-ben-zenediol/gelatin solution gelled at a slower ratethan the pure gelatin solution, and the 1,2-ben-zenediol/gelatin solution had the slowest gelationprocess. When the viscosity of each of the threesamples reached the instrument limit (2.0 3 106

cps), the viscosity was seen to be still on the riseand the formed gel had not reached the equilib-rium state. In the case of the catechin/gelatinsolution the viscosity increased quickly to reach1.0 3 106 cps in less than 10 min after which itincreased more slowly to a constant value of 1.33 106 cps, which indicated that an equilibrium gelstate was reached. Because the gelation mecha-nism of gelatin gel involved the slow associationof three apidly formed single-helix segments,23

the introduction of the phenolic compound maythermodynamically favor the triple-helix forma-tion of the gelation process. But it is an openquestion how the phenolic additive affects thegelation process kinetically. From this investiga-tion, we found that the addition of catechin, apolyphenol, to gelatin favored the gelation processkinetically, and the sample gel reached the equi-librium state (from the standpoint of viscosity)much more quickly than the gelatin gel itself.However, there was a reduced gel strength. Theaddition of 1,4-benzenediol favored the gelationprocess thermodynamically (increase in Tm), butaffected adversely the process kinetically (slowergelation rate). The addition of 1,2-benzenediol in-fluenced negatively the gelation process boththermodynamically (decrease in Tm) and kineti-cally (slower gelation rate).

From the experiments, we believed that, ingeneral, the addition of a phenolic compound such

Figure 5. Comparison of the effects of various phe-nolic additives on gel-melting temperature, gel modu-lus, and gel-aging stability of gelatin gels.

Figure 6. Schematic representation of intra-hydro-gen bonding of 1,2,3-benzenetriol and 1,2,4-benzenet-riol.

Figure 7. Time-dependent changes in viscosity of 10wt % gelatin solutions with various phenolic additives.

GELATION BEHAVIOR OF GELATIN GELS 229

as 1,2-benzenediol and 1,4-benzenediol might re-tard the gelatin chains from approaching eachother kinetically during gelation. Therefore, ittook a longer time for the gel sample to reach theequilibrium state. However, the thermodynami-cally favorable 1,4-benzenediol/gelatin sampleformed a more stable gel, while the thermo-dynamically unfavorable 1,2-benzenediol/gelatinsample formed a less stable gel (from the previousstudies of Tm and modulus).

The situation for the catechin/gelatin samplewas different. The equilibrium state was reachedwith a much faster rate than from the gelatin gelitself, although the final gel strength was lower.The chemical structure of catechin was shown inFigure 8, which indicated meta- and ortho-hy-droxyl groups on different benzene rings. It isknown that proteins and polyphenols can com-bine to form soluble complexes.19 The catechinmolecule, which was a polyphenol from the struc-ture, may also be able to form complexes with thegelatin chain, but we do not know the mechanismhow the catechin molecule affected the gelationprocess kinetically at this time.

Gel-Melting Rate

In a previous section, we discussed the effects ofphenolic compounds on the gel modulus and gel-aging stability. To further investigate this issuewe have focused on the gel-melting kinetics atroom temperature. The preset (at 4 °C for 24 h)gelatin gel was put in 22 °C water bath, and thegel strength was measured as a function of time.The results were shown in Figure 9. Three gelsamples were tested including 2% gelatin gel, 2%(gelatin 1 10% 1,2-benzenediol) gel and 2% (gel-atin 1 10% 1,4-benzenediol). The protein concen-tration was chosen at 2% simply due to the con-venient experimental time range. As we can seehere, initially (time 5 0) the three gel sampleshad nearly same strength around 200 g, whichwas consistent with the previous gel modulusstudy that the phenolic additives did not changethe initial gel strength. After that, the gelstrength of each sample was found to be de-

creased with time. However, the gel strength de-creased at different rates. The gel sample contain-ing 1,2-benzenediol dropped the fastest, while thegel sample containing 1,4-benzenediol droppedthe slowest, with the pure gelatin gel right in themiddle. The choice of 20-gram gel strength as thelower threshold above which the gel maintainedits integrity is empirical. The gel containing 1,2-benzenediol lost its integrity at around 120 min-utes, while the gel containing 1,4-benzenediolmaintained its integrity after 6 hours. The puregelatin gel lost its integrity at around 180 min-utes. Therefore, the addition of 1,4-benzenediolimproved the gel aging stability (at 22 °C), whilethe addition of 1,2-benzenediol decreased the sta-bility, which was also consistent with the previ-ous DMA study. Both the DMA study and thetexture analyzer study provided the same conclu-sion.

CONCLUSIONS

The effects of phenolic compounds on gelationbehavior of gelatin gels were investigated includ-ing gel-melting temperature, gel-aging stability,gel modulus, and gelation time. It was found thatphenolic compounds containing meta- or para-hydroxyl groups on the benzene ring increasedthe Tm and the gel aging stability of gelatin gel,while phenolic compounds containing ortho-hy-droxyl groups on benzene ring had little or nega-tive effect on both Tm and aging stability. Thephenolic additives had little effect on gel-storagemoduli, and affected the gelation rate differently.Figure 8. Chemical structure of (6) catechin.

Figure 9. Time-dependent changes in gel strength of2 wt % gelatin gels with various phenolic additives.

230 WU ET AL.

The authors wish to thank Kraft Foods, Inc., Mr. EvanTurek, and Mr. Dominic Vellucci of their staffs forfinancial support and valuable insights. Special thanksare due to Professor Edith Turi for using the ProfessorEdith Turi Thermal Analysis Laboratory and ProfessorJovan Mijovic for using his instrument.

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