The significance of cooling rate on water dynamics in porcinemuscle from heterozygote carriers and non-carriers of the

halothane gene—a low-field NMR relaxation study

Hanne Christine Bertrama,b, Anders Hans Karlssona, Henrik Jørgen Andersena,*aDepartment of Animal Product Quality, Danish Institute of Agricultural Sciences, Research Centre Foulum, PO Box 50, DK-8830 Tjele, DenmarkbCentre for Advanced Food Studies, Department of Dairy and Food Science, Food Technology, The Royal Veterinary and Agricultural University,

Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark

Received 6 December 2002; received in revised form 31 January 2003; accepted 31 January 2003

Abstract

The post mortem changes in the chemical/physical state distribution of water were followed in pig muscle (M. longissimus dorsi)from heterozygote (n=12) and non-carriers (n=12) of the halothane gene exposed to two different cooling profiles using con-

tinuous low-field NMR relaxation measurements. T2 relaxation data were analyzed using distributed exponential fitting analysis.Independent of genotype post mortem changes were observed in the two water populations characterizing water within the myofi-brillar space (T21) and the extra-myofibrillar space (T22), respectively, as a function of chilling regime. The effect was most pro-

nounced in samples from heterozygote carriers of the halothane gene. The obtained results strongly suggest that improved water-holding capacity of muscles upon fast chilling can be ascribed to a reduced accumulation of extra-myofibrillar water in the meatpost mortem, and it is hypothesized that differences in the accumulation of extra-myofibrillar water post mortem can be ascribed

largely to the time at which disruption of cell membrane integrity takes place.# 2003 Elsevier Ltd. All rights reserved.

Keywords: NMR T2 relaxation; Water-holding capacity; Water distribution; Chilling; Membrane integrity; Pork

1. Introduction

During the post mortem period, several physical andbiochemical processes are taking place which character-ize the conversion of muscles to meat. These processesare generally believed to affect the distribution of waterin the muscle, presumably being decisive for the WHCof the meat (Offer & Cousins, 1992; Offer & Knight,1988). However, the chemical/physical state and dis-tribution of water during the post mortem period, andthe precise mechanisms determining the WHC of meat,are far from understood at present. Accordingly, infor-mation about chemical/physical states and distributionof water during the post mortem period is essential for afurther understanding of the main mechanisms deter-mining the WHC of the meat.

In the late 1960s and the early 1970s, it was demon-strated that NMR relaxation provides informationabout the state of water in muscle tissue (Cope, 1969;Finch, Harmon, & Muller, 1971; Hazlewood, Nichols,& Chamberlain, 1969). Subsequently, low-field NMR(LF-NMR) relaxation measurements on meat have beenshown to be able to predict the WHC of the meat (Ber-tram, Andersen, & Karlsson, 2001; Brøndum et al.,2000; Brown et al., 2000; Tornberg, Andersson, Gor-ansson, & von Seth, 1993). In addition, the potent non-destructive and non-invasive nature of NMR relaxationmeasurement makes it suitable for studies on the postmortem changes in chemical/physical state and dis-tribution of water in muscles.

Three decades ago tunnel chilling was introduced asan alternative to the conventional batch chilling due toits capability to reduce the incidences of porcine meatwith unacceptable WHC, i.e. PSE (pale soft exudative)meat (Taylor, 1971), often associated with pork fromcarriers of the so-called halothane gene (Barton-Gade,

0309-1740/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/S0309-1740(03)00038-X

Meat Science 65 (2003) 1281–1291

www.elsevier.com/locate/meatsci

* Corresponding author. Tel.: +45-89-99-12-41; fax: +45-89-99-

15-64.

E-mail address: henrik.andersen@agrsci.dk (H.J. Andersen).

1984; Cassens, 2000). It has been recognized that thehalothane gene is associated with abnormalities in theCa2+ releasing channels of sarcoplasmatic reticulum,the so-called the ryanodine receptor, which leads todeficiencies in the Ca2+ regulation of the cell, andaccordingly lack of regulatory control of muscle cellvolume (Fujii et al., 1991; Gallant, Godt, & Gronert,1979; Louis, Zualkernan, Roghair, & Mickelson,1992).

The aim of the present study was for the first time tocharacterize the dynamics of water in porcine m. long-issimus dorsi from heterozygote carriers of the halo-thane gene, known to give rise to inferior WHC ofpork, and non-carriers, during the post mortem con-version of muscle to meat as affected by two coolingprofiles using continuous low-field NMR relaxationmeasurements.

2. Materials and methods

2.1. Animals and sampling

Thirty pigs were included in the study. Twenty-four ofthese pigs were crossbreeds of Duroc/Pietrain boarscross-bred with Landrace/Yorkshire sows, and of these,12 animals were heterozygote for the halothane gene(HAL-Nn), while the remaining 12 of these animalswere non-carriers or wildtype (HAL-NN). At the timeof slaughter, the pigs had a live weight between 100 and120 kg. As expression of defects in membrane propertieswas desired for the pigs carrying the halothane gene(HAL-Nn), these pigs were exercised for 10 min on atread mill prior to slaughter in order to induce pre-slaughter stress. The remaining six animals were cross-breeds of Duroc/Landrace boars and Landrace/York-shire sows, and these animals were only used for aconstant temperature experiment (see below).

The pigs were slaughtered in the experimental abat-toir at Research Centre Foulum, and the same slaughterprocedure was used for all animals. The animals werestunned by 80% CO2 for 3 min, after which the pigswere exsanguinated. Immediately after exsanguination,a sample of approx. 5 cm was taken from each pig at thelast rib curvature of M. longissimus dorsi. From thissample, a sub-sample (approx. 5 cm long and 1�1 cm incross-sectional area, weight approx. 5 g) was cut alongthe fibres using a scalpel. The sample was immediatelyplaced in a cylindrical glass tube with the fibre directionperpendicular to the tube wall. In order to avoid exces-sive contraction, the sample was fixed to both ends ofthe cylindrical tube with cyanoacrylate glue (Sekundlim,Dana Lim, DK). The transportation of the samplesfrom the abattoir to the NMR instrument took approx.3 min, and during this period, the samples were storedin water with a temperature of 38 �C.

2.2. pH development in the carcasses

pH was measured 1 min, 15 min, 30 min, 60 min and24 h post mortem with pH-meter (Metrohm AG CH-9101 Herisau, Switzerland) equipped with an insertionglass electrode (LL glass electrode, Methrom, Switzer-land) at the level of the last rib curvature. At the mea-surements 1 min, 15 min, 30 min and 60 min postmortem the electrode was calibrated at a temperatureof 35 �C, while at 24 h post mortem the calibrationtemperature was 4 �C. A two-point calibration wasperformed, and the pH of the calibration buffers was7.000 and 4.005 at 25 �C (Radiometer, Copenhagen,Denmark).

2.3. NMR relaxation measurements

The relaxation measurements were performed on aMaran Benchtop Pulsed NMR Analyzer (ResonanceInstruments, Witney, UK) with a magnetic fieldstrength of 0.47 Tesla, with a corresponding resonancefrequency for protons of 23.2 MHz.

From 20 min post mortem until 18 h post mortem,transverse relaxation, T2, was measured continuouslyevery 10 min using the Carr–Purcell–Meiboom–Gillsequence (CPMG, Carr & Purcell, 1954; Meiboom &Gill, 1958). The T2 measurements were performed witha �-value (time between 90� pulse and 180� pulse) of 150ms. Data were acquired as 16 scan repetitions. Therepetition time between two succeeding scans was 2 s.

The NMR instrument was equipped with an 18 mmtemperature variable probe. By programming the NMRinstrument (in-house-made script), it was possible tohave the temperature regulated automatically during themeasuring period. The two temperature programs usedresulted in two different temperature profiles (calledslow and fast cooling), and meat samples from 12

Fig. 1. Temperature profiles for slow and fast cooling, respectively.

The slow cooling profile corresponds to typical commercial batch

chilling, while fast cooling profile corresponds to typical commercial

tunnel chilling.

1282 H.C. Bertram et al. /Meat Science 65 (2003) 1281–1291

animals (6 HAL-NN+6 HAL-Nn) were cooled witheach program. The temperature profiles are shown inFig. 1. Furthermore, on samples from the remaining sixanimals (crossbreeds of Duroc/Landrace boars andLandrace/Yorkshire sows, see above), the temperaturewas kept constant at either 15 �C (three samples) or35 �C (three samples) during the entire measuringperiod.

Distributed exponential fitting analysis was per-formed on T2 relaxation data using the RI Win-DXPprogram (software release version 1.2.3) released fromResonance Instruments Ltd. The RI Win-DXP programperforms distributed exponential curve fitting. A con-tinuous distribution of exponentials for a CPMGexperiment may be defined by Eq. (1):

gi ¼Xmj¼1

fje�ti=Tj ð1Þ

where gi is the values of the exponential distribution attime ti, fj is the pre-exponential multipliers of the dis-tribution and I is the exponential time constants (the T2

values). The RI Win-DXP program solves this equationby minimizing the function (2):

gi �Xmx¼1

fxe��i=Tx

!2

þlXmx¼1

f 2x ð2Þ

where lPm

x¼1 f2x is a linear combination of functions,

added to the equation in order to perform using a zeroorder regularization as described by Press, Teukolsky,Vetterling, and Flannery (1992).

From distributed analysis, average time constants foreach population were calculated from peak position,and the corresponding areas was determined by cumu-lative integration using an in-house program written inMatlab (The Mathworks Inc., Natick, MA, USA).

2.4. Determination of water-holding capacity (WHC)on NMR pork samples

The water-holding capacity was determined by cen-trifugation at 24 h post mortem on the samples used forrelaxation measurements. From each sample, five sub-samples, approx. 1 cm long and having a cross-sectionalarea of approx. 3�3 mm (weight approx. 0.3–0.5 g),were cut out parallel to the fibre direction. The sub-samples were weighed and placed in tubes (Mobicolsfrom MoBiTec, Gottingen) with a filter (pore size 90mm) in the bottom of the tubes to separate the meatfrom the expelled liquid. The samples were then cen-trifuged at 40 g for 1 h at a temperature of 4 �C. Aftercentrifugation the samples were weighed again, and thecentrifugation loss was calculated as the percentage dif-ference in weight before and after centrifugation.

2.5. Statistical analysis

Statistical analysis was carried out with the StatisticalAnalysis System (SAS Institute, 1991), using correlationanalysis (Proc CORR) and analysis of variance (ProcGLM). The statistical model included the fixed effect ofcooling regime and HAL-genotype.

3. Results

3.1. pH measurements in carcasses

Muscle pH 1, 15, 30 and 60 min post mortem wasfound to be significantly lower in the muscle of the het-erozygotes (HAL-Nn) compared with genotype HAL-NN, with pH60min being 5.7 and 6.2, respectively. pHmeasured 24 h post mortem was not found to differsignificantly between the two genotypes (5.4 versus 5.5).

3.2. Relaxation characteristics for muscle samples

Continuously distributed exponential curve fittingwas performed on the obtained relaxation data frommuscle samples. Independently of the nature of themuscle sample, three components were detected, aminor component between 1 and 10 ms, in the followingcalled T2B, a major component between 30 and 60 ms,in the following called T21, and finally, a componentbetween 100 and 400 ms, in the following called T22.Fig. 2a and b show typical progresses in these T2

relaxation characteristics during the post mortem periodof muscle samples exposed to slow and fast coolingfrom wild-type pigs. Fig. 3a and b show the changes inrelaxation characteristics post mortem when the tem-perature was kept constant at 15 and 35 �C, respec-tively. Independently of fixed or changing temperatureprofile, an equal progress pattern in relaxation char-acteristics post mortem was observed despite a dramaticdifference in the degree of change. Moreover, at 35 �Cthe T22 population became less defined and began tomerge into the T21 component with concomitant for-mation of an extra component around 800 ms, whichrepresents initial expelling of water. The conditionreflects severe PSE (Pale Soft Exudative) meat, whereidentical relaxation characteristics are often observed(T2 relaxation characteristics in typical PSE meat aredisplayed at the top of Fig. 3b).

Fig. 4a–c displays detailed changes during the postmortem period in the T2B, T21 and T22 time constantsfor the two genotypes and chilling regimes. For allcombinations of genotype and chilling regime, T2B timeremained constant at approx. 1–2 ms.

An initial increase in the T21 time constant, corre-sponding to slower relaxation, was observed indepen-dently of cooling regime and genotype. However, the

H.C. Bertram et al. /Meat Science 65 (2003) 1281–1291 1283

rate by which this initial increase progressed was affec-ted by the cooling regime with fast cooling acceleratingthe rate. Moreover, the genotype slightly affected theextent of the increase in T21, with muscles from het-erozygotes being characterized by slower relaxationcompared with muscles of wildtypes at this stage.The latter effect was most pronounced at fast cool-ing. In continuation of the initial increase in T21, aminor decrease in T21 proceeded, being most severefor muscle samples from heterozygotes exposed tofast chilling.

Independently of genotype, only a decrease in the T22

time constant was observed for the slow cooling profile.In contrast, an initial increase was observed for the fastcooling profile for muscle samples from heterozygotepigs until a decrease set in, which was much more pro-nounced compared with muscle samples from wild-typepigs exposed to fast cooling.

The registered changes in the population of relaxationdescribed by the T2B, T21 and T22 time for the two gen-otypes and chilling regimes are shown in Fig. 5a–c. Postmortem progresses in T21 and T22 water populations

Fig. 2. Typical example of three-dimensional plot showing the post mortem progress of T2 relaxation after distributed exponential fitting analysis of

data for the (a) slow and (b) fast cooling profile. Time post mortem (p.m.) in min on X-axis, relaxation time in ms on Y-axis and signal intensity (I)

on Z-axis.

1284 H.C. Bertram et al. /Meat Science 65 (2003) 1281–1291

were highly dependent on genotype and chilling regime.Muscle samples exposed to fast chilling displayed aninitial decrease in the T22 population, reaching theminimum approx. 1.5–2 h post mortem. Subsequentlyan increase in the T22 population appeared, this increasewas most severe for muscle samples originating from

heterozygotes. Muscle samples from wild-type pigsexposed to slow chilling progressed with a minordecrease in the T22 population followed by an identicalmoderate increase as seen in muscle samples of samegenotype exposed to fast cooling. In contrast, the com-bination of muscle samples from heterozygotes and slow

Fig. 3. Typical example of three-dimensional plot showing the post mortem progress of T2 relaxation after distributed exponential fitting analysis of

data (a) At a constant temperature of 15 �C, (b) At a constant temperature of 35C. Time post mortem (p.m.) in min on X-axis, relaxation time in ms

on Y-axis and signal intensity (I) on Z-axis. Insert: T2 relaxation measured in typical PSE meat (drip loss=14%).

H.C. Bertram et al. /Meat Science 65 (2003) 1281–1291 1285

chilling procedure gave rise to a dramatic initial increasein the T22 population followed by a more moderateincrease throughout the period.

WHC determined by centrifugation and T2 relaxationcharacteristics for the two genotypes and the two chil-ling regimes 24 h post mortem are shown in Table 1. A

significant effect of both genotype and chilling regimeon WHC was found. Meat from heterozygotes had sig-nificantly higher centrifugation loss compared withmeat from wildtypes, and slow chilling gave significantlyhigher centrifugation loss compared with fast chilling.An identical pattern between genotypes and cooling

Fig. 4. The post mortem progression in the time constants (a) T2B, (b) T21 (c) T22 in muscle samples from genotype HAL-Nn and HAL-NN exposed

to slow and fast cooling, respectively. LSMeans are given. Bars show standard errors.

Table 1

Least-squares mean values and standard error (S.E.) for WHC, determined by centrifugation and T2 parameters for the two genotypes and cooling

profiles. All traits were determined 24 h post mortem

HAL-NN

Slow cooling

(n=6)

HAL-NN

Fast cooling

(n=6)

HAL-Nn

Slow cooling

(n=6)

HAL-Nn

Fast cooling

(n=6)

Centrifugation loss (%)

5.2 (0.5)bcd 4.6 (0.5)cd 7.1 (0.5)ab 6.2 (0.5)abc

Relaxation

T22 (ms)

136.8 (9.9) 158.5 (9.9) 163.0 (9.9) 135.2 (9.9)

pT22 (%)

4.9 (0.5)abc 3.6 (0.5)bc 5.7 (0.5)ab 4.7 (0.5)abc

T21 (ms)

42.9 (0.6) 43.8 (0.6) 44.4 (0.5) 42.8 (0.5)

pT21 (%)

92.4 (0.7) 93.4 (0.7) 92.5 (0.7) 92.2 (0.7)

T2B (ms)

1.6 (0.3)ab 1.9 (0.3)ab 2.3 (0.3)ab 1.3 (0.3)ac

pT2B (%)

2.7 (0.3)ab 2.1 (0.3)b 2.8 (0.3)ab 3.1 (0.3)a

Different letters (a,b,c,d) in rows indicate significant differences (P<0.05).

1286 H.C. Bertram et al. /Meat Science 65 (2003) 1281–1291

profiles was observed for the T22 characteristics of themuscle samples.

4. Discussion

Meat quality is affected by environmental and geneticfactors (for a review, see Rosenvold & Andersen, 2003),and two major genes have been identified within pigpopulations, the so-called halothane-gene (Ludvigsen,1954) and the so-called RN-gene (Rendement Napole)(Naveau, 1986). The heterozygote carriers of the halo-thane gene (HAL-Nn) are known to have a defect in theryanodone receptor in the sarcoplasmatic reticulum(Fujii et al., 1991; Gallant et al., 1979; Louis et al.,1992), which disturbs muscle cell homeostasis and integ-rity upon exposure to stressors. This typically causesaccelerated post mortem glycolysis and simultaneousdecrease in pH upon slaughter, which results in inferiorwater-holding capacity (WHC) (Busk, Karlsson, & Her-tel, 2000; De Smet et al., 1996; Larzul et al., 1997; Lund-strom, Essen-Gustavsson, Rundgren, Edfors-Lilja, &Malmfors, 1989), as also found in meat from heterozygote

pigs compared with wild-type pigs in the present study(Table 1).

WHC is a major quality attribute of fresh meat as itdetermines potential drip loss, technological quality andappearance of the meat. Previous surveys have in gen-eral shown a moderate improvement in WHC usingtunnel chilling compared with batch chilling (Gigiel,Butler, & Hudson, 1989; Jones, Jeremiah, & Robertson,1993; Taylor, 1971; van der Wal, Engel, van Beek, &Veerkamp, 1995). The present study supports thisresults, independent of genotype exposure of musclesamples to conditions corresponding to tunnel chillinggives rise to a reduction of 0.6% in centrifugation loss24 h post mortem, compared with conditions corre-sponding to batch chilling (Table 1).

The reduced WHC in meat from halothane carriers ismainly explained by denaturation of muscle proteinsinduced by the combination of high temperature andlow pH (Offer, 1991), while the effect of acceleratedcarcass chilling has been explained by a decrease in therate of glycolysis post mortem according to the Arrhe-nius equation (Arrhenius, 1889) resulting in a slowerdecrease in pH. However, a recent study showed that

Fig. 5. The post mortem course of the population of relaxation described by (a) T2B, (b) T21, (c) T22 for genotype HAL-Nn and HAL-NN and slow

and fast cooling, respectively. LSMeans are given. Bars show standard errors.

H.C. Bertram et al. /Meat Science 65 (2003) 1281–1291 1287

the slower decrease in pH in porcine muscles exposed tofast chilling compared with muscles exposed to slowcooling is merely a result of a direct temperature effecton the buffering capacity of muscle than an effect on therate of metabolism post mortem (Bertram, Dønstrup,Karlsson, Andersen, & Stødkilde-Jørgensen, 2001). Inaddition, the properties of water in muscle tissue areknown to be temperature dependent (Belton, Jackson,& Packer, 1973; Fung & McGaughy, 1979; Peemoeller& Pintar, 1989), and therefore the improved WHCobtained upon tunnel chilling may be explained merelyby a temperature effect on the chemical/physical stateand distribution of water in the muscles.

In the present study dynamic NMR relaxationmeasurement has been introduced to follow waterdynamics in porcine muscle during its conversion tomeat. Distributed exponential fitting analysis of T2

relaxation data revealed three different relaxation com-ponents, a minor component with very fast relaxation(T2�1–10 ms), T2B, an intermediate component(T2�30–60 ms), T21, and a slower component(T2�100–400 ms), T22, and in accordance with an ear-lier 1H NMR study on muscle samples from halothanegene carriers (Renou, Kopp, Gatellier, Monin, &Kozak-Reiss, 1989), the present study revealed sig-nificant differences in the T2 relaxation characteristicspost mortem in muscles of halothane gene carrierscompared with muscles of wild-type pigs. The char-acteristics of the T21 and T22 components changed dur-ing the initial post mortem period (Figs. 4 and 5). Theserather dramatic shifts correspond to changes in waterenvironments and reveal that changes in distributionand chemical/physical state of water are taking placewithin the muscle during its conversion to meat. Thefact that the observed changes in relaxation parametersat fixed temperatures (Fig. 3) resemble the patternsfound using different cooling regimes shows that theobserved progress in T2 characteristics during chillingis not solely a result of temperature. The temperatureonly determines the rate and extent of the T2 patternchanges.

In the living muscle, it has been suggested that the T21

time constant reflects intra-cellular water, while the T22

time constant reflects extra-cellular water, as the cellmembrane is believed to act as a physical barrier forrapid exchange (Cole, LeBlanc, & Jhingran, 1993;Hazlewood, Chang, Nichols, & Woessner, 1974; Mauss,Grucker, Fornasiero, & Chambron, 1985; Rumeur, deCertaines, Toulouse, & Rochcongar, 1987). Moreover,the T2B time constant is suggested to reflect watertightly associated with the macromolecules (Hazlewoodet al., 1974). Earlier studies on meat also reveal thepresence of 2–4 water fractions that have been ascribedthe corresponding T21 and T22 populations in meat tointra- and extra-cellular water, respectively (Tornberg etal., 1993; Tornberg, Wahlgren, Brøndum, & Engelsen,

2000). However, recent data have demonstrated thatrelaxation behaviour in meat cannot strictly beexplained by the presence of intra- and extra-cellularspaces, as disruption of the macroscopic structure inmeat and thereby disintegration of membrane structuresdoes not change the relaxation characteristics (Bertram,Karlsson et al., 2001). Accordingly, the T21 time con-stant is more likely to reflect water located within highlyorganized protein structures, e.g. water in tertiary andquaternary protein structures and spatials with highmyofibrillar protein densities including actin and myo-sin filament structures, while the T22 time constantreflects extra-myofibrillar water. Likewise muscle, theT2B time constant probably relates to water tightlyassociated with macromolecules. This complies withboth impedance (Bertram, Schafer, Rosenvold, &Andersen, 2003) and microscopic (Dutson, Pearson, &Merkel, 1974; Feldhusen, Konigsmann, Kaup, Drom-mer, & Wenzel, 1992) studies, which reveal destabiliza-tion and damage of the cell membrane system duringthe post mortem processes, and the reason for the fail-ure of the intra-/extra-cellular model in meat is prob-ably disruption of the cell membrane during theconversion of muscle to meat. The present findings aretherefore most likely to reflect a change from T21 andT22 characteristics in vivo resembling intra- and extra-cellular water due to intact cell membranes to T21 andT22 characteristics resembling intra- and inter-myofi-brillar ‘‘trapped’’ water in the meat.

The observed decrease in the T2B population earlypost mortem probably reflects the decrease in pH fallpost mortem, as a change in pH towards the isoelectricpoint, due to reduced net charge, can be expected toreduce the hydration shell around the proteins.

The observed increase in the T21 time constant earlypost mortem has likewise been reported in earlier stud-ies on both muscle (English, Roy, & Henkelman, 1991;Tornberg et al., 2000) and liver tissue (Moser, Winkl-mayr, Holzmuller, & Krssak, 1995) at a constant mea-suring temperature. Accordingly, the increase cannotsolely be explained by a temperature effect on the T21

time constant, which was further supported by the con-stant temperature experiments carried out in connectionwith the present study (data not shown). This decreasein relaxation rate of the T21 component, found to beindependent of genotype (Fig. 4b) might instead beexplained by swelling of the myofilament spacing trig-gered by an increase in intracellular osmolarity andconcomitant dilution of protein concentration uponswelling of the muscle cell, which should coincide with asimultaneous increase in the T21 population. However,the fact that an increase in the T21 population immedi-ately post mortem did not appear in slowly cooledsamples (Fig. 5b) reveals that the increase in the T21

time constant cannot solely be explained by cellularswelling. Consequently, other factors must contribute to

1288 H.C. Bertram et al. /Meat Science 65 (2003) 1281–1291

this initial increase in the T21 time constant, e.g. changesin pH as suggested by Tornberg et al. (2000). Thisagrees with the fact that we noticed comparable differ-ences in T2 relaxation rates in pure protein (bovineserum albumin) model systems as a function of pH(data not shown). The observed subsequent modestdecrease in the T21 time constant may be explained byphysical changes caused by contraction and a pH-induced transversal myofibrillar shrinkage, which isexpected to take place at this stage of the post mortemperiod, and furthermore to be accelerated in meat fromheterozygotes, as also seen in Fig. 4b when comparingidentical cooling rates. In addition, this is in accordancewith recent studies revealing that the T21 time constantdecreases with increased contraction (shorter sarcomerelengths) (Bertram, Purslow, & Andersen, 2002), andthat decrease in the T21 time constant takes placewithin the same period as maximum contraction occurs(Bertram et al., 2003).

It is worth noticing that almost identical develop-ments in T21 time constant patterns in samples fromboth genotypes and exposed to different cooling regimes(Fig. 4b) were not directly reflected in identical devel-opments in corresponding T21 water population pat-terns (Fig. 5b). This stresses that factors affecting theT21 time constant have one or several derived effects,which subsequently may affect the size of the corre-sponding T21 population without affecting the relaxa-tion environment. A more thorough analysis of thedevelopment in the T21 populations reveals that thecooling rate is directly reflected in the progression in theT21 population in contrast to the progression in T21 timeconstant. This especially accounts for the progression inthe T21 population in muscle from wild-types, as thesebecome similar at the time where the difference betweenthe two cooling profiles is just about eliminated (8–9 hpost mortem). In contrast, the T21 population for therapidly cooled muscle samples from heterozygotesbegins to differ from the counterparts from wild-typepigs by a more severe drop in the T21 population at thetime where the temperatures for the two cooling ratesbecome identical. This difference might be explained bya more severe protein denaturation (decreasing themyofibrillar protein network’s ability to hold water) inmuscle samples from carriers of the halothane gene dueto a more rapid decrease in pH at a time when the tem-perature-induced buffering capacity (Bertram, Dønstrupet al., 2001) cannot eliminate pH-induced protein dena-turation. Moreover, this complies with the observedprogress in the T21 population in both slow-cooledmuscle samples from wild types and heterozygotes, asthe physical counteracting in pH due to the moderatecooling rate cannot delay the pH-induced protein dena-turation in samples from heterozygotes, and therefore adifference in the T21 populations between samples fromwild types and heterozygotes arises immediately post

mortem in the slowly cooled samples, in contrast to therapidly cooled samples.

Taking into consideration that changes in the T22

population early post mortem reflect changes in theamount of extra-cellular water characteristics, theresults reveal an initial decrease in extra-cellular water(with a parallel increase in the population of waterdescribed by the T21 time constant), followed by a smallbut steady increase in the T22 population, independentof genotype when muscle samples were exposed to fastcooling. This subsequent increase in the T22 populationmay be explained by redistribution of water stores fromintra-/extracellular to intra-/extramyofibrillar, as thisprogresses around the expected time for loss in mem-brane integrity (�1,5–2 h post mortem), as found in arecent study (Bertram et al., 2003). In contrast to waterdynamics upon fast cooling, no initial decrease in theT22 population took place upon slow cooling, and inaddition, a pronounced difference in the T22 time con-stant of muscle samples from the halothane carrierscompared with non-carriers was observed. The con-siderable increase in the T22 population observed inslowly cooled muscle samples from carriers of the halo-thane gene suggests that an initial swelling of musclecells immediately post mortem does not take place inthese, as suggested above for rapidly cooled musclesamples, and if swelling of muscle cells in slow-cooledsamples from wild-types takes place, this is only to alimited extent compared with fast-cooled samples. Con-sequently, temperature seems critical for the progress inT22 characteristics. As mentioned above, fast cooling isknown to reduce the rate of decrease in pH in muscle(Bertram, Dønstrup et al., 2001), and is moreoverexpected to decrease the rate of post mortem glycolysis.Accordingly, fast cooling may counteract the expectedfast post mortem glycolysis rate and simultaneousdecrease in pH, which would normally trigger rapid lossin membrane integrity, and in combination with theearly rigor development, squeeze water out from themyofibrillar lattice and into the extra-myofibrillar spacegiving rise to a rapid increase in the T22 population. Onthe contrary, the combined effect of the presence of thehalothane gene and slow cooling stimulates these eventsand gives rise to the observed rapid increase in the T22

population immediately post mortem in muscle samplesfrom heterozygote carriers of the halothane gene. Inaddition, an intermediate situation was observed inmuscle samples from wild-types exposed to slow cool-ing. Further studies are in progress to substantiate theproposed role of membrane integrity on water dynamicsin post mortem muscle.

Noticeably, the difference in the T22 population inmuscle samples from wild types exposed to the two dif-ferent cooling profiles are established within the first 3 hpost mortem, as the remaining post mortem progres-sions in the T22 population become identical thereafter.

H.C. Bertram et al. /Meat Science 65 (2003) 1281–1291 1289

In contrast, the difference in the T22 population in slow-cooled muscle samples from carriers and non-carriers ofthe halothane gene, respectively, first became evidentapproximately 7 h post mortem. This difference may beexplained by the expected more pronounced proteindenaturation in muscle samples from carriers of thehalothane gene due to more rapid decrease in pH, whichslowly results in decreased ability of the myofibrillarproteins to hold water, similar to the explanation for theprogression of T21 characteristics discussed earlier.

Finally, the T22 population was reduced by approxi-mately 1.0–1.3% upon fast cooling 24 h post mortem.In view of the fact that the T22 population has beenfound to be closely related to potential drip loss of pork(Bertram, Dønstrup, Karlsson, & Andersen, 2002), fastcooling seems to change the water dynamics in postmortem muscle in a way that reduces water movementsto the extra-myofibrillar space and thereby limitingaccumulation of potential drip, as also partly reflectedin the WHC of the muscle samples (Table 1).

5. Conclusion

The present study demonstrates significant differencesin water dynamics in pig muscle during its conversion tomeat from carriers and non-carriers of the halothanegene. In addition, the cooling rate of samples was foundto be decisive for the water dynamics, as fast coolingwas found to result in a reduced accumulation of extra-myofibrillar water, which nearly eliminated the effect onwater distribution in meat from halothane carrierscompared to non-carriers. This cooling effect on waterdynamics in muscle from halothane carriers mightexplain why accelerated cooling of carcasses of halo-thane carriers have been reported to improve the WHCof meat.

Acknowledgements

The authors are grateful to the Danish Veterinary andAgricultural Research Council (SJVF) and to the Dan-ish Directorate for Development (Product Quality Pro-ject to H.J.A.) for financial support to this project.Moreover, Mr. Sune Dønstrup (M.Sc.) is greatlyacknowledged for creating the program used in Matlab(The Mathworks Inc., Natick, MA, USA) for determi-nation of time constants and corresponding areas.

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