Stress Relaxation Behaviour and Structural Changes of Muscle Tissues From Gilthead Seabream Following High Pressure Treatment

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Stress relaxation behaviour and structural changes of muscle tissues fromGilthead Sea Bream (Sparus aurata L.) following high pressure treatment

Marco Campus *, Maria Filippa Addis, Roberto Cappuccinelli, Maria Cristina Porcu, Luca Pretti,Vittorio Tedde, Nicola Secchi, Giuseppe Stara, Tonina RoggioPorto Conte Ricerche Srl, Tramariglio, Alghero (Sassari), Italy

a r t i c l e i n f o

Article history:Received 25 May 2009Received in revised form 13 July 2009Accepted 16 July 2009Available online 21 July 2009

Keywords:High pressure treatmentSea breamStress relaxation testWater holding capacityImmunoblottingDesmin

a b s t r a c t

Sea Bream muscle tissue was subjected to different high pressure treatments, and rheological changeswere monitored during storage by means of the stress relaxation test. The best t was obtained by appli-cation of the three term Maxwell exponential model, followed by the Nussinovich model. The applicationof 300 and 400 MPa pressures appeared to enable preservation of elasticity and stiffness of sh muscleduring storage, compared to untreated samples. On the contrary, samples treated at 200 MPa underwenta decrease in elasticity during storage. The water holding capacity of dorsal muscle was also assessed, andit was found to decrease with increasing pressures. Immunoblot studies performed on the main struc-tural proteins revealed that a pronounced time-dependent degradation of desmin, observed in untreatedsamples, could be prevented by treatment at 400 MPa. Taken together, our results strongly suggest thathigh pressure treatments inactivate degrading enzymes acting on proteins that are related to tissueintegrity preservation, texture quality, and water holding capacity.

2009 Elsevier Ltd. All rights reserved.

1. Introduction

Quality of sh as a food has been dened as a combination ofsuch characteristics as wholesomeness, integrity, and freshness(Martin, 1988). Freshness is one of the most important factors forevaluating sh quality since it can be stated directly throughappearance, texture and taste.

High pressure processing is a preservation technology which al-lows decontamination of foods at low or moderate temperature,with the major advantage of extending shelf life through microbialinactivation with minimum loss of quality (Cheftel and Culioli,1997). Studies on the application of this technology to sh havebeen focused on the effects of treatments on muscle structural pro-teins, on oxidative stability (Chret et al., 2006; Sequeira-Munozet al., 2006), inactivation of proteolytic enzymes related to quality(Ashie and Simpson, 1996), changes in physicochemical parame-ters (Lakshmanan et al., 2007; Chret et al., 2005), and high pres-sure assisted thawing (Rouill et al., 2002; Schubring et al.,2003). Texture Prole Analysis (TPA) (Angsupanich and Ledward,1998; Chret et al., 2005) and puncture test (Ashie and Simpson,1996) have been used to evaluate the textural changes in sh fol-lowing high pressure treatment. Nevertheless, both methods pres-ent drawbacks, since they are destructive and time consuming.Therefore, non-destructive methods are needed in order to evalu-

ate changes introduced in sh muscle by high pressure treatments.In this respect, the stress relaxation test can be used for qualityassurance of high pressure treated sh, being fast, robust, simple,and non-destructive (Herrero et al., 2004).

The stress relaxation test (or step strain test) is one of the mostimportant evaluation tools used for determining viscoelastic prop-erties of materials. In a stress relaxation test, the sample is given aninstantaneous strain and the stress required to maintain the defor-mation is observed as a function of time. A generalised Maxwellmodel is frequently used to interpret stress relaxation data(Herrero et al., 2004; Jain et al., 2007; Ma et al., 1996). The modelconsists of n Maxwell elements and a free spring in parallel, eachelement consisting in 1 spring and 1 damper in series. (Steffe,1992). The generalised Maxwell model can be written as follows:

rt Xn

i1Ciet=si re 1

where r is the stress (N) at a given time, t (seconds), Ci are stressrelaxation constants (N), re is the equilibrium stress (N), si (sec-onds) are the relaxation times of the Maxwell elements.

A simplied version of the Maxwell model is the model of Nus-sinovich (Nussinovich et al., 1989). In this model, the relaxationtimes of Maxwell elements (si) are given as constants. A three ele-ments Nussinovich model can be represented as follows:

rt A0A1et=100 A2et=10 A3et=1 2

0260-8774/$ - see front matter 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.jfoodeng.2009.07.013

* Corresponding author. Tel.: +39 079 998 400; fax: +39 079 998 567.E-mail address: [email protected] (M. Campus).

Journal of Food Engineering 96 (2010) 192198

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where r is the stress (N) at a given time, t (seconds), A0, A1, A2, A3are constants (N). The model allows to obtain relaxation data inshort terms, avoiding the reaching of an equilibrium stress to endthe test.

A third model (Peleg, 1980) is used for linear tting of the stressrelaxation data:

r0tr0 r k1 k2t 3

where r0 is the initial stress (N), r (N) is the decaying stress at a gi-ven time, t (seconds), K1, and K2 are dimensionless constants. 1/K1gives the initial relaxation rate, while 1/K2 represents the propor-tion of relaxed initial force and gives a measure of the solidityof the material (Gamero et al., 1993). 1 1/K2 is the magnitude ofthe asymptotic residual modulus and gives a measure of the stressthat remains unrelaxed (Nussinovich et al., 1989).

Textural changes are related to evolution of muscle constitu-ents, primarily proteins, with the endogenous calpain systemplaying a major role in proteolysis of muscle proteins underpost-mortem conditions (Lonergan et al., 2001; Maddock et al.,2005). High pressure treatments may affect muscle constituents,primarily large molecules as proteins. Covalent bonds in proteinsare not affected by high pressure while ionic and hydrogen bondsand then tertiary structure can change noticeably. Particularly,modications in enzymes structure and compartmentation maybe of chief importance because of their role in post-mortem pro-teolysis. Also, the ability of muscle to retain water is strictly re-lated to the post-mortem events such as pH decline, proteolysisand protein oxidation (Huff-Lonergan et al., 1996), and it is veryimportant in sh both from a quality and, consequently, commer-cial point of view. Aim of the present work was to evaluate thechanges induced by high pressure treatments in sea bream mus-cle, by relating modications in texture and water holding capac-ity to changes in myobrillar and cytoskeletal proteins. Therheological properties of high pressure treated farmed headed/gutted Gilthead Sea Bream during chilled storage were assessedby stress relaxation test. The data generated were tted withthree different models, in order to allow the best possible inter-pretation of the relaxation curves obtained. Water Holding Capac-ity was evaluated by centrifugal methods. The status of structuralproteins was investigated by means of electrophoresis and wes-tern immunoblotting.

2. Materials and methods

2.1. Samples and high pressure treatment

Sixty farmed Sea Breams were obtained from an aquacultureplant located in South-western Sardinia (Italy). After capture, sh(18 months of age, average length 26 cm, average weight 262.3 g)were kept in melting ice, transported to the laboratories, andstored in ice for 24 h. Then, sh were manually headed, gutted,washed in cold water (+3 C) and vacuum sealed individually inplastic bags (co-extruded PA/PE-20/70; O2 transmission rate:3050 cm3/m224 h-atm; water vapour transmission rate: 2.6 g/m2-atm; CO2 transmission rate 150 cm3/m2-24 h-atm; N2 trans-mission rate 10 cm3/m2-24 h-atm, at 23 C and 50% RH). Sampleswere divided in four groups. One group (not pressurised) waskept as a control in chilled conditions (+3 C), while the remain-ing were subsequently pressurised. Prior to high pressure treat-ments, packed samples were kept on an ice bath (02 C) inorder to prevent adverse effects induced by temperature. Treat-ments were carried out with a Pilot scale HPP410100 IsostaticPress (Flow Autoclave Systems Inc, Columbus, Ohio, USA). Internaldiameter of the vessel was 100 mm, inside length was 254 mm,

internal volume was 2L, pressure transmitting medium was glycol(Houghton-Safe 620 TY, Houghton, Toronto, Ontario)/distilledwater solution 50/50-v/v. Two samples were pressurised at onetime. The pressures applied were 200, 300, and 400 MPa with set-tings of 20 C for 10 min for the thermostatic bath. The pressurecome-up time was 5 MPa/s, and the pressure release time was10 s. Control and pressurised samples were stored in chilled con-ditions (+3 C) in the dark and analysed at 0, 7, and 13 d of stor-age at 3 C.

2.2. Microbial analysis

Samples were analysed for Total Aerobic Count and psychro-trophic bacteria, at 0d and after 13d. Samples were taken asepti-cally (5 g), sampling the skin and the underlying dorsal muscleportion, from the anterior-dorsal part of each sh, then stomachedfor 2 min in 0.1% sterile peptone water. Ten fold dilutions of thesamples were made using 0.1% sterile peptone water and 0.1 mlaliquots of the appropriate dilutions were plated on Plate CountAgar (Merck, Danstadt, Germany). Plates were incubated at 30 Cfor 72 h, for aerobic total count, and 4 C for 10 days for psychro-trophic bacteria.

2.3. Water holding capacity

The ability of muscle to retain water was expressed as waterholding capacity and determined according to Skipnes et al. (2007).

2.4. Stress relaxation test

Stress relaxation tests were performed with a TA.XT2i SMS Sta-ble Microsystems Texture Analyzer. (Stable Microsystems Ltd., Sur-rey, England) using a 5 kg load cell. Samples were compressed by5% with a 10 mm cylinder probe at a crosshead speed of 10 mm/s. The compression was kept constant for 100 s, allowing the stressto reach equilibrium. Measurements were taken from between thedorsal n and the lateral line, parallel to the contact surface of theprobe, taking three measures from each sh (Fig. 1). For each stor-age time (0, 7, 13 days), 5 sh for each treatment condition wereanalysed (control, 200, 300, 400), making a total of 20 sh per timeof analysis. Texture Expert Exceed Software was used to acquirethe data output.

Fig. 1. Diagram showing the experimental setup and compression areas measuredin each sh.

M. Campus et al. / Journal of Food Engineering 96 (2010) 192198 193


2.5. Protein extracts

A fragment of dorsal muscle was weighed, minced with a sterilescalpel and placed in an Eppendorf safe-lock tube (Eppendorf,Hamburg, Germany). The tissue was then immersed at a 5% w/v ra-tio in 20 mM Tris HCl pH 8.8, 2% CHAPS, 8 M urea (lysis solution),and subjected to three cycles of 3 min at 30 cycles/s in a TissueLy-ser mechanical homogenizer (Qiagen, Hilden, Germany), withfreezing at 80 C after each passage. Extracts were then clariedfor 15 min at 12,000g at 4 C, quantied by the BCA method(Pierce, Rockford, MD), and stored at -80 C until needed.

2.6. SDS-PAGE and western immunoblotting

Protein extracts were subjected to SDS-PAGE according to Lae-mmli (1970). Briey, 20 lg or 5 lg of proteins for Coomassie stain-ing and immunoblotting, respectively, were re-suspended inLaemmli buffer and then loaded in a mini format polyacrilamidegel with the miniProtean Tetra Cell (BioRad, Hercules, CA). Afterthe electrophoretic separation, proteins were stained with the Coo-massie Blue stain and digitalised with an ImageScanner (GEHealthcare, Little Chalfont, UK). For western immunoblots, gelswere subjected to western immunoblotting with the mini-Trans-Blot apparatus (BioRad, Hercules, CA). Once transferred to nitrocel-lulose, proteins were blocked with Phosphate Buffered Saline(PBS), 0.05% Tween 20 (PBS-T), plus 5% skim milk for 1 h to over-night, and then incubated from 2 h to overnight with the primaryantibodies. Rabbit polyclonal antibodies (SigmaAldrich, St. Louis,MO) against actin (1:50,000), tropomyosin (1:5000), and desmin(1:5000) were used. Membranes were then washed ve times withPBS-T and incubated with HRP-conjugated anti-rabbit antibodies.After ve washes with PBS-T, reactivity was visualised with achemiluminescent peroxidase substrate (SigmaAldrich, St. Louis,MO). Blot images were digitalised with a VersaDoc 4000MP andprocessed with QuantityOne (Bio-Rad Hercules, CA).

2.7. Statistical analysis

Regression analysis was performed on stress relaxation datawith Table curve 2D v.5.01 (Systat Software Inc., San Jos, Califor-nia, USA), using the Levemberg-Marquardt regression analysismethod. Percentage maximum relative difference (MRD%) and per-cent explained variation (R2) were used to determine which modeltted best to the relaxation curves. The effects of high pressuretreatment, storage time, and their interaction, were tested bytwo-factor analysis of variance (ANOVA) using the statistic soft-ware Statgraphics plus v 5.1 (StatPoint Technologies Inc., Warren-ton, VA). Signicant effects were compared using Fishers leastsignicant difference (LSD) test at P = 0.05.

3. Results and discussion

3.1. Microbial analysis

The microora of temperate saltwater sh is dominated by psy-chrotrophic gram-negative bacteria belonging to various genera,although gram-positive bacteria can also be found in various por-tions (Gram and Huss, 1996). Microorganisms in sh are presenton the skin and in the digestive system, and can contaminate mus-cle after the sh dies. Starting Total Aerobic Count on untreatedsamples was 3.82 103 cfu/g, which rose to 2.1 105 cfu/g after13 days of storage (Fig. 2). Treatments at 200, 300 and 400 MPafor 10 min resulted in a reduction in aerobic total count by 0.27,2, and 2.2 log10, maintaining microbial counts under 7 103,2 103, and 3.2 102 cfu/g, respectively, after 13 days of storage.Psychrophiles in control samples at 0 days were 9.5 102 cfu/g,and were reduced by 0.8log10 after treatment at 200 MPa, whilein samples treated at 300 and 400 MPa counts were less then1 cfu/g. At the end of storage, psychrotrophic bacteria countsreached 1.7 105 cfu/g in untreated samples, while in samplestreated at 200, 300, and 400 MPa counts were of 9.5 103,1.4 103 and 1 102 cfu/g, respectively. Microorganisms inactiva-tion by high pressure has been widely reported in food and modelsystems (Cheftel, 1995). Studies performed on other species of shindicate a consistent decrease in microbial loads after treatmentsabove 300 MPa, with a shelf life extension of at least 1 week(Chret et al., 2005).

3.2. Water holding capacity

The water holding capacity was found to decrease signicantly(P < 0.05) with increasing pressures, while it did not vary signi-cantly with storage time (Fig. 3). Very few data can be found in lit-erature on the effect of high pressures on water holding capacity ofsh and, in general, on muscle foods. Lakshmanan et al. (2007), re-corded a minor increase on WHC in fresh salmon subjected to apressure of 150 MPa for 10 min. This study was carried out usingboth centrifugal methods and nuclear magnetic resonance spec-troscopy (1H NMR). Hedges and Goodband (2003) used HP to in-crease the water holding capacity of fresh cod at 200 MPa, andtheir results are in contrast with ours. In general, water holdingcapacity is related to protein denaturation, and evidences supportthe idea that proteolysis of cytoskeletal proteins, such as the inter-mediate lament protein desmin, may be related to uid retentionby myobrils.

Changes in the intracellular architecture of brils can be in-duced by high pressure and can inuence the ability of muscle cellsto retain water. As rigor progresses, the space for water to be heldin the myobrils is reduced, and uid can be forced into the extra-

Fig. 2. Total aerobic count and psychrotrophic bacteria count of control and pressurised samples at and after 13 days of chilled storage. Results are shown as the averagevalue (n = 5).

194 M. Campus et al. / Journal of Food Engineering 96 (2010) 192198


myobrillar spaces where it is more easily lost as drip as a conse-quence of lateral shrinkage of the myobrils occurring during rigor,which can be transmitted to the entire cell if proteins that linkmyobrils together and myobrils to the cell membrane (such asdesmin) are not degraded (Kristensen and Purslow, 2001; Melodyet al., 2004). These proteins have been shown to be degraded earlypost-mortem in some muscles. Degradation of these proteins atsuch an early time post-mortem would certainly allow water to re-main in the cell for a longer period of time. Proteins other than des-min may be involved in such mechanism. High pressure treatmentmay affect, of course, the degradation of key proteins involved inthe mechanism. The molecular aspects will be elucidated hereinaf-ter in this article.

3.3. Stress relaxation test

Curves obtained were tted considering the relaxation part(Fig. 4), taking not into account the rst part of the curve, namelyfrom the start of the test to the reaching of the maximum force.Generalised Maxwell, Nussinovich and Peleg models were usedto describe the viscoelastic behaviour of samples. From prelimin-ary analyses we found that three terms were optimal for the Max-well model, and also that the Nussinovich and the Peleg models asrepresented by Eqs. (2) and (3) were adequate to describe theexperimental data. The Maxwell model showed the best(R2 > 0.99 and maximum relative difference < 7) representation ofexperimental data.

The stress relaxation constants, C1 and s1, are often used foranalysing results, since the rst of the three terms of Maxwellmodel has a major contribution to the total modulus (Baragale

et al., 1994; Hassan et al., 2005). Regression analysis results areshown in Table 1. C1 values of untreated samples and of samplestreated at 200 MPa were not signicantly different just after treat-ments, while, in contrast, they were signicantly lower in samplestreated at 300 and 400 MPa. Notably, after 7 days of storage, C1 val-ues of control and of samples treated at 200 MPa decreased signif-icantly, while in samples treated at 300 and 400 the magnitude ofthis parameter, although decreasing, is kept signicantly higherduring storage in chilled conditions. Taking the values of C1 of freshcontrol samples at 0 days as reference, control and samples treatedat 200 MPa retained 1.35% and 3.2% of this value after 13 days,while samples treated at 300 and 400 MPa maintained 16.9% and20.9% of this value, respectively. The decay forces of the Maxwellmodel (Ci) represent the elastic components, and thus provide ameasure of the elasticity of the material (Khazaei and Mann,2005). Values of s1 are affected by treatments as well. In fact, s1values decrease drastically in untreated samples after 13 days ofchilled storage, and this is consistent with changes found in ice-stored cod (Herrero et al., 2004), while in high pressure treatedsamples the values of the parameter are kept more constant, espe-cially in samples treated at 300 and 400 MPa. After 13 days ofchilled storage, samples treated at 300 and 400 MPa showed highervalues of s1, compared to samples treated at 200 MPa and un-treated samples. Higher values of s1 show that the muscle is rmerand more elastic (Herrero et al., 2004).

Immediately after treatment (T0) at 200 MPa, values of re,namely the residual modulus or the amount of initial stress that re-mained unrelaxed, are signicantly lower. During storage, themagnitude of this parameter decreases, but it is kept higher insamples treated at 300 and 400 MPa, after 7 and 13 days in chilledconditions. The magnitude of re can be taken as a measure of thestiffness of the material. The results obtained by analysing thecoefcients of the Nussinovich model are basically in accordancewith those obtained with the generalised Maxwell model (Table1). The values for the residual modulus, A0, are signicantly higherin samples treated at 300 and 400 MPa after 13 days of storage.Moreover, the values of the rst term A1, after 13 days of storage,are signicantly higher in samples treated at 300 and 400 MPa,and the effect increases with increasing pressures.

The constants of the Peleg model (Table 1) are less reliable forthe interpretation of stress relaxation data, as showed by the highvalues obtained for MRD%. This is because the model showed a lackof t in the initial portion of the curve, as stated by other authors(Gamero et al., 1993). Nevertheless, after 13 days of storage, we ob-served that samples treated at 400 MPa showed the lower values ofparameter 1/K2, the proportion of relaxed initial force, and highervalues of 1 1/K2, that is, the magnitude of the asymptotic residualmodulus, similar to the constant A0 of Nussinovich model, and ameasure of the stiffness and strength of the material.

Other authors reported textural changes in sh subjected tohigh pressure treatments. Ashie and Simpson (1996) performed aPuncture test and reported a decrease in strength values whenBlue sh is subjected to pressure above 200 MPa beyond 10 min,and also in sh treated at pressure levels above 300 MPa. Also,the authors reported a decrease of elasticity with increasing pres-sures just after the treatment.

Notably, the values of the rst term of the Maxwell model de-creased with increasing pressure just after the treatment, althoughduring storage samples treated at 300 and 400 MPa showed highervalues for C1, while in untreated samples and in samples treated at200 MPa there was a dramatic decrease in this parameter.

In this study, changes in the coefcients obtained throughregression analysis of stress relaxation data showed that samplestreated at 300 and 400 MPa retain more elasticity and solidityduring storage, compared to untreated control and 200 MPa trea-ted samples. This could be related to changes in muscle constitu-

Fig. 3. Changes in water holding capacity during storage in untreated (control) andtreated samples. Results are shown as the average value with the standarddeviation (n = 5).

Fig. 4. Example of relaxation curve obtained through the stress relaxation test.



ents, as showed hereinafter by SDS-PAGE and westernimmunoblotting.

3.4. SDS-PAGE and Immunoblotting

In order to investigate the status of cytoskeletal proteins, SDS-PAGE and immunoblotting were performed. The SDS-PAGE proles(Fig. 5A) obtained did not show evident changes. Except for a slightalteration of few bands, mostly in the 100 kDa and 80 kDa area,there were no substantial differences in intensity of protein bands

as a consequence of treatments and of storage. In general, the ma-jor documented changes during post-mortem storage are weaken-ing of the Z-line, degradation of titin, nebulin, dystrophin, desmin,as well as release of a-actinin from the Z-line, and breakdown ofcollagen junctions between myotomes (Papa et al., 1997;Delbarre-Ladrat et al., 2006).

Several years ago, other authors (Cheftel and Culioli, 1997) re-viewed solubilisation of sh myobrillar proteins in the range be-tween 150 and 300 MPa, and attributed the appearance of intensebands to depolymerisation of myobrillar proteins. Similar results

Table 1Constants of the generalised Maxwell model, Nussinovich model, and Peleg model for controls and samples treated at 200, 300, and 400 MPa for 10 min at 20 C. The mean of 15measures (3 for each sh on 5 sh) is reported.

Sample Days re (N) C1 (N) C2 (N) C3 (N) s1 (s) s2 (s) s3 (s) R2 MRD (%)

Generalised Maxwell modelControl 0 0.0700a 0.0736a 0.0347a 0.0311 2.6413 6.4240 2.5618 0.9990 3.0620Control 7 0.0110a 0.0085a 0.0002a 0.0079 2.7753 1.5099 2.8330 0.9860 6.544Control 13 0.013ab 0.001ab 0.006ab 0.010 0.0670a 2.362 28.213 0.996 3.9198

200 MPa 0 0.0493b 0.0634ab 0.0377a 0.0101 2.8139 2.3636 4.3936 0.9987 3.6447200 MPa 7 0.0140ab 0.0052a 0.0016a 0.0022 2.1600 2.8364 2.8452 0.9944 6.3351200 MPa 13 0.0087a 0.0024a 0.0057a 0.0036 1.9086b 3.3108 0.8917 0.9904 6.0321

300 MPa 0 0.0730a 0.0224b 0.0329a 0.0510 2.9676 2.9662 2.8037 0.9968 3.0492300 MPa 7 0.0247b 0.0173b 0.0090ab 0.0099 2.7503 34.1914 34.2135 0.9911 5.2801300 MPa 13 0.0202bc 0.0125c 0.0052b 0.0067 2.5067c 36.5174 36.4858 0.9946 4.1452

400 MPa 0 0.0809a 0.0515b 0.0296b 0.0306 2.5758 1.1959 7.0410 0.9959 4.2030400 MPa 7 0.0500c 0.0236b 0.0192b 0.0197 2.8489 27.8991 27.8991 0.9954 2.7171400 MPa 13 0.0240c 0.0154c 0.0085b 0.0074 2.8237c 61.4732 61.5668 0.9984 1.3399

P HPP 0.0000 0.0104 0.0105 0.0809 0.0489 0.1476 0.2653P storage 0.0000 0.0557 0.0494 0.02437 0.0664 0.5819 0.6759P HPPx st. 0.0423 0.0102 0.0103 0.0236 0.1964 0.3151 0.1773

Nussinovich modelA0 A1 A2 A3 R

2 MRD (%)

Control 0 0.0490b 0.0719 0.0653 0.0247 0.9974 5.1970Control 7 0.0092a 0.0062a 0.0085 0.0041a 0.9810 7.6336Control 13 0.0100a 0.0080a 0.0076 0.0047a 0.9934 3.6718

200 MPa 0 0.0292a 0.0515 0.0549 0.0258 0.9975 4.5644200 MPa 7 0.0128a 0.0030a 0.0035 0.0039a 0.9419 7.1589200 MPa 13 0.0066a 0.0056a 0.0039 0.0043a 0.9911 4.4977

300 MPa 0 0.0566b 0.0446 0.0511 0.0287 0.9941 5.7927300 MPa 7 0.0179a 0.0184b 0.0144 0.0105b 0.9895 8.5479300 MPa 13 0.0163b 0.0115b 0.0089 0.0086b 0.9956 3.9749

400 MPa 0 0.0609b 0.0506 0.0517 0.0307 0.9920 6,9273400 MPa 7 0.0352b 0.0412c 0.0150 0.0158c 0.9952 2,7504400 MPa 13 0.0214b 0.0130b 0.0090 0.0091b 0.9979 1,6388

P HPP 0.0008 0.0788 0.52 0.0004P storage 0.0000 0.0000 0.60 0.0000P HPPx st. 0.3653 0.0069 0.4317 0.83

Peleg modelK1 K2 1/K1 1/K2 1 1/K2 R2 MRD(%)

Control 0 21.25a 1.38a 0.0471 0.7246 0.2754 0.9956 66.99Control 7 14.52ab 1.6a 0.0689 0.6250 0.3750 0.9931 60.66Control 13 26.87 1.74 0.0372 0.5747 0.4253 0.9949 20.88

200 MPa 0 14.25ab 1.31a 0.0702 0.7634 0.2366 0.9984 58.72200 MPa 7 10.67a 2.47b 0.0937 0.4049 0.5951 0.9921 61.8200 MPa 13 25.69 1.82 0.0389 0.5495 0.4505 0.9917 41.09

300 MPa 0 15.17b 1.58b 0.0659 0.6329 0.3671 0.9983 51.72300 MPa 7 20.75b 1.53a 0.0482 0.6536 0.3464 0.9903 50.03300 MPa 13 25.11 1.84 0.0398 0.5435 0.4565 0.9932 38.69

400 MPa 0 17.63a 1.57b 0.0567 0.6369 0.3631 0.9974 61.36400 MPa 7 32.17c 1.61a 0.0311 0.6211 0.3789 0.9801 67.06400 MPa 13 36.10 1.99 0.0277 0.5025 0.4975 0.9908 43.32

P HPP 0.0270 0.0169P storage 0.5496 0.0000P HPPx st. 0.0343 0.0000

a,b,cMeans with different letters at the same day are signicantly different (p < 0.05) among high pressure treatments.PHPP: P value of the effect of high pressure treatment; Pstorage: P value of the effect of storage; PHPPxstorage: P value of interaction between high pressure treatment and chilledstorage.



were obtained by Sequeira-Munoz et al. (2006) on carp llets, andthe authors interpreted the more intense banding at low molecularweights (around 24 KDa) as degradation products of myobrillarproteins. However, Chret et al. (2006) in a study on sea bass mus-cle, reported that actin, myosin and troponin T did not show evi-dent signs of degradation of as a consequence of high pressuretreatment.

More sensitive techniques based on SDS-PAGE can provide bet-ter information on the presence of specic proteins and their deg-radation as a consequence of treatments. Consequently, animmunoblotting study was carried out on key cytoskeletal proteinssuch as actin, tropomyosin, and desmin. Actin and tropomyosin didnot show evident alterations upon short and medium-term storagein refrigerated conditions or following different pressure treat-ments (Fig. 5B and C). The antibody-reactive bands did not revealpresence of degradation products, according to the SDS-PAGEprole.

Immunoblot analysis of desmin, on the other hand, indicatedthe occurrence of degradation during storage in untreated samples(Fig. 5D). In fact, the reactive band of intact desmin was visible onlyin the sample at 0 days of refrigerated storage, and disappearedcompletely at 7 days in untreated samples; upon treatment at200 MPa, desmin could be detected at 0 and 7 days, but not at13 days. On the contrary, at 300 and 400 MPa the protein appearedto remain intact until the end of storage (13 days), with more in-tense bandings at 400 MPa compared to 300 MPa. As shown bythe stress relaxation test, treatments at 300 and 400 MPa are morepreservative of muscle elasticity. In light of the immunoblot re-sults, such observation could in part be explained by the higher de-gree of desmin integrity at the higher pressure treatments.Nanomechanical studies (Kiss et al., 2006) indicated that desminplays an important role in integrity and elasticity of muscle cells.Also, a decrease of WHC was observed with increasing pressures.A decrease of water holding capacity has been positively correlatedwith conservation of desmin laments in post-mortem muscle(Zhang et al., 2006).

Desmin is a major component of intermediate laments, and islocated primarily on the periphery of disk Z. It is reported that des-min is cleaved by calpains, m and l calpain, present in the sarco-

plasm, and also by the lysosomal protease cathepsin B, asassessed through experiments carried out in vitro (Baron et al.,2004). Calpains are active at early stages, while the action ofcathepsin B is related to the degree of post-mortem lysosomalmembrane breakdown, which occurs due to permeabilisation ofthe lysosomal membrane related to depletion of ATP reservesand malfunctioning of ionic pumps (Sentandreu et al., 2002).Ohmori et al. (1992) indicated that application of high pressuresto fresh meat induces release of cathepsins from lysosomes, pro-ducing an enhancement of proteolytic activity. The magnitude ofthis increase is the balance of two contrasting phenomena: the re-lease of cathepsins from the lysosomes and the inactivation of theenzymes by pressure. On the other hand, calpains from sea basssubjected to high pressure treatment are readily inactivated at300 MPa, probably due to structural modication and dissociationof calpain subunits (Chret et al., 2006). Desmin is a known calpainsubstrate (Huff-Lonergan et al., 1996). Calpain inactivation by highpressures might partially explain the differences in desmin degra-dation seen between samples which, in turn, may inuence the dif-ferences in drip loss and WHC. The preservation of desminintegrity in samples treated at 300 and 400 MPa could be trackeddown to denaturation and inactivation of calpains leading to block-ade of proteolytic processes acting on desmin, which could allowthe maintenance of elasticity, as assessed by stress relaxation tests,and leads to a decrease in water holding capacity. Similar resultswere reported for enhanced pork loins (Davis et al., 2004) wherereduced degradation of desmin was associated with increasedpurge loss, i.e. to a decrease in Water Holding capacity.

4. Conclusions

The stress relaxation test can be used successfully for assessingrheological changes in high pressure treated Sea Bream muscle tis-sue. High pressures showed a positive effect on tissue texturemaintenance: in fact, treatments at 300 and 400 MPa allowed thepartial preservation of elasticity and stiffness of samples duringstorage. However, Water Holding Capacity was negatively affectedby high pressure, showing a decrease with increasing pressures. By

Fig. 5. SDS-PAGE and immunoblotting of dorsal muscle proteins. A. Coomassie blue staining of whole muscle extracts. B. Immunoblot results for actin. C. Immunoblot resultsfor tropomyosin. D. Immunoblot results for desmin. Lane 1, Markers; Lanes 2, 3, 4, Untreated samples after 0, 7, 13 days of storage; Lanes 5, 6, 7, samples treated at 200 MPaafter 0, 7, 13 days of storage; Lane 8, Markers; Lanes 9, 10, 11, samples treated at 300 MPa after 0, 7, 13 days of storage; Lanes 12, 13, 14, samples treated at 400 MPa after 0, 7,13 days of storage.



application of SDS-PAGE and immunoblotting, it was found thattreatment at 400 MPa allowed preservation of the cytoskeletal pro-tein desmin integrity during 13 days of chilled storage, probablydue to denaturation and loss of activity of desmin-degrading en-zymes. Preservation of desmin might play a key role in the effectexerted by the high pressure treatments, by contributing to main-tenance of tissue elasticity and stiffness during storage, althoughreducing the water holding capacity.

Acknowledgement

This work was supported by Sardegna Ricerche.

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Stress Relaxation Behaviour and Structural Changes of Muscle Tissues From Gilthead Seabream Following High Pressure Treatment