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Post mortem energy metabolism and pH development in porcine M. longissimus dorsi as affected by two different cooling regimes. A 31P-NMR spectroscopic study
Magnetic Resonance Imaging 19 (2001) 993–1000
Post mortem energy metabolism and pH development in porcine M. longissimus dorsi as affected by two different cooling regimes. A 31P-NMR spectroscopic study Hanne Christine Bertrama,*, Sune Dønstrupa, Anders Hans Karlssona, Henrik Jørgen Andersena, Hans Stødkilde-Jørgensenb a
Department of Animal Product Quality, Danish Institute of Agricultural Sciences, Research Centre Foulum, P.O. Box 50, DK-8830 Tjele, Denmark b The MR Research Centre, Aarhus University Hospital, Skejby Sygehus, DK-8200 Århus N, Denmark Received 15 December 2000; accepted 7 June 2001
Abstract 31
P-NMR spectroscopy was carried out on M. longissimus dorsi samples chilled by two different cooling profiles corresponding to commercial batch and tunnel chilling. The half-life of post mortem phosphocreatine (PCr) degradation was found to be significantly less in muscle samples exposed to tunnel chilling (rapid) compared with muscle samples exposed to batch chilling (soft) conditions, while no difference in the post mortem ATP degradation was found. Moreover, the post mortem pH development in the muscle samples differed considerably between the two cooling regimes. A maximum difference of approx. 0.25 pH units between the two cooling profiles was observed around 150 min post mortem. Theoretical calculations of the registered pH difference between rapid and soft chilling of muscle samples revealed that the temperature effect on the buffer capacity of muscle is the major determining factor in the detected difference in intracellular pH between the two cooling profiles, while any contribution from a temperature-induced delayed progress in the lactate formation post mortem seems negligible. Moreover, calculations on the effect of the registered pH difference between rapid and soft chilling of muscle samples resemble a 2.5 times greater denaturation of myosin in samples which were chilled softly compared with samples chilled more rapidly. Finally, the relationship to the functionality of meats from soft and rapid chilled pork carcasses is discussed. © 2001 Elsevier Science Inc. All rights reserved. Keywords:
31
P-NMR spectroscopy; Temperature; Intracellular pH; Water-holding capacity; Pork
1. Introduction Water holding capacity (WHC) is of major concern in the meat industry, as it affects both economic and sensory attributes of the meat. High incidences of porcine meat with unacceptable/reduced WHC, so-called PSE (Pale Soft Exudative) meat, some decades ago led to the introduction of tunnel chilling as an alternative to the conventional batch chilling [1]. Several studies have documented a positive effect of tunnel chilling on WHC [1– 4]. However, the explanation of why tunnel chilling improves WHC of the meat is far from understood. The improved WHC may either be explained by a biochemical effect, i.e., by a direct temperature effect on post mortem energy metabolism, by a
* Corresponding author. Tel.: ⫹45 89 99 15 06; fax: ⫹45 89 99 15 64. E-mail address: [email protected] (H.C. Bertram).
structural effect, i.e., a temperature-induced effect on the mobility and distribution of water in the muscles, or by a combination of both. Moreover, even though the combination of low pH and high temperature may cause protein denaturation and result in reduced WHC [5–7], a further understanding of how the chilling regime affects protein denaturation post mortem is necessary in order to be able to optimize the chilling procedure. The major reasons why a more thorough understanding of the effect of different chilling regimes on post mortem energy metabolism is not available, are the lack of suitable methods for these investigations. Due to the non-invasive and non-destructive character of NMR spectroscopy, 31P-NMR spectroscopy is a suitable method in the study of post mortem energy metabolism in muscle tissue [8 –10]. 31P-NMR spectroscopy makes it possible to follow the time course of the degradation of phosphocreatine (PCr), ATP and phosphomonoesters (PME), and together with the simultaneous formation of inorganic
0730-725X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 0 - 7 2 5 X ( 0 1 ) 0 0 4 1 2 - X
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phosphate (Pi), it gives valuable information about energy status in post mortem muscle tissue. Furthermore, intracellular pH can be calculated from 31P-NMR data. The present study aims to investigate how a commercial (rapid) tunnel chilling affects the post mortem energy metabolism and pH development in pig muscle tissue compared with a commercial (soft) batch chilling using NMR 31 P-spectroscopy. Furthermore, the influence of cooling regime on protein denaturation was considered from a theoretical point of view in relation to WHC of meat.
2. Materials and methods 2.1. Animals and sampling Sixteen female Danish Landrace pigs with a live weight of approx. 80 kg at the time of slaughter was used in the present experiment. Approx. 24 h before slaughter, the pigs were transported from the producer’s farm to the Institute of Experimental Clinical Research at Skejby Hospital, Denmark, where the slaughtering was carried out. The pigs were killed by i.v. injection of pentobarbital (50 mg/kg live weight) after i.v. injection of midazolam (Dormicum®, Alpharma A/S Oslo, Norway, 0.6 mg/kg live weight) used as premedication. Immediately after killing, a sample of approx. 6 cm was taken at the level of the last rib from M. longissimus dorsi. From this sample, a sub-sample with a cross-sectional area of approx. 2.5 ⫻ 2.5 cm2 and a length of approx. 5 cm was cut out along the fiber direction and placed in the tube used for NMR measurements. 2.2. pH-measurements pH was measured with an insertion electrode (Metrohm AG CH-9101 Herisau) equipped with an insertion glass electrode (Hamilton Tiptrode P/N: 238⬘080, Switzerland) at 45 min and 24 h post mortem in the remaining sample (see above) taken at the level of the last curvature of M. longissimus dorsi. The electrode was calibrated at a temperature of 22°C. A two-point calibration was performed, and the pH of the calibration buffer used was 7.000 and 4.005 at 25°C (Radiometer, DK-Copenhagen).
Fig. 1. Temperature profiles of M. longissimus dorsi for the fast and slow cooling. The fast cooling profile corresponds to a commercial tunnel chilling, and the slow cooling profile corresponds to a commercial batch chilling.
points per FID and a spectral width of 7000 Hz. The first spectrum was taken between 18 and 30 min post mortem, and thereafter spectra were recorded continuously until 9 –13 h post mortem, resulting in between 120 and 170 spectra per animal. Volume selection with a size of 12 ⫻ 12 ⫻ 12 mm was performed using the ISIS-method [11]. Thermostatic control and regulation of the samples was ensured by an air flow (24 l/min) controlled by a temperature variable unit (FTS Systems, model TP-88, Stone Ridge, New York, USA). By programming the temperature unit, it was possible to have the temperature regulated automatically during the measuring period. Two temperature programs, resulting in two different cooling profiles corresponding to a commercial tunnel (fast) and batch (slow) chilling, were developed. Development of thermal equilibrium in the muscle samples during exposure to the profiles was assured in initial investigations. The temperature profiles are shown in Fig. 1. Accordingly, the samples were placed in the NMR-probe during the whole measuring period, and exactly the same sample position was measured under each acquisition. Of the 16 samples included in the experiment, 8 samples were exposed to each cooling profile. The spectra were phased manually, and chemical shifts and areas of the signals were calculated using a least-square regression program (included in the Varian software), fitting the spectrum to Lorentzian lines. Chemical shifts were reported in ppm relative to the PCr signal.
2.3. NMR measurements 31
P-NMR spectra were recorded at 121.4 MHz on a 7 Tesla Sisco 300/173 spectrometer (Varian Associated, Inc., Palo Alto, CA) equipped with a 30 mm diameter in-house build single circular probe. After fine tuning of the probe with the sample installed, the magnetic field homogeneity was optimized on the FID of water protons. A half-width of PCr signal in the 31P-spectrum in the range 35–75 Hz was obtained. Each spectrum was an average of 32 transients with a recycle time of 8 s, accumulated in a total time of 4 min and 16 s. The acquisition parameters used were 7168
2.4. Temperature dependence of saturation effects (longitudinal relaxation time) and saturation transfer effects The non-invasive and non-destructive character of 31PNMR spectroscopy makes the technique very suitable for studying the post mortem metabolism in muscles, as changes in phosphor metabolites can be registered continuously. However, using 31P-NMR spectroscopy to study temperature effects on post mortem metabolism includes some further considerations, as relaxation mechanisms and
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thereby signal intensity depend on temperature. It is well recognized that an underestimation of the signal may occur because of partial saturation, if the nuclears are not fully relaxed before the succeeding excitation. However, longitudinal relaxation (T1) is temperature dependent, and therefore different degrees of saturation effects may turn out at different temperatures. Moreover, exchanges between P nucleus of PCr and of the ␥-phosphate of ATP may generate saturation transfer effects [12–13], which can also be expected to be temperature dependent. Consequently, temperature dependence on saturation effects (longitudinal relaxation time) and saturation transfer effects were initially determined on muscle samples exposed to the two cooling profiles. For this purpose a special protocol was developed. Succeeding scans as an average of 16 transients with alternately a recycle time of 8 and 24 s were recorded continuously. A recycle time of 24 s allows more than 99% of the original magnetization to recover for all metabolites [14], as T1 for Pi, which is the metabolite having the longest T1, was found to be approx. 5 s in muscle tissue. The areas of signals obtained with a recycle time of 8 s were compared with areas of signals from spectra obtained with a recycle time of 24 s, and the ratios between the two areas were used to correct for saturation and saturation transfer effects.
5 solutions of 50 mM phosphate buffer (pH ⫽ 7.4, ion strength I ⫽ 0.16) containing a) 20 mM PCr (Boehringer Mannheim, GmbH, Germany) and 4 mM ATP (Boehringer Mannheim, GmbH, Germany); b) 15 mM PCr and 3 mM ATP; c) 10 mM PCr and 2 mM ATP; d) 5 mM PCr and 1 mM ATP; e) 0 mM PCr and 0 mM ATP. The 31P-NMR spectra were recorded with the same acquisition parameters as those used on muscle samples (see above) at a constant temperature of 20°C. 2.7. pH determination pH was calculated from the chemical shift of Pi using the Henderson-Hesselbach equation 2 [15]:
冉
pH ⫽ pKa ⫹ log
M⫽
N ␥ 2h 2I共I ⫹ 1兲 Bo 3K BT
␦O ⫺ ␦A ␦B ⫺ ␦O
冊
(2)
where Ka is the second dissociation constant for H3PO4, and ␦O,␦A and ␦B are the chemical shifts of Pi, H2PO⫺ 4 and HPO2⫺ 4 , respectively. As both pKa and chemical shift values are temperature dependent, the following temperaturecorrected algorithm was used to calculate pH from the chemical shift of Pi 3 [16]: pH ⫽
1979.5 ⫺ 5.4409 ⫹ 0.018567共T ⫹ 273兲 T ⫹ 273
2.5. Temperature dependence of magnetization Theoretically, the magnetization (M) of a set of N nuclei exposed to a magnetic induction field of strength Bo is given by equation 1 [14]:
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⫹ log
冋
␦ O ⫺ 3.280 ⫹ 0.003579T 5.625 ⫹ 0.001888T ⫺ ␦ O
册
(3)
where T is the temperature expressed in°C. 2.8. Statistical analysis
(1)
where KB is the Boltzmann constant, T the temperature, ␥ the gyromagnetic ratio, h the Planck constant and I is the nuclear spin quantum number. Accordingly, the magnetization is inversely proportional to the temperature and an increase in signal intensity is expected, when the temperature is decreased. If this parameter is not considered, the content of metabolites will be overestimated at lower temperatures. Consequently, the temperature effect on magnetization was initially determined, and also included in the calculation of the concentrations of the different metabolites. For this purpose, 31P-spectra were recorded on muscle samples exposed to the two cooling profiles 24 – 48 h post mortem, at which time the biochemical processes has ended, and where changes in the metabolites therefore no longer will take place. A recycle time of 32 s was used. 2.6. Investigation of linear response of concentration The linearity of concentration determinations of PCr and ATP was investigated with concentrations equal to those found in muscle tissue. 31P-NMR spectra were recorded on
Analysis of variance was carried out on the response variables using the Proc GLM from the Statistical Analysis System package [17]. The statistical model included the fixed effect of chilling regime. Furthermore, the Proc NLIN was used for break-point analysis on ATP data. In breakpoint analysis, data are fitted to a horizontal line, and a line with a decreasing slope [18].
3. Results 3.1. Linear response of concentration Fig. 2a and b show the correlations between concentration and area of signal from PCr and phosphate groups in ATP, respectively. Due to the high off-set frequency of the -phosphate resonance, the selected volume was partially outside the meat sample, which accounts for the low intensity of this resonance. A linear correlation coefficient of r ⫽ 0.98 was found between concentration and area of signal from PCr. For the phosphate groups in ATP, the correlation depended on the particular phosphate group. The strongest
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Fig. 3. Typical stacked plot of spectra recorded continuously from 20 min post mortem until 12 h post mortem on a muscle sample. Each spectrum is average of 32 scans accumulated after 90° pulses at 8 s intervals.
Fig. 2. Relationship between concentration and area of signal measured on solutions of phosphate buffer (pH ⫽ 7.4, I ⫽ 0.16) containing PCr and ATP. 31P-NMR spectra were recorded at 20°C. a) area of signal from PCr, b) area of signal from ␣-, - and ␥-phosphate groups in ATP.
linear correlation was found for the ␥-phosphate group (r ⫽ 0.92), and the weakest linear correlation was found for the -phosphate group (r ⫽ 0.69). 3.2. pH measurements on muscle samples In all muscle samples, pH45 min was measured with electrode to validate the variation in energy metabolism among the samples. Table 1 shows that only minor variations were found between samples. Three muscle samples had a pH45 min below 7.0, while the rest had a pH at 45 min above 7.0. The three muscle samples having a pH below 7.0 were considered outliers and left out of further analysis. In order to evaluate the glycogen stores and the extent of glycolysis post mortem, muscle pH was also measured 24 h post mortem (Table 1). No significant difference between muscle samples used in the two cooling profiles was observed.
Table 1 pH values for all animals included in analysis and for fast and slow cooling. LSMeans, standard errors (SE) and level of significance within row are shown
pH, 45 min pH, 24 h
All animals (n ⫽ 13)
Fast cooling (n ⫽ 7)
Slow cooling (n ⫽ 6)
Significance level
7.19 (0.07) 5.64 (0.12)
7.20 (0.03) 5.66 (0.05)
7.17 (0.03) 5.63 (0.05)
p ⫽ 0.52 p ⫽ 0.74
3.3. NMR measurements on muscle samples Fig. 3 shows a typical time course in the phosphorus spectra recorded continuously from 20 min until 12 h post mortem. From the left (⬃7 ppm), an increasing signal from PME is seen. At approx. 5 ppm, the signal from Pi is seen. Besides increasing in intensity, this signal also moves toward lower chemical shifts, which reflects a decrease in intracellular pH during the post mortem period. The signal from PCr is positioned at approx. 0 ppm and is inversely correlated to an identical and simultaneous increase in the Pi signal. Finally, at approx. ⫺2, ⫺7.5 and ⫺16 ppm the resonances from the ␥-, ␣-, and -phosphate groups in ATP are registered. The total amount of phosphor metabolites in the muscle tissue was assumed to be 50 mol/g muscle due to earlier results [9]. Subsequently, the amount of the different phosphor metabolites was calculated after correction for saturation and saturation transfer effects and temperature dependence on magnetization using the relative area of signals. Fig. 4 shows typical changes in the different phosphor metabolite concentrations during the post mortem period. In agreement with earlier studies [9,19], an exponentially decline in PCr during the post mortem period was found. This decline corresponded to a first order reaction, which could be described by equation 4: PCr ⫽ K PCre 共⫺bPcrtime兲
(4)
where KPCr and bPCr are coefficients. For an expression for the rate of PCr break-down, the half-life of PCr was calculated as ln 2/bPCr. Moreover, the Pi was found to grow exponential toward a steady state level, and the progress in Pi could be described by the following equation 5:
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Table 2 Results from analysis of Pi, PCr and ATP-data. LSMeans, standard errors (SE) and level of significance within rows are shown Fast cooling Slow cooling Significance (n ⫽ 7) (n ⫽ 6) level Half-life, PCr (min) 79.3 (5.08) Initial growth rate, Pi (min⫺1) 0.15 (0.003) ␥-ATP, break point (min) 259 (12.8)
Fig. 4. Typical post mortem changes in the concentration of phosphocreatine (PCr), inorganic phosphate (Pi), phosphomonoesters (PME) and ATP. Absolute concentrations are determined on the assumption that the total amount of phosphor metabolites is 50 mol/g muscle in the first spectrum recorded.
Pi ⫽ K Pi共1 ⫺ e 共⫺bPitime兲兲
62.1 (5.48) p ⫽ 0.04* 0.018 (0.003) p ⫽ 0.51 281 (13.8) p ⫽ 0.27
the two cooling profiles, however, a pronounced difference appeared soon after slaughter (approx. 40 – 60 min post mortem). pH was significantly higher during fast cooling compared with the slow cooling from 100 to 300 min post mortem. A maximum difference between the two cooling profiles of approx. 0.25 pH-units was observed approx. 150 min post mortem.
(5)
where KPi and bPi are constants. For an expression for the rate of formation of Pi, KPi is given as the initial growth rate of Pi. Finally, ATP was found to be constant during the initial stage of the post mortem period. Subsequently, a generally linear decrease in the ATP responds was observed. In conjunction with the observed ATP course, break-point analysis [18] was carried out on ATP data (Fig. 5). Table 2 shows Pi, PCr and ATP data. The ␥-phosphate resonance from ATP was used to calculate the ATP concentration, as linear response analysis showed highest linearity for this group (Fig. 2b). The post mortem half-life of PCr was found to be significantly different for the two cooling profiles, with slow cooling significantly reducing the half-life of PCr. Initial growth rate of Pi was not significantly lower for the slow cooling profile. Break-point analysis of ATP data revealed no significant differences in the initiation of ATP break-down between the two cooling profiles. In Fig. 6 the calculated pH courses for the two cooling profiles, corrected for temperature, are shown. Initial pH (approx. the first 20 – 40 min post mortem) was identical for
Fig. 5. Typical picture of the time course of ATP degradation and breakpoint analysis (see ref. 12) of the ATP-data.
4. Discussion The present study demonstrated that a significant reduction in the post mortem processes is achieved by fast (tunnel) chilling compared with slow (batch) chilling, using 31 P-NMR spectroscopy. This was mainly reflected in the rate of post mortem degradation of PCr, which was found to be significantly lower upon fast (tunnel) chilling compared with slow (batch) chilling. In contrast, no significant effect of chilling rate was registered on ATP depletion. This might be due to lack of sensitivity in the used method, as reflected by the moderate linearity of ATP concentration compared with the PCr concentration (Fig. 2). A remarkable difference in pH progress within muscles was registered depending on the cooling regime used. Approx. 150 min post mortem, the pH-difference in muscles cooled by the two different rates reached a maximum of 0.25 pH units. This time point corresponds to a temperature difference of approx. 12°C between the two cooling re-
Fig. 6. Typical pH courses for the slow and fast cooling profile. pH is calculated from the chemical shifts of PCr and Pi and calibrated for temperature according to [14].
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Fig. 7. Effect of pH dependence of temperature on the post mortem pH courses for the two cooling regimes. Shown are; post mortem pH courses calculated on the assumption that only directly temperature dependence on pH affects pH (0.016 pH-unit/°K) for 1) fast and 2) slow cooling, the observed pH courses for 3) fast and 4) slow cooling, and the observed pH courses after subtraction of temperature dependence on pH (0.016 pH-unit/ °K) for 5) fast and 6) slow cooling.
gimes. The observed difference in pH is in accordance with earlier in vivo studies in muscle tissue, where a temperature dependence of 0.016 pH-units per °C temperature has been reported [20 –22]. It has been proposed that a temperature dependence of the intracellular acid-base regulation (buffering capacity) in muscle tissue exists with the purpose of maintaining a constant fractional dissociation of intracellular protein and preservation of enzymes [21]. This is called the alphastat hypothesis [23]. In order to elucidate the contribution of the above-mentioned temperature effect on intracellular pH in muscles exposed to the two cooling profiles, the theoretical progress in pH, as affected by the known temperature dependence on pH in muscle tissue, was constructed for the two cooling regimes. Subsequently, these constructed pH progresses were subtracted from the pH progresses based on 31P-NMR measurements to differentiate between metabolically induced pH decrease and influence of the temperature effect on muscle pH (Fig. 7). Noteworthy, the progress in metabolically induced pH decrease seems to be independent of the used cooling regimes. Consequently, the temperature effect on the buffer capacity of muscle is the major determining factor in the detected difference in intracellular pH between the two cooling profiles, while any contribution from a temperature-induced delayed progress in the lactate formation post mortem seems negligible. This is in contrast to earlier suggestions on the effect of chilling regime on post mortem metabolism and pH development, where the pH difference between different cooling profiles was supposed to be a direct decrease in the rate of glycolysis post mortem with lower temperatures and a simultaneous decrease in the formation of lactate [3,24]. In fact, the present study completely reverses this suggestion, as subtraction of the temperature effect on muscle buffer capacity not only eliminates the difference in pH between the two cooling profiles (Fig. 7), but even indicates that the initial post mortem pH decrease is faster during fast cooling compared
with slow cooling. This also agrees with the faster observed degradation of PCr upon slow cooling of the muscle, as increased [H⫹] consumption initially post mortem by the creatine kinase reaction must be expected upon slow chilling compared with a faster regime, which decreases the turn-over of the creatine kinase. Results regarding early pH measurements in post mortem muscle as affected by different chilling regimes are sparse in the literature [3]. This is most probably due to practical problems in a continuous measurement of pH in the carcass from the final cutting via the chilling systems and until temperature equilibration. However, 31P-NMR spectroscopic studies equal to those presented in the present study show the possibility of performing continuous measurement of pH post mortem and understand how the post mortem metabolism and its consequences are affected by different chilling regimes. It has been recognized for decades that the combination of low pH and high temperature post mortem may reduce the water holding capacity (WHC) of the meat due to protein denaturation [5–7]. Consequently, the differences in the pH development post mortem between the two cooling regimes in the present study may explain the improved WHC obtained using fast (tunnel) chilling instead of slower (batch) chilling, as reported elsewhere [1– 4]. Moreover, in a recent NMR proton relaxation study [26], where muscle samples were exposed to almost identical cooling profiles as in the present study, water mobility and distribution were found to differ significantly between the two cooling profiles. As the water mobility and distribution depend on micro-structural changes, which again is related to the pH development post mortem, this difference can probably also partly be explained by the difference in pH development post mortem as observed in the present study. Offer [27] has used the kinetics of the inactivation of myosin ATPase [25] to set up a relationship between the rate of inactivation of myosin ATPase (K), temperature and pH, equation 6:
冉
K ⫽ Ko exp
冊
⫺43500 10 ⫺1.3pH RT
(6)
where R is the gas constant, T is the temperature in degrees K, and Ko is an empiric constant, assumed to be 2.13 ⫻ 1034 from earlier results [25,28]. When K is known, the fraction of inactivated myosin can be determined by integration. Loss in myosin ATPase activity has not been demonstrated to directly reflect denaturation affecting the WHC of the meat; however, loss of myosin ATPase activity may be an indicator of protein denaturation [27]. In order to estimate how the different progresses in pH and temperature due to the two chilling regimes applied in the present study affect the degree of protein denaturation, the fraction of inactivated myosin was calculated on the basis of the obtained temperature and pH profiles (Fig. 1 and 6). The fraction of inactivated myosin was estimated to be approx. 2.5 times larger using the slow cooling profile
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compared with the fast cooling profile. The result of these calculations indicates that the observed differences in pH and temperature in muscles exposed to these two cooling regimes may be expected to have a huge influence on the degree of protein denaturation and thereby WHC of the meat. However, further studies are needed to support this stated relationship between degree of protein denaturation and WHC. Nevertheless, such information would be crucial to further understand the contributions of pH and temperature on the WHC of meat. Relatively large biologic variations in muscle energy status at the time of slaughter have been found in several surveys [29 –30], but also under standardized conditions [31]. This can be explained by differences in the behavioral pattern between animals when exposed to stress [32]. In the present study the pigs were anesthetized before killing, which minimizes the variation between animals and thereby reduces the ante mortem stress. This was reflected in the muscle pH measured at 45 min post mortem, which was high and almost the same between animals compared with what is reported in pigs slaughtered under traditional conditions [29,33–34]. It can of course be questioned if the use of anesthesia is comparable to any practical post mortem circumstances, where the post mortem processes will be accelerated. The present study revealed that a temperature effect on muscle pH in muscles is mainly responsible for the difference in post mortem pH progress between the two chilling regimes. Consequently, a linear temperature dependence on pH in the concerned pH interval is a prerequisite for obtaining the same effect. However, the temperature dependence on pH has only been investigated between pH 6.9 and 7.2 [21]. Hence, further studies on pigs killed without anesthesia would be valuable to demonstrate whether the same chilling effects on pH progress are obtained at higher rates of the post mortem glycolysis. In conclusion, the present study has demonstrated that a significant reduction in the post mortem processes can be achieved under conditions corresponding to tunnel chilling compared with batch chilling conditions, as the half-life of PCr post mortem was found to be significantly shorter when simulating batch chilling compared with simulated tunnel chilling. Moreover, the pH development post mortem differed considerably between the two cooling profiles, which could almost exclusively be ascribed to a temperature dependence on muscle pH.
Acknowledgments The authors are grateful to the Danish Veterinary and Agricultural Research Council (SJVF) and to the Danish Directorates for Development (Product Quality Project to H.J.A.) for financial support of this project. Moreover, Dr. Wolf Dresher, Department of Orthopaedics, Aarhus Municipal Hospital, Denmark, and co-workers, Dr. Karen Weigert and Mr. Mathias Bu¨ nger, are greatly acknowledged for
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[22] Binzoni T, Hiltbrand E, Terrier F, Cerretelli P, Delpy D. Temperature dependence of human gastrocnemius pH and high-energy phosphate concentration by noninvasive techniques. Magn Reson Med 2000;43: 611– 4. [23] Reeves RB. An imidazole alphastat hypothesis for vertebrate acidbase regulation: tissue carbon dioxide content and body temperature in bullfrogs. Respir Physiol 1972;14:219 –236. [24] Marsh BB. Rigor mortis in beef. J Science Food Agric 1954;5:70 – 4. [25] Penny IF. The influence of pH and temperature on the properties of myosin. Biochem Journal 1967;104:609 –15. [26] Bertram HC, Karlsson, AH, Andersen HJ. Intramuscular water mobility and distribution in porcine M. longissimus dorsi as influenced by two cooling regimes. A low-field NMR relaxation study. Submitted to J Agricultural Food Chem 2001. [27] Offer G. Modelling of the formation of pale, soft and exudative meat: effects of chilling regime and rate and extent of glycolysis. Meat Science 1991;30:157– 84. [28] Blum JJ. Interaction between myosin and its substrates. Arch Biochem Biophys 1960;87:104 –19.
[29] Kauffman RG, Sybesma W, Smulders FJM, Eikelboom G, Engel B, van Laack RJLM, Hoving-Bolink AH, Sterrenburg P, Nordheim EV, Walstra P, van der Wal PG. The effectiveness of examining early post mortem musculature to predict ultimate pork quality. Meat Science 1993;34:283–300. [30] Bertram HC, Petersen JS, Andersen HJ. Relationship between RN⫺ genotype and drip loss in meat from Danish pigs. Meat Science 2000;56:49 –55. [31] Henckel P, Karlsson A, Oksbjerg N, Petersen JS. Control of post mortem pH decrease in pig muscles: experimental design and testing of animals models. Meat Science 2000;55:131– 8. [32] Hessing MJC, Hagelsø AM, Schouten WGP, Wiepkema PR, van Beck JAM. Individual behaviour and physiological strategies in pigs. Physiol Behaviour 1994;55:39 – 46. [33] Bendall JR, Swatland HJ. A Review of the relationships of pH with physical aspects of pork quality. Meat Science 1988;24:85–126. [34] Long VP, Tarrant PV. The effect of pre-slaughter showering and post-slaughter rapid chilling on meat quality in intact pork sides. Meat Science 1990;27:181–95.
Post mortem energy metabolism and pH development in porcine M. longissimus dorsi as affected by two different cooling regimes. A 31P-NMR spectroscopic study Hanne Christine Bertrama,*, Sune Dønstrupa, Anders Hans Karlssona, Henrik Jørgen Andersena, Hans Stødkilde-Jørgensenb a
Department of Animal Product Quality, Danish Institute of Agricultural Sciences, Research Centre Foulum, P.O. Box 50, DK-8830 Tjele, Denmark b The MR Research Centre, Aarhus University Hospital, Skejby Sygehus, DK-8200 Århus N, Denmark Received 15 December 2000; accepted 7 June 2001
Abstract 31
P-NMR spectroscopy was carried out on M. longissimus dorsi samples chilled by two different cooling profiles corresponding to commercial batch and tunnel chilling. The half-life of post mortem phosphocreatine (PCr) degradation was found to be significantly less in muscle samples exposed to tunnel chilling (rapid) compared with muscle samples exposed to batch chilling (soft) conditions, while no difference in the post mortem ATP degradation was found. Moreover, the post mortem pH development in the muscle samples differed considerably between the two cooling regimes. A maximum difference of approx. 0.25 pH units between the two cooling profiles was observed around 150 min post mortem. Theoretical calculations of the registered pH difference between rapid and soft chilling of muscle samples revealed that the temperature effect on the buffer capacity of muscle is the major determining factor in the detected difference in intracellular pH between the two cooling profiles, while any contribution from a temperature-induced delayed progress in the lactate formation post mortem seems negligible. Moreover, calculations on the effect of the registered pH difference between rapid and soft chilling of muscle samples resemble a 2.5 times greater denaturation of myosin in samples which were chilled softly compared with samples chilled more rapidly. Finally, the relationship to the functionality of meats from soft and rapid chilled pork carcasses is discussed. © 2001 Elsevier Science Inc. All rights reserved. Keywords:
31
P-NMR spectroscopy; Temperature; Intracellular pH; Water-holding capacity; Pork
1. Introduction Water holding capacity (WHC) is of major concern in the meat industry, as it affects both economic and sensory attributes of the meat. High incidences of porcine meat with unacceptable/reduced WHC, so-called PSE (Pale Soft Exudative) meat, some decades ago led to the introduction of tunnel chilling as an alternative to the conventional batch chilling [1]. Several studies have documented a positive effect of tunnel chilling on WHC [1– 4]. However, the explanation of why tunnel chilling improves WHC of the meat is far from understood. The improved WHC may either be explained by a biochemical effect, i.e., by a direct temperature effect on post mortem energy metabolism, by a
* Corresponding author. Tel.: ⫹45 89 99 15 06; fax: ⫹45 89 99 15 64. E-mail address: [email protected] (H.C. Bertram).
structural effect, i.e., a temperature-induced effect on the mobility and distribution of water in the muscles, or by a combination of both. Moreover, even though the combination of low pH and high temperature may cause protein denaturation and result in reduced WHC [5–7], a further understanding of how the chilling regime affects protein denaturation post mortem is necessary in order to be able to optimize the chilling procedure. The major reasons why a more thorough understanding of the effect of different chilling regimes on post mortem energy metabolism is not available, are the lack of suitable methods for these investigations. Due to the non-invasive and non-destructive character of NMR spectroscopy, 31P-NMR spectroscopy is a suitable method in the study of post mortem energy metabolism in muscle tissue [8 –10]. 31P-NMR spectroscopy makes it possible to follow the time course of the degradation of phosphocreatine (PCr), ATP and phosphomonoesters (PME), and together with the simultaneous formation of inorganic
0730-725X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 0 - 7 2 5 X ( 0 1 ) 0 0 4 1 2 - X
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phosphate (Pi), it gives valuable information about energy status in post mortem muscle tissue. Furthermore, intracellular pH can be calculated from 31P-NMR data. The present study aims to investigate how a commercial (rapid) tunnel chilling affects the post mortem energy metabolism and pH development in pig muscle tissue compared with a commercial (soft) batch chilling using NMR 31 P-spectroscopy. Furthermore, the influence of cooling regime on protein denaturation was considered from a theoretical point of view in relation to WHC of meat.
2. Materials and methods 2.1. Animals and sampling Sixteen female Danish Landrace pigs with a live weight of approx. 80 kg at the time of slaughter was used in the present experiment. Approx. 24 h before slaughter, the pigs were transported from the producer’s farm to the Institute of Experimental Clinical Research at Skejby Hospital, Denmark, where the slaughtering was carried out. The pigs were killed by i.v. injection of pentobarbital (50 mg/kg live weight) after i.v. injection of midazolam (Dormicum®, Alpharma A/S Oslo, Norway, 0.6 mg/kg live weight) used as premedication. Immediately after killing, a sample of approx. 6 cm was taken at the level of the last rib from M. longissimus dorsi. From this sample, a sub-sample with a cross-sectional area of approx. 2.5 ⫻ 2.5 cm2 and a length of approx. 5 cm was cut out along the fiber direction and placed in the tube used for NMR measurements. 2.2. pH-measurements pH was measured with an insertion electrode (Metrohm AG CH-9101 Herisau) equipped with an insertion glass electrode (Hamilton Tiptrode P/N: 238⬘080, Switzerland) at 45 min and 24 h post mortem in the remaining sample (see above) taken at the level of the last curvature of M. longissimus dorsi. The electrode was calibrated at a temperature of 22°C. A two-point calibration was performed, and the pH of the calibration buffer used was 7.000 and 4.005 at 25°C (Radiometer, DK-Copenhagen).
Fig. 1. Temperature profiles of M. longissimus dorsi for the fast and slow cooling. The fast cooling profile corresponds to a commercial tunnel chilling, and the slow cooling profile corresponds to a commercial batch chilling.
points per FID and a spectral width of 7000 Hz. The first spectrum was taken between 18 and 30 min post mortem, and thereafter spectra were recorded continuously until 9 –13 h post mortem, resulting in between 120 and 170 spectra per animal. Volume selection with a size of 12 ⫻ 12 ⫻ 12 mm was performed using the ISIS-method [11]. Thermostatic control and regulation of the samples was ensured by an air flow (24 l/min) controlled by a temperature variable unit (FTS Systems, model TP-88, Stone Ridge, New York, USA). By programming the temperature unit, it was possible to have the temperature regulated automatically during the measuring period. Two temperature programs, resulting in two different cooling profiles corresponding to a commercial tunnel (fast) and batch (slow) chilling, were developed. Development of thermal equilibrium in the muscle samples during exposure to the profiles was assured in initial investigations. The temperature profiles are shown in Fig. 1. Accordingly, the samples were placed in the NMR-probe during the whole measuring period, and exactly the same sample position was measured under each acquisition. Of the 16 samples included in the experiment, 8 samples were exposed to each cooling profile. The spectra were phased manually, and chemical shifts and areas of the signals were calculated using a least-square regression program (included in the Varian software), fitting the spectrum to Lorentzian lines. Chemical shifts were reported in ppm relative to the PCr signal.
2.3. NMR measurements 31
P-NMR spectra were recorded at 121.4 MHz on a 7 Tesla Sisco 300/173 spectrometer (Varian Associated, Inc., Palo Alto, CA) equipped with a 30 mm diameter in-house build single circular probe. After fine tuning of the probe with the sample installed, the magnetic field homogeneity was optimized on the FID of water protons. A half-width of PCr signal in the 31P-spectrum in the range 35–75 Hz was obtained. Each spectrum was an average of 32 transients with a recycle time of 8 s, accumulated in a total time of 4 min and 16 s. The acquisition parameters used were 7168
2.4. Temperature dependence of saturation effects (longitudinal relaxation time) and saturation transfer effects The non-invasive and non-destructive character of 31PNMR spectroscopy makes the technique very suitable for studying the post mortem metabolism in muscles, as changes in phosphor metabolites can be registered continuously. However, using 31P-NMR spectroscopy to study temperature effects on post mortem metabolism includes some further considerations, as relaxation mechanisms and
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thereby signal intensity depend on temperature. It is well recognized that an underestimation of the signal may occur because of partial saturation, if the nuclears are not fully relaxed before the succeeding excitation. However, longitudinal relaxation (T1) is temperature dependent, and therefore different degrees of saturation effects may turn out at different temperatures. Moreover, exchanges between P nucleus of PCr and of the ␥-phosphate of ATP may generate saturation transfer effects [12–13], which can also be expected to be temperature dependent. Consequently, temperature dependence on saturation effects (longitudinal relaxation time) and saturation transfer effects were initially determined on muscle samples exposed to the two cooling profiles. For this purpose a special protocol was developed. Succeeding scans as an average of 16 transients with alternately a recycle time of 8 and 24 s were recorded continuously. A recycle time of 24 s allows more than 99% of the original magnetization to recover for all metabolites [14], as T1 for Pi, which is the metabolite having the longest T1, was found to be approx. 5 s in muscle tissue. The areas of signals obtained with a recycle time of 8 s were compared with areas of signals from spectra obtained with a recycle time of 24 s, and the ratios between the two areas were used to correct for saturation and saturation transfer effects.
5 solutions of 50 mM phosphate buffer (pH ⫽ 7.4, ion strength I ⫽ 0.16) containing a) 20 mM PCr (Boehringer Mannheim, GmbH, Germany) and 4 mM ATP (Boehringer Mannheim, GmbH, Germany); b) 15 mM PCr and 3 mM ATP; c) 10 mM PCr and 2 mM ATP; d) 5 mM PCr and 1 mM ATP; e) 0 mM PCr and 0 mM ATP. The 31P-NMR spectra were recorded with the same acquisition parameters as those used on muscle samples (see above) at a constant temperature of 20°C. 2.7. pH determination pH was calculated from the chemical shift of Pi using the Henderson-Hesselbach equation 2 [15]:
冉
pH ⫽ pKa ⫹ log
M⫽
N ␥ 2h 2I共I ⫹ 1兲 Bo 3K BT
␦O ⫺ ␦A ␦B ⫺ ␦O
冊
(2)
where Ka is the second dissociation constant for H3PO4, and ␦O,␦A and ␦B are the chemical shifts of Pi, H2PO⫺ 4 and HPO2⫺ 4 , respectively. As both pKa and chemical shift values are temperature dependent, the following temperaturecorrected algorithm was used to calculate pH from the chemical shift of Pi 3 [16]: pH ⫽
1979.5 ⫺ 5.4409 ⫹ 0.018567共T ⫹ 273兲 T ⫹ 273
2.5. Temperature dependence of magnetization Theoretically, the magnetization (M) of a set of N nuclei exposed to a magnetic induction field of strength Bo is given by equation 1 [14]:
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⫹ log
冋
␦ O ⫺ 3.280 ⫹ 0.003579T 5.625 ⫹ 0.001888T ⫺ ␦ O
册
(3)
where T is the temperature expressed in°C. 2.8. Statistical analysis
(1)
where KB is the Boltzmann constant, T the temperature, ␥ the gyromagnetic ratio, h the Planck constant and I is the nuclear spin quantum number. Accordingly, the magnetization is inversely proportional to the temperature and an increase in signal intensity is expected, when the temperature is decreased. If this parameter is not considered, the content of metabolites will be overestimated at lower temperatures. Consequently, the temperature effect on magnetization was initially determined, and also included in the calculation of the concentrations of the different metabolites. For this purpose, 31P-spectra were recorded on muscle samples exposed to the two cooling profiles 24 – 48 h post mortem, at which time the biochemical processes has ended, and where changes in the metabolites therefore no longer will take place. A recycle time of 32 s was used. 2.6. Investigation of linear response of concentration The linearity of concentration determinations of PCr and ATP was investigated with concentrations equal to those found in muscle tissue. 31P-NMR spectra were recorded on
Analysis of variance was carried out on the response variables using the Proc GLM from the Statistical Analysis System package [17]. The statistical model included the fixed effect of chilling regime. Furthermore, the Proc NLIN was used for break-point analysis on ATP data. In breakpoint analysis, data are fitted to a horizontal line, and a line with a decreasing slope [18].
3. Results 3.1. Linear response of concentration Fig. 2a and b show the correlations between concentration and area of signal from PCr and phosphate groups in ATP, respectively. Due to the high off-set frequency of the -phosphate resonance, the selected volume was partially outside the meat sample, which accounts for the low intensity of this resonance. A linear correlation coefficient of r ⫽ 0.98 was found between concentration and area of signal from PCr. For the phosphate groups in ATP, the correlation depended on the particular phosphate group. The strongest
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Fig. 3. Typical stacked plot of spectra recorded continuously from 20 min post mortem until 12 h post mortem on a muscle sample. Each spectrum is average of 32 scans accumulated after 90° pulses at 8 s intervals.
Fig. 2. Relationship between concentration and area of signal measured on solutions of phosphate buffer (pH ⫽ 7.4, I ⫽ 0.16) containing PCr and ATP. 31P-NMR spectra were recorded at 20°C. a) area of signal from PCr, b) area of signal from ␣-, - and ␥-phosphate groups in ATP.
linear correlation was found for the ␥-phosphate group (r ⫽ 0.92), and the weakest linear correlation was found for the -phosphate group (r ⫽ 0.69). 3.2. pH measurements on muscle samples In all muscle samples, pH45 min was measured with electrode to validate the variation in energy metabolism among the samples. Table 1 shows that only minor variations were found between samples. Three muscle samples had a pH45 min below 7.0, while the rest had a pH at 45 min above 7.0. The three muscle samples having a pH below 7.0 were considered outliers and left out of further analysis. In order to evaluate the glycogen stores and the extent of glycolysis post mortem, muscle pH was also measured 24 h post mortem (Table 1). No significant difference between muscle samples used in the two cooling profiles was observed.
Table 1 pH values for all animals included in analysis and for fast and slow cooling. LSMeans, standard errors (SE) and level of significance within row are shown
pH, 45 min pH, 24 h
All animals (n ⫽ 13)
Fast cooling (n ⫽ 7)
Slow cooling (n ⫽ 6)
Significance level
7.19 (0.07) 5.64 (0.12)
7.20 (0.03) 5.66 (0.05)
7.17 (0.03) 5.63 (0.05)
p ⫽ 0.52 p ⫽ 0.74
3.3. NMR measurements on muscle samples Fig. 3 shows a typical time course in the phosphorus spectra recorded continuously from 20 min until 12 h post mortem. From the left (⬃7 ppm), an increasing signal from PME is seen. At approx. 5 ppm, the signal from Pi is seen. Besides increasing in intensity, this signal also moves toward lower chemical shifts, which reflects a decrease in intracellular pH during the post mortem period. The signal from PCr is positioned at approx. 0 ppm and is inversely correlated to an identical and simultaneous increase in the Pi signal. Finally, at approx. ⫺2, ⫺7.5 and ⫺16 ppm the resonances from the ␥-, ␣-, and -phosphate groups in ATP are registered. The total amount of phosphor metabolites in the muscle tissue was assumed to be 50 mol/g muscle due to earlier results [9]. Subsequently, the amount of the different phosphor metabolites was calculated after correction for saturation and saturation transfer effects and temperature dependence on magnetization using the relative area of signals. Fig. 4 shows typical changes in the different phosphor metabolite concentrations during the post mortem period. In agreement with earlier studies [9,19], an exponentially decline in PCr during the post mortem period was found. This decline corresponded to a first order reaction, which could be described by equation 4: PCr ⫽ K PCre 共⫺bPcrtime兲
(4)
where KPCr and bPCr are coefficients. For an expression for the rate of PCr break-down, the half-life of PCr was calculated as ln 2/bPCr. Moreover, the Pi was found to grow exponential toward a steady state level, and the progress in Pi could be described by the following equation 5:
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Table 2 Results from analysis of Pi, PCr and ATP-data. LSMeans, standard errors (SE) and level of significance within rows are shown Fast cooling Slow cooling Significance (n ⫽ 7) (n ⫽ 6) level Half-life, PCr (min) 79.3 (5.08) Initial growth rate, Pi (min⫺1) 0.15 (0.003) ␥-ATP, break point (min) 259 (12.8)
Fig. 4. Typical post mortem changes in the concentration of phosphocreatine (PCr), inorganic phosphate (Pi), phosphomonoesters (PME) and ATP. Absolute concentrations are determined on the assumption that the total amount of phosphor metabolites is 50 mol/g muscle in the first spectrum recorded.
Pi ⫽ K Pi共1 ⫺ e 共⫺bPitime兲兲
62.1 (5.48) p ⫽ 0.04* 0.018 (0.003) p ⫽ 0.51 281 (13.8) p ⫽ 0.27
the two cooling profiles, however, a pronounced difference appeared soon after slaughter (approx. 40 – 60 min post mortem). pH was significantly higher during fast cooling compared with the slow cooling from 100 to 300 min post mortem. A maximum difference between the two cooling profiles of approx. 0.25 pH-units was observed approx. 150 min post mortem.
(5)
where KPi and bPi are constants. For an expression for the rate of formation of Pi, KPi is given as the initial growth rate of Pi. Finally, ATP was found to be constant during the initial stage of the post mortem period. Subsequently, a generally linear decrease in the ATP responds was observed. In conjunction with the observed ATP course, break-point analysis [18] was carried out on ATP data (Fig. 5). Table 2 shows Pi, PCr and ATP data. The ␥-phosphate resonance from ATP was used to calculate the ATP concentration, as linear response analysis showed highest linearity for this group (Fig. 2b). The post mortem half-life of PCr was found to be significantly different for the two cooling profiles, with slow cooling significantly reducing the half-life of PCr. Initial growth rate of Pi was not significantly lower for the slow cooling profile. Break-point analysis of ATP data revealed no significant differences in the initiation of ATP break-down between the two cooling profiles. In Fig. 6 the calculated pH courses for the two cooling profiles, corrected for temperature, are shown. Initial pH (approx. the first 20 – 40 min post mortem) was identical for
Fig. 5. Typical picture of the time course of ATP degradation and breakpoint analysis (see ref. 12) of the ATP-data.
4. Discussion The present study demonstrated that a significant reduction in the post mortem processes is achieved by fast (tunnel) chilling compared with slow (batch) chilling, using 31 P-NMR spectroscopy. This was mainly reflected in the rate of post mortem degradation of PCr, which was found to be significantly lower upon fast (tunnel) chilling compared with slow (batch) chilling. In contrast, no significant effect of chilling rate was registered on ATP depletion. This might be due to lack of sensitivity in the used method, as reflected by the moderate linearity of ATP concentration compared with the PCr concentration (Fig. 2). A remarkable difference in pH progress within muscles was registered depending on the cooling regime used. Approx. 150 min post mortem, the pH-difference in muscles cooled by the two different rates reached a maximum of 0.25 pH units. This time point corresponds to a temperature difference of approx. 12°C between the two cooling re-
Fig. 6. Typical pH courses for the slow and fast cooling profile. pH is calculated from the chemical shifts of PCr and Pi and calibrated for temperature according to [14].
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Fig. 7. Effect of pH dependence of temperature on the post mortem pH courses for the two cooling regimes. Shown are; post mortem pH courses calculated on the assumption that only directly temperature dependence on pH affects pH (0.016 pH-unit/°K) for 1) fast and 2) slow cooling, the observed pH courses for 3) fast and 4) slow cooling, and the observed pH courses after subtraction of temperature dependence on pH (0.016 pH-unit/ °K) for 5) fast and 6) slow cooling.
gimes. The observed difference in pH is in accordance with earlier in vivo studies in muscle tissue, where a temperature dependence of 0.016 pH-units per °C temperature has been reported [20 –22]. It has been proposed that a temperature dependence of the intracellular acid-base regulation (buffering capacity) in muscle tissue exists with the purpose of maintaining a constant fractional dissociation of intracellular protein and preservation of enzymes [21]. This is called the alphastat hypothesis [23]. In order to elucidate the contribution of the above-mentioned temperature effect on intracellular pH in muscles exposed to the two cooling profiles, the theoretical progress in pH, as affected by the known temperature dependence on pH in muscle tissue, was constructed for the two cooling regimes. Subsequently, these constructed pH progresses were subtracted from the pH progresses based on 31P-NMR measurements to differentiate between metabolically induced pH decrease and influence of the temperature effect on muscle pH (Fig. 7). Noteworthy, the progress in metabolically induced pH decrease seems to be independent of the used cooling regimes. Consequently, the temperature effect on the buffer capacity of muscle is the major determining factor in the detected difference in intracellular pH between the two cooling profiles, while any contribution from a temperature-induced delayed progress in the lactate formation post mortem seems negligible. This is in contrast to earlier suggestions on the effect of chilling regime on post mortem metabolism and pH development, where the pH difference between different cooling profiles was supposed to be a direct decrease in the rate of glycolysis post mortem with lower temperatures and a simultaneous decrease in the formation of lactate [3,24]. In fact, the present study completely reverses this suggestion, as subtraction of the temperature effect on muscle buffer capacity not only eliminates the difference in pH between the two cooling profiles (Fig. 7), but even indicates that the initial post mortem pH decrease is faster during fast cooling compared
with slow cooling. This also agrees with the faster observed degradation of PCr upon slow cooling of the muscle, as increased [H⫹] consumption initially post mortem by the creatine kinase reaction must be expected upon slow chilling compared with a faster regime, which decreases the turn-over of the creatine kinase. Results regarding early pH measurements in post mortem muscle as affected by different chilling regimes are sparse in the literature [3]. This is most probably due to practical problems in a continuous measurement of pH in the carcass from the final cutting via the chilling systems and until temperature equilibration. However, 31P-NMR spectroscopic studies equal to those presented in the present study show the possibility of performing continuous measurement of pH post mortem and understand how the post mortem metabolism and its consequences are affected by different chilling regimes. It has been recognized for decades that the combination of low pH and high temperature post mortem may reduce the water holding capacity (WHC) of the meat due to protein denaturation [5–7]. Consequently, the differences in the pH development post mortem between the two cooling regimes in the present study may explain the improved WHC obtained using fast (tunnel) chilling instead of slower (batch) chilling, as reported elsewhere [1– 4]. Moreover, in a recent NMR proton relaxation study [26], where muscle samples were exposed to almost identical cooling profiles as in the present study, water mobility and distribution were found to differ significantly between the two cooling profiles. As the water mobility and distribution depend on micro-structural changes, which again is related to the pH development post mortem, this difference can probably also partly be explained by the difference in pH development post mortem as observed in the present study. Offer [27] has used the kinetics of the inactivation of myosin ATPase [25] to set up a relationship between the rate of inactivation of myosin ATPase (K), temperature and pH, equation 6:
冉
K ⫽ Ko exp
冊
⫺43500 10 ⫺1.3pH RT
(6)
where R is the gas constant, T is the temperature in degrees K, and Ko is an empiric constant, assumed to be 2.13 ⫻ 1034 from earlier results [25,28]. When K is known, the fraction of inactivated myosin can be determined by integration. Loss in myosin ATPase activity has not been demonstrated to directly reflect denaturation affecting the WHC of the meat; however, loss of myosin ATPase activity may be an indicator of protein denaturation [27]. In order to estimate how the different progresses in pH and temperature due to the two chilling regimes applied in the present study affect the degree of protein denaturation, the fraction of inactivated myosin was calculated on the basis of the obtained temperature and pH profiles (Fig. 1 and 6). The fraction of inactivated myosin was estimated to be approx. 2.5 times larger using the slow cooling profile
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compared with the fast cooling profile. The result of these calculations indicates that the observed differences in pH and temperature in muscles exposed to these two cooling regimes may be expected to have a huge influence on the degree of protein denaturation and thereby WHC of the meat. However, further studies are needed to support this stated relationship between degree of protein denaturation and WHC. Nevertheless, such information would be crucial to further understand the contributions of pH and temperature on the WHC of meat. Relatively large biologic variations in muscle energy status at the time of slaughter have been found in several surveys [29 –30], but also under standardized conditions [31]. This can be explained by differences in the behavioral pattern between animals when exposed to stress [32]. In the present study the pigs were anesthetized before killing, which minimizes the variation between animals and thereby reduces the ante mortem stress. This was reflected in the muscle pH measured at 45 min post mortem, which was high and almost the same between animals compared with what is reported in pigs slaughtered under traditional conditions [29,33–34]. It can of course be questioned if the use of anesthesia is comparable to any practical post mortem circumstances, where the post mortem processes will be accelerated. The present study revealed that a temperature effect on muscle pH in muscles is mainly responsible for the difference in post mortem pH progress between the two chilling regimes. Consequently, a linear temperature dependence on pH in the concerned pH interval is a prerequisite for obtaining the same effect. However, the temperature dependence on pH has only been investigated between pH 6.9 and 7.2 [21]. Hence, further studies on pigs killed without anesthesia would be valuable to demonstrate whether the same chilling effects on pH progress are obtained at higher rates of the post mortem glycolysis. In conclusion, the present study has demonstrated that a significant reduction in the post mortem processes can be achieved under conditions corresponding to tunnel chilling compared with batch chilling conditions, as the half-life of PCr post mortem was found to be significantly shorter when simulating batch chilling compared with simulated tunnel chilling. Moreover, the pH development post mortem differed considerably between the two cooling profiles, which could almost exclusively be ascribed to a temperature dependence on muscle pH.
Acknowledgments The authors are grateful to the Danish Veterinary and Agricultural Research Council (SJVF) and to the Danish Directorates for Development (Product Quality Project to H.J.A.) for financial support of this project. Moreover, Dr. Wolf Dresher, Department of Orthopaedics, Aarhus Municipal Hospital, Denmark, and co-workers, Dr. Karen Weigert and Mr. Mathias Bu¨ nger, are greatly acknowledged for
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