Proteolytic enzyme and inhibitor levels in reindeer { Rangifer tarandus tarandus L . ) vs . bovine longisszmus muscle , as they relate to ageing rate and response

E ight reindeer bulls (age 1.5 years) and six Friesian bulls (age 1.5 years) were included in the study for comparison of tenderness. The reindeer were slaughtered at a commercial reindeer slaughter plant in northern Sweden and the Friesian bulls at a commercial slaughter plant i n The Netherlands. Samples for determination of calpain/calpastatin activity were taken from the M. longtssimus (LO) w i t h i n 1 h post mortem (p.m.), and at various times p . m . p H and temperature were registered in L O ; ultimate p H values were measured at 24 h p . m . for beef and at 35 h p . m . for reindeer. A t day 1 p . m . , samples of L O from both carcass sides were excised, divided in two parts, vacuum packaged and stored at 0-2 °C. One part of each muscle was randomly sampled at 1, 3, 7 and 14 days p . m . for determination of shear force, proteolytic enzyme activity, myofibrillar protein degradation, collagen content and heat solubil i ty. p H and temperature fall was faster in reindeer than in beef. Collagen content in reindeer muscle was found to be low but collagen was 4 times less soluble as compared w i t h beef. Reindeer L O was found to be extremely tender, at 3 days p . m . shear force val ues were only 2-3 kg/cm 2 (8-12 kg/cm 2 for beef L O ) . In reindeer meat, the jJ.-calpain levels dropped to about 5 5 % w i t h in 3 days. Troponin T and 30 k D a values were not related to changes in tenderness in reindeer meat. Cathepsin act ivities in reindeer were up to ten times higher than in beef. As in beef, cathepsin B + L levels in reindeer increased during storage, which is probably associated w i t h a decrease in cystatin-like inhibi tor levels. K e y words: collagen, meat quality, proteases, shear force. Rangifer, 19(1): 13-18


Introduction
As reviewed by Ouali (1990) and Smulders et al. (1991), meat tenderisation is a variable process depending on a number of biological factors (e.g.species, age, sex and muscle type) and environmental factors (nutrition, ante mortem stress, slaughterand chilling conditions and ageing).
Modern reindeer management often includes various stressful pre-slaughter conditions, such as gathering, herding and long distance animal transport.These conditions can promote the occurrence of DFD (Dark, Firm, Dry) meat (Wiklund etal., 1995;1996a;1996b;1997a).The incidence of DFD in reindeer meat has been measured in Sweden (DFD Rangifer, 19 (1), 1999 limit pH 6.2 in M. longissimus), where 6% of 3400 reindeer carcasses were classified as DFD (Wiklund et al, 1995).Usually, reindeer meat is consumed after no more than 2-3 days of ageing, and allegedly is appreciated particulary for its tenderness; this suggests that reindeer muscle might have a higher ageing rate than beef (Wiklund et al, 1997b).The comparison of their proteolytic capacity and tenderness could be helpful in challenging this hypothesis and identifying the underlying muscle biological mechanisms.
The objective of this study was to compare tenderness development and proteolytic enzyme levels and their inhibitors in bovine and reindeer longissimus muscle.

Reindeer
In Sweden, eight reindeer bulls (age 1.5 years, average carcass weight 33 kg with standard deviation J-= 13 kg), were slaughtered at a commercial slaughter plant.The animals had been exposed to gathering and herding to a corral, a selection procedure, road transport over a distance of about 400 km (i.e.5-6 hours driving) and overnight rest before slaughter with free access to hay and water.The animals were stunned with a captive bolt, bled and skinned.The pH and temperature in the centre of M. longissimus of the righthand carcass side, were measured at 1, 3, 5 and 35 h post mortem (p.m.).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 & Co, Germany) equipped with a polymere electrode (Xerolyt, lot 406 M-6, Ingold Messtechnik AG, Switzerland).Samples for determination of calpain/calpastatin activity were taken within 1 h p.m.At 1 day p.m., samples of M. longissimus from both carcass sides were excised, divided in two parts, vacuum packaged and stored at 0-2 °C.One part of each muscle was randomly sampled at 1, 3, 7 and 14 days p.m. for determination of shear force, proteolytic enzyme activity, myofibrillar protein degradation, collagen content and heat solubility.All samples were frozen in liquid nitrogen (-196 °C) and transported at -80 °C to laboratory facilities in the Netherlands.

Pilot experiment
From the same commercial slaughter plant as mentioned above, muscle samples (Al.longissimus) from 14 six reindeer bulls (age 1.5 years) were collected.The animals had been exposed to the same type of preslaughter handling and treatment as described above.Samples for determination of calpain/calpastatin and cathepsin B+L/cystatin activity were taken as soon as possible after slaughter (i.e. 20 min post mortem).

Beef
Six Friesian bulls (age 1.5 years, average carcass weight 360, s -33, kg) were slaughtered at a commercial slaughter plant in the Netherlands.The pH and temperature in the centre of M. longissimus of the righthand carcass side, were measured at 45 min, 1.5, 3 and 24 h p.m. Temperature was measured with a digital thermometer (Tastotherm D700, Germany) and pH values were measured with a portable pH meter (type CG 818, Schott Geräte, Hofheim, Germany) equipped with a polymere electrode (Xerolyt, lot 406-M6-DXK S7/25, Ingold, Germany).Samples for determination of calpain/calpastatin activity were taken within 1 h p.m.At 1 day p.m., samples of Al. longissimus from the lefthand carcass side were excised, divided in four parts, vacuum packaged and stored at 0-2 °C.At random, one part of each muscle was sampled at 1, 3, 7 and 14 days p.m. for determination of shear force, proteolytic enzyme activity, myofibrillar protein degradation, collagen content and heat-solubility.Samples fot determination of calpain/calpastatin and cystatin-like inhibitor activities were frozen in liquid nitrogen (-196 °C) and stored at -80 °C until determination.

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 cm 2 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 (1.2 mm thick) was used at a crosshead speed of 298 mm/min.

SDS gel electrophoresis 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 al. (1983) on a 12.5% acrylamide separating gel.The intensity of the protein bands was measured with a LKB Ultrascan XL Enhanced Laser Densitometer.

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 & Stalder (1967) with minor modifications described by Barnier (1995).

Cathepsin B+L and their inhibitors
In reindeer meat, determination of total cathepsin B -+L activity was performed on frozen meat according to Etherington et al. (1987).In beef the determination of free cathepsin B+L activity was performed on fresh meat samples according to Wu et al. (1985), subsequently aliquots were frozen in liquid nitrogen (-196 °Q and stored at -80 °C.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), described by Barnier (1995).

Statistical analyses
Significance of difference between ageing times was tested with a one-way analysis of variance (SPSS/PC+, Chicago, IL).

pH, temperature, collagen content and heat solubility
Due to their low dressed carcass weights (Table 1), the temperature fall was relatively fast in reindeer (Fig. lb).The pH fall was also rather fast in reindeer in comparison with beef (Fig. la).The present results are in good agreement with earlier studies, where consistently a rapid pH fall in reindeer meat was found (Wiklund et al., 1995;Gundersen & Nummedal, 1996).Collagen content in reindeer muscle was rather low but the collagen was quite insoluble (4 times less soluble as compared with beef, Table 1).

Tenderness, proteolysis and myofibrillar protein degrada tion
In the present study, reindeer longissimus muscle was found to be extremely tender.As early as 3 days post mortem, shear force values were only 2 to 3 kg/cm 2 , which was very low compared with measured shear force values in beef (values around 10 kg/cm 2 ) after the same ageing time (Table 2).The speed of p.m. proteolysis in reindeer muscle appears to be very fast, therefore.Whereas initial values of JJ.-calpain assessed in reindeer meat in a pilot experiment on very early p.m. samples (20 min p.m.) were in the 42 -79 unit range (» = 6), these levels dropped to about 55% within 3 days (Fig. 2a).As |X-calpain, once activated, gradually loses its activity through an autolytic process, a decrease in |J.-calpain activity would mean that the enzyme has been active and possibly caused tenderisation.During p.m. storage, muscle calpastatin levels were lower in reindeer than beef (Fig. 2b).Assuming a similar enzyme autolysis/activation mechanism for both beef and reindeer, this finding suggests that most of the proteolysis in reindeer meat has already taken place within the first few days after slaughter.
Degradation of troponin T during p.m. storage and consequent appearance of a 30 kDa breakdown product (Ho et al., 1994) have been reported as an indicator of beef tenderness (Uytterhaegen et al., 1992).Surprisingly, troponin T and 30 kDa values (Table 2), following a similar pattern as in beef, are difficult to relate to tenderness changes in reindeer meat in the present study.
Remarkably, cathepsin activities in reindeer meat in the present study were up to ten times higher than in beef (Fig. 3).As in beef (Barnier et al., 1993;Barnier, 1995), reindeer cathepsin B+L levels increased during storage of the meat, which is probably associated with a decrease in cystatin levels  (Fig. 4).The results of the present study support this hypothesis and are consistent with the latter author's data on beef.

Conclusions
In reindeer longissimus muscle ultimate tenderness was almost reached at 3 days p.m. Reindeer meat samples had much higher proteolytic enzyme activ- Fig. 1. a) pH and b) temperature (°C) decline in beef (« = 6) and reindeer (n -8) M. longissimus (means and standard deviations).
ity and lower inhibitor levels as compared with beef.One should be cautious, however, to extrapolate muscle physiological observations in beef to other ruminant species.As no relationship between the degradation of troponin T and tenderness (shear force) was found in reindeer meat in the present study, further studies on the degradation pattern of high molecular weight proteins and the changes occurring at an ultrastuctural level during the post mortem tenderisation process are required.The observations made in the present experiment justify further muscle proteolytic enzyme-and tenderness studies in reindeer as this species might prove to be of interest for model studies of a comparative nature.