Proteolysis and tenderisation in reindeer (Rangifer tarandus tarandus L.) bull longissimus thoracis muscle of varying ultimate pH

PII:

SO309-1740(97)00015-6

Meat Science, Vol. 46, No. 1, 33-43, 1997 6 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0309-1740/97 $17.00+0.00

ELSEVIER

Proteolysis and Tenderisation in Reindeer (Rangifeer tarandus tarandus L.) Bull longissimus thoracis Muscle of Varying Ultimate pH E. Wiklund,“” V. M. H. Barnier,b F. J. M. Smulderqc K. Lundstriim” & G. Malmfors” “Swedish University of Agricultural Sciences, Department of Food Science, PO Box 7051, S-750 07 Uppsala, Sweden ‘Utrecht University, Department of the Science of Food of Animal Origin, PO Box 80 175, 3508 TD Utrecht, The Netherlands %stitut fur Fleischhygiene, Fleischtechnologie und Lebensmittelkunde der Veterinarmedizinischen Universitat Wien, Josef Baumanngasse 1, A-1210 Wien, Austria

(Received 26 October 1996; revised version received 31 January 1997; accepted 4 February

1997)

ABSTRACT Proteolysis and tenderisation in reindeer (Rangifer tarandus tarandus L.) M. longissimus thoracis were studied. Mm. longissimus (the part cut out between vertebrae thoracales 6-7 and vertebrae lumbales 5-6) from 12 reindeer bulls (age 1 %years) were, after ultimate pH and temperature measurements, excised and then sampled at various times post mortem for determination of sarcomere length, Warner-Bratzler shear force, calpain and calpastatin activities, cathepsins B+ L activities, active site titration of cystatin-like inhibitors, myofibrillar protein degradation, collagen content and heat solubility. Upon measurement of ultimate pH, the carcasses were divided into two pH groups; normalpH (565 5 pH 5 5.79) and high pH (pH 2 5.80). Temperature and pH fall were relatively rapid in all reindeer carcasses. Sarcomere lengths tended to be shorter in the carcasses of the high pH group. In the three carcasses with the highest ultimate pH values (6.11, 6.34 and 6.38)) sarcomere lengths were around or below 40% of resting length (1,37pm, 1.25 pm and 1.25pm, respectively) which is likely associated with the occurrence of heat shortening. Total collagen content was higher and heat solubility lower in the high pH group, which could have masked d@erences in tenderness. However, all reindeer longissimus muscle samples were found to be extremely tender regardless of ultimate pH. By 3 days post mortem Warner-Bratzler shear force values were varying between 2.1 - 4.9 kg cm2. There was a signt$cantly higher activity of p-calpain in the high pH group at 1 day post mortem. No dtflerences in shear force, myofibrillar protein degradation as observed by SDS-PAGE, m-calpain and calpastatin activities, cathepsins B+ L activities or the levels of cystatin-like inhibitors were found between the two pH groups. 0 1997 Elsevier Science Ltd

*To whom correspondence Eva.Wilkund@,lmv.slu.se

should be addressed. Tel. + 46-l 8-67 1949; Fax. + 46- 18-672995; E-mail:

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E. Wiklundet al.

INTRODUCTION Tenderness is regarded as the most important sensory attribute affecting meat acceptability (Ouali, 1990; Warkup et al., 1995). As stated in reviews by Ouali (1990) and Smulders et al. (1991), meat tenderisation is a multifactorial process dependent on a number of biological (e.g. species, age, sex and muscle type) and environmental factors (nutrition, ante-mortem stress, slaughter and chilling conditions, and ageing). Current evidence suggests that proteolysis of key myofibrillar and associated proteins is the cause of meat tenderisation (Koohmaraie, 1996). The function of these proteins is to maintain the structural integrity of myofibrils; degradation of these proteins would therefore cause weakening of myofibrils and, consequently, tenderisation (Price, 1991). There has been a debate about the specific proteases responsible for these changes. Several arguments supporting, or opposing, a single role or synergistic action of the calpain/calpastatin or the cathepsin/cystatin systems in meat tenderisation have been put forward (Barnier, 1995; Go11 et al., 1995; Roncal& et al., 1995). However, there is overwhelming evidence in support of the former system as the primary mechanism of post-mortem proteolysis (Koohmaraie, 1996). Generally speaking, reindeer meat is tender, especially in view of the short ageing times - usually no more than 2 days storage before cutting and/or freezing (Wiklund, 1995). Most of the produced reindeer meat is sold frozen and it is usually consumed immediately after thawing. This suggests that post-mortem metabolism is rapid and the efficiency of muscle proteolysis in reindeer meat is high. Even though electrical stimulation is not used for reindeer carcasses in Sweden, the post-mortem pH decline is generally very rapid: ultimate pH values could be reached as early as 15 hr post mortem. Other important parameters for reindeer meat quality, such as muscle glycogen content and ultimate pH, have been studied in various muscles from a large number of animals of different sex and age (Wiklund et al., 1995, 1996a, 1996b). Sensory quality characteristics of reindeer meat have also been described (Wiklund et al., 19963). From these studies, mainly concentrated on the effects of various pre-slaughter handling routines on reindeer meat quality, it was concluded that ultimate pH and muscle glycogen content varied considerably between different animal groups (Wiklund et al., 19956, 1996), due to a mixture of traditional and modern reindeer husbandry (and slaughter) procedures, including pre-slaughter supplementary feeding, selection of individual animals by use of a lasso, long distance lorry transport and industrialized slaughter (Malmfors and Wiklund, 1996). There are numerous reports that such variations in muscle pH and glycogen content give rise to considerable variation in tenderness in beef (e.g. Smulders et al., 1990). Therefore, the purpose of the present investigation was to study the effect of ultimate pH on tenderness (Warner-Bratzler shear force) of reindeer meat. In addition, the effect of pH on proteolytic enzyme levels and ageing rate in reindeer meat was investigated.

MATERIALS

AND METHODS

A total of 12 reindeer bulls (age 1% years) were included in the study. They were slaughtered in April and had been exposed to a pre-slaughter handling including gathering in the mountains and herding to a selection corral, a selection procedure and subsequent lorry transport to a stationary slaughterhouse. At slaughter, the reindeer were stunned with a captive bolt. Temperature and pH values were measured in the M. longissimus thoracis (at the last rib) at 1, 3, 5 and 35 hours post mortem. At 1 day post mortem, M. longissimus thoracis (the part between vertebrae thoracales 6-7 and vertebrae lumbales 5-6) from both right- and left-hand carcass side were excised,

Proteolysis and tenderisation in reindeer

35

whereafter each muscle was divided into two parts and vacuum packaged. One part of each muscle was randomly assigned to a 1, 3, 7 or 14 day ageing period (2-4C). Each muscle was sampled at 1 day post mortem to determine sarcomere length and on days 3, 7 and 14 post mortem for shear force measurements. Samples for determination of proteolytic enzyme activity were taken 1, 3 and 14 days post mortem, subsequently frozen in liquid nitrogen (-196°C) and stored at -80°C until analysis. On days 1, 3, 7 and 14 post mortem, samples were also taken for measurement of myofibrillar protein degradation. An official DFD pH ‘limit’ has not yet been established for reindeer meat. Upon measurement of the ultimate pH (35 hr post mortem) the animals were therefore divided into two pH groups: normal pH (5.65 5 pH <_ 5.79, n= 5) and high pH (pH 2 5.80, n=7) on the basis of the fact that in Swedish industry practice, ultimate pH values beyond 5.8 are associated with an increased occurrence of dark, firm, dry (DFD) meat in beef M. longissimus (Fabiansson et al., 1984). Temperature and pH

Temperature was measured with a digital thermometer (Ama-digit ad 40 th, Amarell electronic, Germany) and pH values were measured with a portable pH meter (Portamess 651-2, Knick Elektronische Messgerate GmbH and Co., Germany) equipped with an Xerolyte electrode (lot 406 M-6, Ingold Messtechnik AG, Switzerland). Sarcomere length

Sarcomere length was determined using the laser-diffraction method described by Koolmees et al. (1986). Muscle samples were fixed in glutaraldehyde and, from each sample, bundles of muscle fibres were dissected so that 10 specimens per sample could be provided. Shear force

Warner-Bratzler shear force was determined on samples heated in polyethylene bags in a waterbath at 75°C until a core temperature of 70°C was reached, whereafter they were chilled in running tap water for 40 min (Boccard et al., 1981). Ten rectangular samples of 1 cm2 cross-section were cut out from each cooked sample, parallel to the muscle fibre direction. Shear force was measured using a draw bench (Adamel Lhomargy, Division d’Instruments S. A. Paris, France) equipped with a Warner-Bratzler shearing device. A triangular blade (1.2mm thick) was used at a crosshead speed of 298 mm min-‘. Determination

of calpain and calpastatin

activity

Separation of calpains and calpastatin was performed according to Etherington (1987) with minor modifications as described by Geesink (1993). SDS gel electrophoresis

et al.

and densitometry

Myofibrils were prepared according to the method of Ouali et al. (1983). Degradation of myofibrillar proteins was assessed by means of SDS-PAGE according to Greaser et af. (1983) on a 12.5% acrylamide separating gel. The intensity of the protein bands was measured with an LKB Ultrascan XL Enhanced Laser Densitometer.

36

E. Wiklundet al.

Determination of collagen content and heat solubility The extraction of heat soluble collagen was performed according to Hill (1966) and acid hydrolysis according to Bauer (1991) with a microwave oven. Hydroxyproline was measured according to the method of Stegemann and Stalder (1967) with minor modifications described by Barnier (1995). Cathepsins B + L and their inhibitors Determination of total cathepsin B + L activity was performed on frozen meat according to Etherington et al. (1987). Muscle samples were prepared according to Bige et al. (1985) to extract the cysteine proteinase inhibitors, omitting the ultimate purification step. The amount of cystatin-like inhibitors was determined using a papain active-site titration based on the method of Anastasi et al. (1983). Statistical analyses The statistical analysis was carried out with the Statistical Analysis System (SAS Institute Inc., 1995) using the MIXED and GLM procedures. The model for comparing sarcomere length, collagen content and heat solubility included the fixed effect of pH group. When the enzyme activities, shear forces, 30kDa peptide and troponin T were compared during storage (1, 3, 7 and 14 days), the fixed effect of storage time, the random effect of animal, and the interaction (pH group x storage time) were also included in the model. Since the interactions (pH group x storage time) were non-significant for all traits, the comparisons of storage times were carried out using the pooled dataset for the whole animal material (n = 12) and not the division into pH groups.

RESULTS Temperature and pH decline No significant differences in temperature measured at 1, 3, 5 and 35 hr post mortem were found between the pH groups which is why, in Fig. 1, the temperature decline has been presented as the pooled dataset for the whole animal material (n= 12). At 1 hr post mortem, no significant difference in pH values was found between the two pH groups, but at 3, 5 and 35 hr, pH values from the two groups differed significantly (Fig. 1). Sarcomere length, collagen content and heat solubility The difference in sarcomere length between the two pH groups was negligible (p>O.O5) (Table 1). However, the correlation between sarcomere length and ultimate pH was significant (r = - 0.8) suggesting that high ultimate pH value associated with short sarcomere length (Fig. 2). In the high-pH group, total collagen content was significantly higher and collagen solubility was significantly lower than in the normal pH group (Table 1). Shear force and intensity of troponin T and 30kDa peptide Since there were no significant differences between the two pH groups in shear force or the degree of post-mortem proteolysis, as reflected by the intensity of the 30 kDa peptide,

37

Proteolysis and tenderisation in reindeer

Temp., “C ‘30

PH 6.7 ’

6.1

5.5

IO 3

5

35

Time post mortem, hr Fig. 1. Temperature and pH decline in reindeer bull longissmus thoracis muscle, measured at 1, 3, 5 and 35 hr post mortem (least-squares means). Temperature decline is presented as the pooled dataset for the whole animal material (n = 12), while the pH decline in the normal pH group (5.65 < pH 5 5.79, n = 5) and the high pH group (pH L 5.80, n = 7) are presented separately.

comparisons of storage times were also made on the pooled dataset for the whole animal material (n = 12) (Table 2). After 3 and ‘7days of storage, shear force values of both pH

groups were similar (p > O.OS), but at 14 days of storage, significantly lower shear force values were found (Table 2). A significant degradation of troponin T and, concomittantly, the appearance of a 30 kDa breakdown product was observed during storage. Proteolytic enzyme activities m-Calpain activity, cathepsin B + L activities and their endogenous inhibitors calpastatin and cystatin-like inhibitors, respectively, did not differ significantly between the two pH groups, while ,u-calpain activity was higher in the high pH group at 1 day post mortem (Table 3). During post-mortem storage, both CL- and m-calpain activities decreased TABLE 1 Sarcomere Length, Collagen Content and Heat Solubility of Reindeer Longissimus thoracis Muscle (least-squares means i standard errors) from Two Different pH-groups; Group 1 (5.65 5 pH < 5.80, n = 5), Group 2 (pH 2 5.80, n = 7), and the Degree of Significance for the Effect of pH-group Trait

Normal pH group

Sarcomere length (pm) Collagen content (pg hydroxyproline Heat solubility (%)

gg’ muscle)

1.60*0.08 443a f 22 3.11”*0.2

High pH group

Degree of significance*

1.45 f 0.07 527b* 18 2.36b f 0.2

ns. *

*n.s. = p > 0.05; * = p 5 0.01. Within-trait means having the same superscript are not significantly different (p > 0.05). l

l

l

*

38

E. Wiklund

et al.

Sarcomere length, pm

:ir&&q /

-

‘+

I

1.6 /-

++ 1.2 5.6

5.7

5.8

5.9

6.0

6.1

6.2

6.3

6.4

Ultimate pH

Fig. 2. Relationship between sarcomere length and ultimate pH in reindeer bull longissimus thoracis muscle from animals in two pH groups; normal pH group (5.65 5 pH 5 5.79, n = 5) and high pH group (pH > 5.80, n = 7).

significantly. Calpastatin activity was not affected by post-mortem storage. Cathepsin B + L activity increased significantly, while the level of cystatin-like inhibitors decreased significantly between 3 and 14 days post mortem.

DISCUSSION The diet and nutritional status of reindeer vary considerably over the year (Ahman, 1994) and, hence, also ultimate pH values and glycogen content in the muscles (Petajija, 1983; Wiklund et al., 1995, 1996a). Of the potentially stressful pre-slaughter handling routines so far studied for reindeer (lorry transport, lairage, helicopter herding and two different selection techniques), only the traditional selection technique of using a lasso caused meat quality deterioration (i.e. high ultimate pH values and low muscle glycogen content) (Wiklund et al., 1995, 1996a, 19966). In earlier studies, it was indicated that pH fall in reindeer meat was surprisingly rapid (Wiklund et al., 1995; Gundersen and Nummedal, 1996) compared with other ruminants and it was suggested that measurement of ultimate pH as early as 15 hr post mortem was justified (Wiklund et al., 1995). The present results confirmed that glycolysis in reindeer meat had proceeded rapidly, as the pH values measured in the two pH groups at 3 hr postmortem were as low as 5,87 and 6.14, respectively. The fibre profile of reindeer muscles has been investigated and M. longissimus dorsi has been found to contain a large proportion of IIB fibres (Kiessling and Rydberg, 1983; Es&n-Gustavsson and Rehbinder, 1984). Whereas this fibre type is usually characterised by its fast contractile properties and glycolytic capacity, the IIB fibres in reindeer muscles are both highly glycolytic and remarkably oxidative (Kiessling and Kiessling, 1984; Es&n-Gustavsson and Rehbinder, 1985). A

39

Proteolysis and tenderisation in reindeer

TABLE 2

Shear Force at 70°C Intensity of Troponin T (% of Troponin T Relative to Actin) and 30kDa Peptide (% of 30 kDa Peptide Relative to Actin) in Reindeer Bull Longissimus thoracis Muscle (least-squares means f standard errors) from Two pH Groups (normal pH: 5.65 5 pH 5 5.79, n= 5; high pH: pH > 5.80, n=7) and from the Pooled Dataset from the Whole Animal Material (n= 12) after Different Storage Times (1, 3, 7 and 14 days at +2”C to +4”C), and the Degree of Significance for the Effects of pH Group and Storage Time Normal pH group

High pH group

Degree of significance

Pooled dataset (degree of sign$cancP)

Shear force (kg cm2) 3 days I days 14 days

3.4kO.3 2.6*0.2 2.2~kO.3

2.4ztO.4 2.1 f 0.4 1.7zto.4

n.s. n.s. ns.

2.9a f 0.2 2.6af0.2 1.9bh0.3 **

30kDa 1 day 3 days 7 days 14 days

n.m.+ 2.2& 1.0 7.4rt 1.0 9.7 + 1.0

n.m. 4.7k 1.8 5.4+ 1.8 13.8zt 1.8

nm. n.s. ns. ns.

n.m. 3.4”* 1.0 6.4b f 1.0 11.7Ck 1.0 ***

11.9* 1.2 7.0* 1.2 3.2~t 1.2 n.m.

13.2*2.0 4.1 i 2.0 0.7 f 2.0 n.m.

n.s. ns. n.s. n. m.

12.5” zt 1.2 5.6b* 1.2 2.0”11.2 ***

Trait

Troponin T 1 day 3 days 7 days 14 days

“ns. = p>O.O5; ** = p 0.05).

hypothesis has been put forward to explain the large amount of IIB fibres in reindeer muscle, which is attributed to the reindeer’s need of this tissue for survival as it constitutes a source of energy reserve during the forced starvation in winter (Kiessling and Rydberg, 1983). The muscle fibre-type composition has been shown to determine the rate of tenderisation, as ageing seems to occur more rapidly in fast-twitch-glycolytic bovine muscles than in slow-twitch-oxidative muscles (Gann and Merkel, 1978; Ouali et al., 1983; Geesink et al., 1992~). The physiological significance of the particular properties of reindeer IIB fibres might be that they act as fast-twitch-glycolytic fibres during post-mortem pH decline, proteolysis and ageing; their high oxidative capacity could be of advantage when reindeer migrate over long distances and also when their diet and the climatic conditions change during the winter; reindeer often have to rely on their lean tissue and fat reserves as energy source. Although one of the objectives of the present study was to collect animal material representative for the broad variation in ultimate muscle pH value, the pH range obtained (566.38) may have been too narrow and the number of animals too few to allow evaluation of variations in tenderness. Furthermore, all measured Warner-Bratzler shear force values in reindeer meat were very low, regardless of ultimate pH and sarcomere length, and already

40

E. Wiklund et al.

TABLE 3 Enzyme Activities (p-, m-calpain, Calpastatin, Cathepsin B + L and Cystatin-like Inhibitors) in Reindeer Longissimus thorucis Muscle (least-squares means * standard errors) from Two pH Groups (normal pH: 5.65 5 pH 5 5.79, n= 5; high pH: pH 2 580, n=7) and from the Pooled Dataset from the Whole Animal Material (n = 12) after Different Storage Times (1, 3, 7 and 14 days at + 2°C to + 4”C), and the Degree of Significance for the Effects of pH Group and Storage Time Trait PH group

High pH group

Degree of significance’

-

Pooled dataset (degree of signiJicance”)

CL-Calpain activity, units 1 day 3 days 14 days

18.4 f 3.5 12.0* 3.5 12.8*3.5

35.9zt6.1

25.7 f 6.1 16.7f6.1

* ns. ns.

m-Calpain activity, units 1 day 3 days 14 days

35.0 f 3.0 40.0*3.0 15.313.0

47.6 f 5.1 34.4f5.1 21.515.1

n.s. n.s. n.s.

41.3”f3.0 37.2a f 3.0 18.4b*3.0 ***

Calpastatin activity, units 1 day 3 days 14 days

286.7 f 23.8 216.3i23.8 192.8 f 23.8

251.Ok41.2 262.7 i 41.2 219.3*41.2

n.s. n.s. n.s.

268.8” f 23.8 239.5a i 23.8 206.0a f 23.8 ns.

Cathepsin B + L activity, units 1 day 3 days 14 days

24.2 f 2.0 25.9k2.0 28.5k2.0

20.8 f 3.4 22.3 f 3.4 29.6 * 3.4

ns. ns. n.s.

22.5” f 2.0 24.1”&2.0 29.0bk 2.0 **

293.2Zt11.3 271.0* 11.3 244.0 f 11.3

295.01 19.5 293,7* 19.5 249.0 f 19.5

n.s. n.s. ns.

294.1a+ 11.3 282.3” f 11.3 246.5b* 11.3 ***

Cystatin-like inhibitors pm01 g-r 1 day 3 days 14 days

27.1”&3.5

18.gabf3.5 14.7b* 3.5 *

%.s. = p>o.o5; * = pso.05; ** = p 0.05). at 3 days post mortem they were varying between 2.1- 4.9 kg cm-*. As regards the order of magnitude of the shear force values, it must be borne in mind that a triangular instead of a rectangular blade was used to shear the rectangular samples. This may have resulted in absolute values lower than may be expected when using the recently recommended procedure described by Chrystall et al. (1994). Due to the small variation in tenderness, it was not possible to find significant correlations between sarcomere length and tenderness or ultimate pH and tenderness, which have earlier been described for beef muscle (Marsh and Leet, 1966; Purchas, 1990; Smulders et al., 1990). However, one significant correlation was found in the present study: between ultimate pH values and sarcomere lengths (r = a-8), suggesting that high pH values were associated with short sarcomere lengths. This relationship has also been found in other species, e.g. beef M. longissimus dorsi

Proteolysis and tenderisationin reindeer

41

(Purchas, 1990) and pork M. longissimus dorsi (Tornberg, 1996). The very short sarcomere lengths (1.37pm, 1.25 pm and 1.25pm) measured in the three carcasses with the highest ultimate pH values in the present study (6.11, 6.34 and 6.38, respectively) may possibly be explained by the phenomenon of ‘rigor shortening’ (Honikel, 1992). In severely shortened sarcomeres ( < 1.5 hm), the thick myosin filaments may have penetrated and disrupted the Z-disk structure, causing an increase in tenderness (Marsh and Carse, 1974). The differences in collagen content and heat solubility observed between the two pH groups in the present study might have affected meat tenderness, though they were not reflected in the Warner-Bratzler shear values of meat cooked at 75°C. Since all measurements were taken completely at random, methodological flaws are unlikely to be the source of this phenomenon. Young and Braggins (1993) found that in ovine M. semimembranosus, the collagen concentration was the more important determinant of eating quality, whereas Warner-Bratzler shear data were more clearly related to collagen solubility. Further studies are needed to determine the influence of animal age on collagen characteristics in reindeer muscles and to establish whether or not collagen concentration as well as solubility is correlated to Warner-Bratzler shear force values. Degradation of troponin T during post-mortem storage and consequent appearance of a 30 kDa breakdown product (Ho et af., 1994) have been proposed as an indicator of beef tenderness (Uytterhaegen et al., 1992). As no significant correlation between the degradation of troponin T and shear force was found in the present study, further investigations are required to ascertain which protein degradation during storage is related to tenderness improvement in reindeer meat. In the present study, a higher activity of @-calpain was found at 1 day post mortem in the high pH group as compared to the normal pH group. This result is not in agreement with those reported by Geesink et al. (19928) in bovine muscles and the calpain-activity model proposed by Dransfield (1994). Dransfield (1994) reported that, for high ultimate pH meat, a rapid rigor development and an early release of calcium ions can produce very active calpains which are short-lived at the prevailing high temperature. Furthermore, the calpain-activity model (Dransfield, 1994) predicts that tenderisation in high pH meat occurs before 24 hr and no ageing occurs. In our study, shear force values were measured not earlier than after 3 days of post-mortem storage, at which time these were similar for both pH groups. In the high pH group, a trend (JJ< 0.07) towards lower shear force values between 7 and 14 days post mortem was observed. From these results, it can be hypothesized that (according to the low shear force values measured at 3 days post mortem) the tenderistation process in reindeer meat was fast and that no significant difference in shear force values between the two pH groups was to be expected. The tendency of an ageing effect observed between 7 and 14 days post mortem is difficult to assign to the calpain system as neither p-calpain nor its endogenous inhibitor calpastatin were affected. In addition, it must be borne in mind that the extraction procedure for the calpain system and the estimation of its potential activity used in this study might have underestimated the proteolytic activity of p-calpain, as indicated by the results of Geesink and Go11 (1995). In the present study, the decline of p-calpain activity during post-mortem storage was rather small, which suggests that early post-mortem activation of p-calpain had taken place, whereafter activity was lost through an autolytic process. As recently reported in a review by Koohmaraie (1996) F-calpain retains some of its activity even after extensive autolysis and prolonged storage at +4”C. It has been reported that during ageing, the expression of activity of cathepsins B + L might increase as a result of a decrease in the level of cystatins (Barnier et al., 1993; Barnier, 1995). The results of the present study support this hypothesis and are consistent with the latter author’s data on beef. Furthermore, the significant increase in cathepsin B+ L activity and the significant decrease of

42

E. Wiklund et al.

cystatin-like inhibitors observed between 3 and 14 days of post-mortem storage are in parallel with the significant decrease in shear force. Reindeer longissimus thoracis muscle was found to be extremely tender regardless of ultimate pH, and no differences in shear force, myofibrillar protein degradation as observed by SDS-PAGE, m-calpain and calpastatin activities, cathepsins B + L activities or the levels of cystatin-like inhibitors were found between the two pH groups. However, there was significantly higher activity of p-calpain in the high pH group at 1 day post mortem. As no significant correlation between the degradation of troponin T and shear force was found, further studies on the degradation pattern of high molecular weight proteins and the changes occurring at an ultrastructural level during the post-mortem tenderisation process are required. Comparable reindeer and beef animal materials should be investigated concerning tenderness development and levels of proteolytic enzymes and their inhibitors as this could be helpful in identifying the underlying muscle biological mechanisms. ACKNOWLEDGEMENTS The authors thank the staff at ‘Tottes slakt AB’ in Harads and Gertrud Andersson for their assistance and co-operation in the collection of samples. We are also grateful to Albert van Dijk, Philip Schippers and Mauro de Rosa for their invaluable assistance in the lab. Financial support for this work was provided by OECD (Organisation for Economic Co-operation and Development, Paris, France) within the programme ‘Co-operative Research Project on Biological Resource Management’, and also by the Swedish Council for Forestry and Agricultural research. REFERENCES Ahman, B. (1994) Radiocaesium in reindeer (Rangifer tarandus tarandus) after fallout from the Chernobyl accident. Dissertation, Department of Clinical Nutrition, Swedish University of Agricultural Sciences, Uppsala, Sweden. Anastasi, A., Brown, M. A., Kembhavi, A. A., Nicklin, M. J. H., Sayers, C. A., Sunter, D. C. and Barrett, A. J. (1983) Journal ofBiochemistry, 211, 129. Barnier, V. M. H. (1995) Determinants and precitors of beef tenderness. Involvement of muscle proteinases and their inhibitors in post mortem tenderisation. Dissertation, Department of the Science of Food of Animal Origin, Utrecht University, The Netherlands. Barnier, V. M. H., van Laack, H. L. J. M. and Smulders, F. J. M. (1993) In 39th International Congress of Meat Science and Technology, Calgary, Canada, p. 148. Bauer, F. (1991) In 37th International Congress of Meat Science and Technology, Kulmbach, Germany, p. 1127. Bige, L., Ouali, A. and Valin, C. (1985) Biochimia et Biophysics Acta, 843, 269. Boccard, J. L., Buchter, L., Casteels, M., Cosentino, E., Dransfield, E., Hood, D. E., Joseph, T. L., MacDougall, D. B., Rhodes, D. N., Schiin, I., Tinbergen, B. J. and Touraille, C. (1981) Livestock Production Science, 8, 385.

Chrystall, B. B., Culioli, J., Demeyer, D., Honikel, K., Merller, A. J., Purslow, P., Schwligele, F., Shorthose, R., Uytterhaegen, L., Bruggemann, D. and Klettner, P. G. (1994) In 40th Znternational Congress of Meat Science and Technology, The Hague, The Netherlands. Dransfield, E. (1994) Meat Science, 36, 105. Es&-Gustavsson, B. and Rehbinder, C. (1984) Rung@, 4, 2. Es&n-Gustavsson, B. and Rehbinder, C. (1985) Comparative Biochemistry and Physiology, 3, 675. Etherington, D. J., Taylor, M. A. J. and Dransfield, E. (1987) Meat Science, 20, 1. Fabiansson, S., Ericksen, I., Laser ReuterswHrd, A. and Malmfors, G. (1984) Meat Science, 10, 21. Gann, G. L. and Merkel, R. A. (1978) Meat Science, 2, 29.

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