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The effect of high hydrostatic pressureon the muscle proteins of rainbow trout(Oncorhynchus mykiss Walbaum) filletswrapped with chitosan-based ediblefilm during cold storage (4±1°C)Ali Günlüa, Sinem Sipahioǧlub & Hami Alpasc

a Department of Fisheries and Seafood Processing Technology,Muǧla Sıtkı Koçman University, Muǧla, Turkeyb Antalya Aquarium, Antalya, Turkeyc Food Engineering Department, Middle East Technical University,Ankara, TurkeyPublished online: 13 Mar 2014.

To cite this article: Ali Günlü, Sinem Sipahioǧlu & Hami Alpas (2014) The effect of high hydrostaticpressure on the muscle proteins of rainbow trout (Oncorhynchus mykiss Walbaum) filletswrapped with chitosan-based edible film during cold storage (4±1°C), High Pressure Research: AnInternational Journal, 34:1, 122-132, DOI: 10.1080/08957959.2014.894510

To link to this article: http://dx.doi.org/10.1080/08957959.2014.894510

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High Pressure Research, 2014Vol. 34, No. 1, 122–132, http://dx.doi.org/10.1080/08957959.2014.894510

The effect of high hydrostatic pressure on the muscle proteins ofrainbow trout (Oncorhynchus mykiss Walbaum) fillets wrappedwith chitosan-based edible film during cold storage (4 ± 1◦C)

Ali Günlüa∗, Sinem Sipahioglub and Hami Alpasc

aDepartment of Fisheries and Seafood Processing Technology, Mugla Sıtkı Koçman University, Mugla,Turkey; bAntalya Aquarium, Antalya, Turkey; cFood Engineering Department, Middle East Technical

University, Ankara, Turkey

(Received 14 November 2013; final version received 11 February 2014)

This study was to determine the effects of changes that occurred in the muscle proteins of fresh rainbowtrout (Oncorhynchus mykiss) fillets during storage at 4 ± 1◦C as a result of packaging in vacuum (C),subject to high pressure after packaging in vacuum high hydrostatic pressue (HHP), packaged in vacuumafter wrapping with chitosan film (CFW) and subject to high pressure after wrapping with chitosan-basedfilm and packaged in vacuum (HHP + CFW). Samples were subjected to SDS-PAGE in four-day intervalsand the densitometric analyses of the gels were carried out. According to the results, minor changes weredetermined in the major bands of the sarcoplasmic and myofibrillar muscle fractions of trouts as a result ofHHP application and CFW. The most important change occurred in the myofibrillar muscle fraction as adecrease in the densities of the bands at 200 and 31.4 kDa after HHP application. Similarly, the sarcoplasmicmuscle fraction of trout fillet decreased in the densities of the bands at 39.3, 26.6 and 23.3 kDa after HHPapplication. In addition, it is thought that the bands that occur at 30 kDa in the myofibrillar muscle fractionand at 20.7 kDa at the sarcoplasmic muscle fraction may be related with the degradation of trouts duringcold storage.

Keywords: Oncorhynchus mykiss; chitosan; high hydrostatic pressure; muscle proteins; SDS-PAGE

Introductıon

Freshness is the most important criterion in the determination of the quality of seafoods. Hence, thedevelopment of reliable methods for the determination of the freshness of fish has been the basisof aquaculture researchers for many years. It is known that protein breakdown (proteolysis) is thebasic foundation of the textural and quality changes that occur in fish muscles during storage.[1–3]Ante mortem muscle biochemistry and postmortem biochemical processes are directly related withthe quality characteristics of the final product. The understanding of postmortem mechanisms is aprerequisite for defining objective identifier and indicators and the accurate control of the qualityof commercialized fish.[4]

The increase in consumer demand for high-quality, reliable fish meat without any additives anda long shelf life have resulted in the development of new seafood product-processing methods.Chitosan has a wide application range since it is natural, nontoxic, degradable in nature and

∗Corresponding author. Email: aligunlu@mu.edu.tr

© 2014 Taylor & Francis

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High Pressure Research 123

commercially obtainable.[5,6] This biopolymer has the ability to provide perfect film and coatingsolution when dissolved in acidic water solutions.[7] Hence, it has a wide potential to be usedas a food packaging material.[8–10] Even though various studies carried out on different fishspecies have stated that chitosan-based film and coatings decreased microorganism development,enhanced fish quality and increased storage time,[9,11–16] no study determined the changes inmuscle proteins as a result of treatment with chitosan.

The high hydrostatic pressue (HHP) process is reliable, does not require the use of additives anddue to these properties it is one of the non-thermal-processing technologies with high consumeracceptance.[17] HHP prevents the development of pathogen vegetative bacteria and providesenzyme inactivation even at low temperatures.[18,19] The positive effects of high pressure processhas been studied in many fish species.[12,20–26] However, the number of studies on the effectsof high pressure process on fish muscle proteins is limited.[27–30] Hendrickx et al. [27] andKo et al. [29] have stated that alternate changes have been observed in the protein structure atlow pressures applied at generally room temperature (100–300 MPa) and that they have causedirreversible protein denaturation at pressures above 300 MPa. Hurtado et al. [28] have stated thatthere is no change in the myosin heavy chain (MHC) band of octopus muscles after being packagedunder vacuum and being subject to a pressure of 400 MPa for 5 min at 7◦C and 40◦C followedby incubation at 40◦C and 60◦C for different periods of time and autolysis decreased as a resultof the denaturations formed due to high pressure in myofibrillar proteins (MFPs). Researchershave stated that HHP can be used effectively for preventing softening of octopus muscles duringstorage.

Rainbow trouts are among the most important aquaculture fish bred widely in the river and damlakes of Turkey which is sold in local markets as fresh frozen or fillet. In addition, it is also widelyexported to EU countries as fresh chilled whole-fillet or hot smoked vacuum packed.[31] Eventhough many studies have been carried out regarding the effects of chitosan-based edible film andhigh pressure application on the prolongation of the shelf life of fish meat; the number of studieson the changes that occur in the muscle proteins during shelf life period is limited. The objectiveof this study is to determine, via the SDS-PAGE method, the changes in the sarcoplasmic andmyofibrillar muscle proteins of rainbow trouts wrapped with chitosan-based edible film and beensubject to high pressure during storage at 4 ± 1◦C.

Material and methods

Samples

In this study, 80 rainbow trouts (O. mykiss) obtained from aquaculture producers (Baysallar,Isparta, Turkey) with an average weight of 250–300 g were used. Eighty rainbow trout were placedin tanks filled with ice-cold water and they were killed rapidly. The fish were put in styrofoamboxes and covered with bags filled with dry ice and then they were taken to a food laboratorywithin 1 h. The internal organs of the samples were cleaned in the laboratory and washed withwater after which their skinless fillets were taken and the fillets were cut from their sides intoorderly rectangles of equal weight (about 10 g).

Preparation of chitosan-based edible films

Chitosan-based edible films were prepared as described by Günlü and Koyun.[16] In order toobtain chitosan-based edible biofilms, commercial chitosan obtained from shrimp shells (SigmaC3646) was used. About 1.5 g of chitosan was weighed and 100 mL of 1% acetic acid was added

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in order to obtain the biofilm. The solution was mixed at 40◦C for 30 min in a magnetic mixer(Wisestir, MSH 20A, Korea) to ensure that the chitosan dissolves completely in acetic acid, afterwhich 1.5 g glycerol (Sigma G8773) was added as plasticizer and the mixing was continued inthe magnetic mixer at the same temperature for the same duration. The solution was transferred tothe buchner funnel in order to remove the air bubbles that had formed inside the obtained ediblefilm solution and the air bubbles were removed via a vacuum pump. Hundred millilitres of thefilm solution was taken and placed on styrofoam plates (20 × 10 cm) which were placed in a dryair sterilizer (Labart, DHG 9140A, Korea) and held at 40◦C for 8 h, cooled at room temperatureand the coating films were taken out of the plates using pincers. The films that are very flexibleand brittle were kept in a dessicator until the wrapping process of the fillet pieces was completed.

Wrapping of the fish with film and trial plan

Fillets that were previously cut into small pieces (10 g) were separated into four groups. The filletpieces in the first group were only packaged using polyethylene film (Ayhan Plastic Limited,Isparta, Turkey) in vacuum (Abant Vacuum Systems, Turkey) (Control group-C). The fillet piecesin the second group were packaged in vacuum and HHP was applied. Whereas the fillet pieces inthe third group were wrapped with chitosan film separately, the tips of the biofilms were squeezedafter which they were packed under vacuum by placing in vacuum bags (CFW). The fillet piecesin the last group were wrapped by chitosan-based film separately, packaged in vacuum and thenwere subject to the high hydrostatic pressure (HHP + CFW) process.

HHP application

HHP treatments were performed in a designed and constructed laboratory-scale unit (capacity:30 cm3, maximum pressure: 500 MPa). The equipment consists of a pressure chamber of cylin-drical design, two end closures, a means for restraining the end closures, a pressure pump and ahydraulic unit to generate high pressure for system compression, and also a temperature controldevice. The pressure vessel was made of hot galvanized carbon steel and the piston was hardchrome-plated and polished to mirror finish (steel-type heat-treated special K) which was pro-cessed into the required sizes at the Electrical and Electronic Engineering Department of MiddleEast Technical University (Ankara, Turkey). The liquid was heated up to the desired temperatureprior to pressure application using an electrical heating system encompassing the cell.

The vacuum-packed fillet pieces which will be subject to the HHP process were wrapped witha stretch film tightly and brought to the METU-Food Engineering Department HHP Laboratory(Ankara, Turkey) within 10 h after being placed in styrofoam boxes with dry ice and were thensubject to the high pressure process in the HHP Unit under 220 MPa at 15◦C for 5 min. Motorinoil was used as fluid in the pressure unit for pressure transmission.

The storage of samples

According to the chemical and microbiological analysis results, Günlü et al. [32] have stated thatthe shelf lives of rainbow trout fillets in the C, HHP, CFW and HHP + CFW groups ended on the12th, 16th, 24th and 36th day, respectively. In accordance with this information, the fillets in alltrial groups were placed in coolers at +4 ± 1◦C and were stored there until the end of their shelflives. Muscle samples of 3 g were taken from each group during cold storage in four-day intervalsfor SDS-PAGE analysis and these samples were stored at −80◦C (Operon DFU 446, Korea) untilprotein extraction.

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Extraction of muscle sarcoplasmic (SPP) and MFP fractions

The extracts of fish sarcoplasmic and MFPs in rainbow trout muscle were prepared according to theprocedure described by Chen and Hwang.[33] For the extraction of sarcoplasmic protein fraction,a 3 g muscle was homogenized with three volumes in chilled low ionic strength phosphate buffer(15.6 mM Na2HPO4–3.5 mM KH2PO4, ionic strength = 0.05, pH 7.5) by using a mechanicalhomogenizer (Heildolph, Germany) at 10,000 rpm for 1 min. Heating of the homogenates wasprevented by keeping samples on ice during the process. The homogenates were centrifugedat 10, 000 × g for 15 min (4 ◦C) (Sigma 2-16K, Germany) and supernatants were collected.Pellets were resuspended in the same low ionic strength buffer and re-extracted as describedabove. Supernatants of both consecutive centrifugations were pooled, and together constitutedthe sarcoplasmic protein fraction.

Remaining pellets were again extracted twice in chilled myofibrillar buffer (0.45 M KCl–15.6 mM Na2HPO4–3.5 mM KH2PO4, ionic strength = 0.5, pH 7.5) as described, and the pooledsupernatants obtained after the double centrifugation were collected as MFP fraction. The con-centration of soluble protein in sarcoplasmic protein fraction extracts was estimated by thespectrophotometric method of Lowry et al. [34] using the procedure protein determination with-out protein precipitation and measuring absorbance at 700 nm. The concentration of the solubleprotein in MFP fraction extracts was estimated by Lowry et al. [34] using protein determinationwith the protein precipitation method and measuring the colour developed at 700 nm. In bothprocedures, bovine serum albumin was used as the standard.

Electrophoretic separation of muscle proteins

SDS-PAGE separation of soluble protein fractions in sarcoplasmic protein fraction and MFPfraction extracts was performed according to Laemmli [35] in a Mini Protean II electrophoresischamber (Bio-Rad, Richmond, CA, USA), using 4% polyacrylamide stacking gels and 7.5% sep-arating gels. The analyses were carried out according to the instructions of the manufacturer. Thestandard protein mixture contained myosin (200 kDa), β-galactosidase (116 kDa), phosphorylaseB (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3P-dehydogenase (36 kDa),carbonic anhydrase (29 kDa), trypsinogen (24 kDa) and trypsin inhibitor (20 kDa) (SigmaMarker,S8445, M3913). Sarcoplasmic and MFPs were run in SDS-PAGE at 20–35 mA for 3–4 h on aver-age (Bio-Rad PowerPac 300, USA). After electrophoresis, the gels were stained with coomassiebrillant blue R-250 overnight. Afterwards, the removal of the residual stain on the surface of gelswas done with a destaining solution.

Image analysis

The gels preserved in 7% acetic acid were photographed on a white illuminated desk in a darkroom. The images analysed with the ImageJ program, version 1.3. Molecular weights of proteinbands were calculated and densitometric analysis was performed.[36]

Results and discussion

Myofibrillar muscle proteins

The protein bands determined in the myofibrillar muscle fraction in rainbow trout fillet as aresult of SDS-PAGE and densitometric analysis are given in Figure 1. According to the resultsof SDS-PAGE and densitometric analysis, 31 different myofibrillar muscle protein bands were

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Figure 1. The myofibrillar muscle protein bands determined in rainbow trout fillet as a result of SDS-PAGE anddensitometric analysis: R, raw rainbow trout and M, wide range standard.

determined. The bands determined in all samples at approximately 200 kDa (very dense), 167.8,144.4, 105.4–99.1 and 40.6 kDa (very dense), 39.5 and 36.9 kDa (very dense), 34.5 kDa (dense)are thought to be MHC, M protein, C protein, α-actinin, actinin, β-tropomyosin, troponin T andα-tropomyocine, respectively (Figure 1). Similarly, Michalczyk and Surówka [37] have reported30 muscle proteins in raw rainbow trout samples and bands were assigned to respective proteins:a band of molecular weight 215 kDa corresponded to MHC; 180 kDa to M protein; 148 kDa to Cprotein; 102 and 107 kDa to α-actinin; 42.7 kDa to actin; 39.5 kDa to β-tropomyosin; 37.9 kDa to

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(a) (b) (c)

(d)

Figure 2. SDS PAGE results for the myofibrillar muscle fraction of rainbow trout during storage 4 ± 1◦C. (a) vacuumpackaged, (b) high pressure applied after vacuum packaged, (c) vacuum packaged and wrapped with the chitosan-basededible film, (d) high pressure aplied after vacuum packaged and wrapped with the chitosan-based edible film. M, widerange standard.

troponin T; 35.6 kDa to α-tropomyosin; and the set of bands from 35 to 17.6 kDa correspondedto myosin light chain. Godiksen et al. [38] reported that the myofibrillar muscle proteins inrainbow trout muscle found 31 different bands and MHC band was at 222 kDa. In addition,glyceraldehyde-3P-dehydrogenase has been determined at 36 kDa, a myosin light chain has beendetermined at 21 kDa and triphosphate isomerase has been determined at 22.5 kDa in the samestudy. The MHC determined by Ladrat et al. [39] and Cheret et al. [40] in the miyofibrillar extractat 200 kDa was determined in our study as 200 kDa and it is thought that the band determinedbetween 51.9–48.9 kDa might be desmin which has been determined by other researchers at 49and 53 kDa.[39,41] It is thought that the wide band determined at 40.6 kDa in all samples withslight differences is actin determined by Ladrat et al. [39] and Chéret et al. [40] in the myofibrillarextract at 42 kDa.

The results of the SDS-PAGE analysis carried out to determine the changes in the proteinsof the myofibrillar muscle fraction due to the cold storage of all trial group rainbow trout filletsare given in Figure 2. According to the results of SDS-PAGE and densitometric analysis, thedensity of the MHC observed at 200 kDa in C and CFW group samples from among MFPs has

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decreased significantly due to cold storage (Figure 2). Similarly, it has been put forth by Linand Park [42] that myosin and actin have broken down (that over 70% of myosin breaks downwhen stored at 0◦C for 72 h) as a result of the effect of the cathepsin B and H enzymes in themuscles of Pacific berlam fish stored at low temperatures. The density of the MHC observedin the C and CFW group samples has decreased significantly in high pressure applied groups(HHP and HHP + CFW) and has remained relatively constant during cold storage. Qiu et al. [43]have determined a significant breakdown in the MHC determined in silver carp at 200 kDa as aresult of 5 h incubation at 55◦C and have also determined various small changes as a result ofthe 5 h incubation at 55◦C following the 10 min HHP (200–300 MPa) process. In addition, it hasbeen stated by the authors that 300 MPa is more effective than 200 MPa pressure in preventingproteolysis in the MFP. Similarly, it has been stated by Ashie et al. [44] that the proteolytic activityin fish muscles was affected by the high hydrostatic pressurization, and the degree of modificationwas largely dependent on the levels and the duration of pressurization, as well as on the speciesunder treatment.

In our study, the density of the bands observed at mild density at approximately 31.4 and19.9 kDa has decreased significantly in samples (HHP and HHP + CFW) on which high pressurewas applied and this decrease has continued especially in the HHP + CFW group in relation tothe storage period (Figure 2). This is proof that the high pressure process increases the breakdownand rearrangement of the large peptides in trout muscles. A similar situation has been stated byChéret et al. [40] in a study carried out to determine the changes in the muscle proteins of seabass stored in refrigerator after high pressure application. It has been determined in this studythat decreases in the densities of MHC, the double band at 150 kDa, troponin T at 37 kDa and theprotein bands at 32 and 20 kDa as a result of 300 MPa pressure application to sea bass muscles.The reasons for this change have been given by Chéret et al. [40] as denaturations in MFPs thatoccur following high pressure or the modifications in their structure. Similarly, Hurdato et al. [28]have stated that autolysis decreased due to denaturations that occur as a result of high pressure inMFPs. Significant new protein bands have been determined in the CFW group on the 16th dayof cold storage, in the HHP group on the 20th day of cold storage, in the CFW group during the16th–24th days and in the HHP + CFW group on the 36th day at 30 kDa (Figure 2). We thinkthat this new protein band may be related with the spoilage that occurred during the cold storageof the rainbow trout.

Sarcoplasmic muscle proteins

The proteins obtained in the sarcoplasmic muscle fraction determined according to the gel and thedensitometric analysis result of the gel obtained in fresh rainbow trout fillets via the SDS-PAGEmethod along with their molecular weights (kDa) are given in Figure 3. According to these results,21 different sarcoplasmic muscle protein bands have been determined in the muscles of rainbowtrouts. Similarly, 26 sarcoplasmic muscle proteins have been determined in a study carried out byGodiksen et al. [38] to determine the relationship between rainbow trout texture and muscle proteinprofile.Whereas Michalczyk and Surówka [37] have determined a total of 30 muscle proteins in thetotal extract obtained from freshly minced rainbow trout samples. Ladrat et al. [39] have stated thatthe wide band determined in the sarcoplasmic extract of sea bass muscles between 41 and 39 kDais the band determined by Nakagawa et al. defined to be formed due to the bonding of creatinekinase and aldolase, whereas the band at 36 kDa is glyceraldehyde-3P-dehydrogenase and thatthe bands at 13 and 12 kDa are parvalbumin.[39] Similarly, we think that the wide bands observedin the rainbow trout muscle sarcoplasmic extract at 42.4 and 39.3 kDa are creatin kinase andaldolase, whereas the band observed at 36 kDa is glyceraldehyde-3P-phosphate dehydrogenaseand that the bands at 14 and 12 kDa are parvalbumin. The triosephosphate isomerase determined

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Figure 3. The sarcoplasmic muscle protein bands determined in rainbow trout fillet as a result of SDS PAGE anddensitometric analysis: R, raw rainbow trout and M, wide range standard.

by Godiksen et al. [38] in the sarcoplasmic extract of rainbow trouts 22.5 kDa is thought to be theband observed at 23.3 kDa in our study.

The SDS-PAGE analysis results carried out to determine the changes in the proteins deter-mined in the sarcoplasmic muscle fraction of rainbow trout fillets due to cold storage are givenin Figure 4. The band that was observed in the control group samples especially at 39.3 kDadecreased significantly following the high pressure process (HHP and HHP + CFW). Similarly,the two bands observed at 26.6 and 23.3 kDa at medium and low density, respectively, decreasedsignificantly in the other groups at the start of storage and some disappeared completely duringcold storage (Figure 4). Similarly, partial losses were determined as a result of the HHP (170and 200 mPa) process in the band observed intensively at 29 kDa in the sarcoplasmic proteinfraction control samples during the study carried out by Ortea et al. [30] to determine the effectsof the HHP process on (chilled) farm coho salmon muscle proteins. Phosphoglycerate mutasewas determined as a result of the mass spectrophotometer analysis of this band, in which partiallosses occurred during the HHP process.[31] The protein band determined at 26.6 kDa accordingto our study results is thought to be the band observed in cod muscles at 29 kDa (Figure 4). A new

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(a) (b) (c)

(d)

Figure 4. SDS PAGE results for the sarcoplasmic muscle fraction of rainbow trout during storage 4 ± 1◦C. (a) vacuumpackaged, (b) high pressure applied after vacuum packaged, (c) vacuum packaged and wrapped with the chitosan-basededible film, (d) high pressure applied after vacuum packaged and wrapped with the chitosan-based edible film. M, widerange standard.

sarcoplasmic protein band was determined at 20.7 kDa in the C and CFW group samples at theend of storage and in the HHP and HHP + CFW groups starting from the eighth day of storage atlow density in our study the density relatively increased in parallel with storage time (Figure 4).Chéret et al. [40] have stated an increase in the density of sarcoplasmic protein bands of sea bassmuscles at 21.5, 51 and 97 kDa molecular weights following the high pressure process (0.1, 100and 300 MPa). The bands that were not observed in the control samples at 20.5 and 30.5 kDaappeared during the second day of storage.[40] Similarly, the study carried out by Angsupanichand Ledward [45] to determine the effect of the high pressure process on morina muscles, it wasstated that the sarcoplasmic protein profile showed similarities with that of the fresh sample at200 MPa pressure and that there were density decreases only in some of the bands. However, asthe applied pressure increased, minor decreases were observed in the densities of sarcoplasmicproteins at 127, 118 and 71 kDa molecular weights, minor decreases were observed for those at133 and 21 kDa and significant increases were observed for those at 51 kDa. The authors havestated that the increase at 51 kDa could be due to the decrease at 71 kDa.

Conclusions

It has been determined that there have been slight changes in the major bands of the rainbow troutsarcoplasmic and myofibrillar muscle fractions due to the high pressure process and chitosan-based

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film wrapping. The most important change occurred in the myofibrillar muscle fraction as adecrease in the densities of the bands at 200 kDa (MHC) and 31.4 kDa after high pressure appli-cation. Similarly, the sarcoplasmic muscle fraction of rainbow trout fillet decreased in the densitiesof the bands at 39.3, 26.6 and 23.3 kDa after high pressure application. In addition, it is thought thatthe bands that occurred in the myofibrillar fraction at 30 kDa for all trial groups and at 20.7 kDain the sarcoplasmic fraction may be related with the spoiling of rainbow trout during cold storage.Hence, it is important that mass spectrophotmetric analyses of the protein bands that have beendetected are carried out along with further studies to define them.

Acknowledgements

This study is a summary of the Master’s Thesis of Sinem Sipahioglu. The authors would like to thank Scientific StudiesManagement Unit Council, Süleyman Demirel University (Isparta-Turkey) for their financial support with 2107-YL-10numbered project.

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