J Sci Food Agric 1998, 78, 522È526 The Eþect of Storage Temperature on Drip Loss from Fresh Beef Marjan J A den Hertog-Meischke,*1 Frans J M Smulderst and Jan G van Logtestijn Department of the Science of Food of Animal Origin, Faculty of Veterinary Medicine, Utrecht University, PO Box 80.175, 3508 TD Utrecht, The Netherlands (Received 28 January 1997 ; revised version received 23 February 1998 ; accepted 6 April 1998) Abstract : The e†ect of post rigor storage temperature on drip loss of two bovine muscles (M longissimus thoracis and M semimembranosus) was examined using 12 Dutch Friesian Holstein bulls. Drip loss of both longissimus and semimembranosus muscles was inÑuenced by post rigor storage temperature. Higher storage temperatures resulted in increased drip losses, probably caused by a decrease in viscosity of the drip. The increase was most marked for the M longissimus thoracis. The relationship between drip loss and storage temperature was not linear ; drip loss was found to be less a†ected by storage temperature as the latter was increased. The observed relationship could not be explained by a decrease in viscosity of the drip at higher temperatures alone. It was thought that faster ageing of the meat at elevated temperatures might increase the myoÐbrillar water-holding capacity, resulting in a decreased drip loss. However, this hypothesis requires further research. ( 1998 Society of Chemical Industry. J Sci Food Agric 78, 522È526 (1998) Key words : beef ; storage temperature ; water-holding capacity ; meat quality INTRODUCTION storage variables, such as method of suspension, cutting, packaging and storage temperature (O†er and Knight 1988). The e†ect of temperature on drip loss was studied by OÏKee†e and Hood (1980È1981). They reported that increasing storage temperature from 0 to 10¡C results in an appreciable rise in the amount of drip lost from beef steaks. The relationship between storage temperature and drip loss was not linear. Drip loss increased more rapidly in the temperature range from 5 to 10¡C than it did from 0 to 5¡C ; this e†ect was most pronounced in bovine M psoas major as compared to M gluteus medius and M semimembranosus. The cause of the temperature e†ect on drip loss is unknown. O†er and Knight (1988) suggested that the lowered viscosity of the water at higher temperatures might be responsible for the elevated drip losses. However, no evidence for this suggestion could be found in the literature. Therefore, the objective of the present experiments was to characterise the relationship between storage temperature and drip loss of bovine M longissimus thoracis and M semimembranosus and to study the possible mechanisms involved. To this end, The ability of fresh meat to hold on to its own water (water-holding capacity) is an important quality aspect, since it a†ects consumer acceptance and Ðnal weight of the product. Water can be lost from meat by evaporation, in the form of drip and Ðnally in the course of cooking. Drip loss is thought to result from lateral shrinkage of myoÐbrils post mortem, causing water to be expelled into the muscleÏs extracellular space (O†er and Knight 1988). After death, large gaps appear between both Ðbre bundles and individual Ðbres, which function as longitudinal channels with outlets at the cut surface of the meat. The Ñuid in these channels may be transported to the surroundings by the action of gravity and hence is the main source of drip (O†er et al 1989). Drip loss from fresh meat is inÑuenced by many * To whom correspondence should be addressed. ” Present address : Institute of Meat Hygiene, Meat Technology and Food Science, Veterinary Medical University of Vienna, Josef Baumanngasse 1, A-1210 Vienna, Austria. 522 ( 1998 Society of Chemical Industry. J Sci Food Agric 0022È5142/98/$17.50. Printed in Great Britain E†ect of storage temperature on drip loss of beef beef steaks were stored at a range of temperatures, covering that found in commercial practice. The viscosity and protein concentration of drip as related to storage temperature was assessed. 523 Protein concentration of drip Protein concentration of drip was measured using the biuret method of Gornall et al (1949), with bovine serum albumin as a standard. MATERIALS AND METHODS Viscosity of drip Experimental Twelve Dutch Friesian Holstein bulls (age 2 years ; carcass weight 400È460 kg) were slaughtered under commercial circumstances. To facilitate skinning, an electrical current was used (250 V, 50 Hz, 2 s). No electrical stimulation was applied. After ^48 h chilling (2¡C, air velocity 0É5 m s~1), M longissimus thoracis (LT) and M semimembranosus (SM) from the left side of the carcass were excised. From each muscle, 5 portions of 100 g were randomly subjected to one of the following storage temperatures : 1, 3, 5, 7É5 or 10¡C. After 3 days storage, amount of drip and its protein concentration were determined. The viscosity of one drip sample was measured at di†erent temperatures. In a second experiment one Dutch Friesian cow (age 7 years ; carcass weight 258 kg) was slaughtered under the above mentioned conditions. From the M longissimus thoracis, 5 steaks of approximately 200 g were cut, which were subsequently stored in plastic trays at temperatures of 1, 3, 5, 7É5 or 10¡C. After 3 days storage, pH, drip loss, myoÐbrillar protein degradation and myoÐbrillar water-holding capacity were assessed. Muscle pH and temperature Muscle pH was measured at 45 min, and 3, 24 and 48 h post mortem and after 3 days storage using a portable pH meter (type CG818, Scott Gerate, Hofheim, Germany), equipped with a combined (glass/reference) electrode (type N48A, Schott Gerate, Hofheim, Germany). Temperature was monitored at the same time with a digital thermometer (Tastotherm D700, Germany). Drip loss In experiment 1, drip loss was determined in slices of muscle ^100 g, which were suspended from Ðsh-hooks in plastic pouches at temperatures of 1, 3, 5, 7É5 or 10¡C. After 3 days storage, weight loss was assessed (Honikel 1987) and drip was collected to determine its protein content. A temperature data logger was used to monitor changes in the applied temperature. During storage, only small variations in temperature (^0É3¡C) occured. In the second experiment on lab scale, drip loss was determined by weighing the steaks before and after storage. In both experiments, drip loss was expressed as (%) of the initial weight of the sample. Viscosity of drip was assessed with a BrookÐeld LVF viscosity meter in conjunction with a UL-adapter. Viscosity was measured at 30 rpm and expressed in cPoise. MyoÐbrillar protein degradation MyoÐbrils were extracted according to the method of Ouali and Talmant (1990). Degradation of myoÐbrillar proteins was assessed with horizontal SDS-PAGE electrophoresis on a 80È180 g litre~1 precast ExcelGel' SDS (Pharmacia Biotech). Samples with 4 mg ml~1 myoÐbrillar protein were diluted to 2 mg ml~1 myoÐbrillar protein with sample bu†er containing 8 M Urea, 2 M thiourea, 0É025 M Tris (pH 6É8), 0É075 M DTT and 0É104 M SDS. Samples were heated at 100¡C for 5 min and then loaded on gel (10 ll). Gels were stained overnight in Coomassie R 350 (1 g litre~1) in glacial acetic acid/water/ethanol, 1 : 5 : 5 (v/v/v) and destained over several days by repeated washes in solutions containing 100 ml litre~1 glacial acetic acid and 250 ml litre~1 ethanol. The intensity of the protein bands was measured with a LKB Ultrascan XL Enhanced Laser Densitometer. Intensity of troponin T and 30 kDa bands was expressed as % relative to actin. MyoÐbrillar water-holding capacity Determination of the myoÐbrillar water-holding capacity was based on the method of Penny et al (1963). Eight grams of muscle were homogenised in 80 ml icecold 0É1 M KCl. The homogenate was kept on ice for 30 min. After washing several times with 0É1 M KCl, a suspension of 6 mg protein ml~1 was made. pH of the suspension was measured with a Radiometer PHM83 Autocal pH meter equipped with a combined (Glass/ reference) electrode (type GK2401C, Radiometer, Denmark). After centrifugation at 1200 ] g for 5 min, the supernatant was discarded and the precipitate weighed. MyoÐbrillar water-holding capacity was expressed as g H 0 g protein~1. 2 Statistical analysis SigniÐcance of di†erences was tested using the ANOVA procedure. Fixed factors in the analysis were temperature of storage and muscle. Means were separated with TukeyÏs method. M J A den Hertog-Meischke, F J M Smulders, J G van L ogtestijn 524 RESULTS AND DISCUSSION The results of the pH and temperature measurements are presented in Table 1. The results of the temperature measurements indicate a slower chilling rate of the SM muscle as compared to the LT muscle. Also, chilling rate within the SM muscles varied, as can be seen from the high standard deviation at 24 h post mortem. The use of electrical current to facilitate skinning resulted in an accelerated glycolysis. The pH values observed at 3 h post mortem, were similar to those often found in electrically stimulated carcasses (for review see Seideman and Cross 1982). SM muscles had a signiÐcantly higher rate of pH decline as compared to the LT muscles. As the rate of glycolysis is temperature dependent (Greaser 1986), the faster pH fall of SM muscles may be associated with the slower chilling rate of this muscle. Ultimate pH was reached within 24 h post mortem, regardless the muscle considered. Di†erences in ultimate pH between LT and SM muscles were found to be negligible. A graphic presentation of the e†ect of storage temperature on drip loss is given in Fig 1. In every temperature treatment group, higher drip loss values were recorded for SM muscles than for LT muscles. This Ðnding was expected as drip loss is increasing with increasing rate of pH fall and decreasing chilling rate (Penny 1977 ; O†er 1991). However, di†erences in muscle Ðbre type, muscle Ðbre size and amount of collagen between LT and SM muscles may also play a role in determining the di†erence in drip loss between the two muscles. In both LT and SM muscles, drip loss increased with increasing storage temperature. The observed e†ect was more pronounced in LT than in SM muscles, which may be caused by di†erences in muscle structure between the two muscles. The relationship between storage temperature and drip loss was not linear ; drip loss was found to be less a†ected by storage temperature as the latter was increased (Fig 1). These results are in contrast with the observations of OÏKee†e Fig 1. E†ect of storage temperature on drip loss of M longissimus thoracis (LT) and M semimembranosus (SM) after 3 days of storage (mean ^ SD). and Hood (1980È1981), who reported that drip loss increased more rapidly with temperature between 5¡C and 10¡C than between 0¡C and 5¡C. It is not clear were this discrepancy results from. Probably, the method of measuring drip loss plays a role. OÏKee†e and Hood (1980È1981) measured drip loss of vacuum packed meat after 4 days of storage. In their experiment, the meat was already aged for 4 days before sampling. However, more research is needed to establish the relationship between storage temperature and drip loss. Protein concentration of drip from SM samples was lower than that from the LT samples (58É9 ^ 6É9 mg ml~1 for SM muscles versus 66É7 ^ 7É7 mg ml~1 for LT muscles ; P \ 0É05). Savage et al (1990) have shown that protein content of drip decreases with increasing amount of drip. They suggested that this may be the result of sarcoplasmic protein precipitation onto the myoÐbrils. This may also explain the observed di†erence in protein concentration between SM and LT samples in the present experiment. Although protein concentration varied between individual samples, di†erences between temperature treatment groups were negligible, indicating that the protein con- TABLE 1 pH and temperature (mean ^ SD) of M longissimus thoracis (LT) and M semimembranosus (SM) at various times post mortema,b T ime post mortem 45 3 24 48 min h h h pH Temperature (¡C) LT SM LT SM 6É78 ^ 0É12 5É99 ^ 0É17a 5É50 ^ 0É04 5É49 ^ 0É03 6É72 ^ 0É07 5É77 ^ 0É06b 5É50 ^ 0É03 5É50 ^ 0É03 38É7 ^ 0É4 30É6 ^ 0É9a 2É0 ^ 0É3a 2É1 ^ 0É1 39É1 ^ 0É5 32É0 ^ 0É9b 8É7 ^ 1É1b 2É2 ^ 0É4 a Experiment 1, n \ 12 per group. b Within traits, within muscles, means lacking a common following letter di†er signiÐcantly (P \ 0É05). E†ect of storage temperature on drip loss of beef 525 centration of drip was not inÑuenced by storage temperature. Penny (1975) studied the e†ect of freezing and centrifuging on the amount of drip and its protein concentration. Both these treatments resulted in a decrease in the protein concentration and an increase of the amount in drip. Penny (1975) postulated that through freezing or centrifuging additional water was removed from those compartments within the meat structure (ie the spaces between the myoÐlaments and those in the tubulus of the sarcoplasmic reticulum) which contain a low amount of soluble proteins. Since in the present experiment no signiÐcant di†erences in protein concentration between temperature treatment groups was recorded, it is suggested that the increase in drip loss with increasing storage temperature is not due to removal of additional water from the spaces within the myoÐlaments or sarcoplasmic reticulum. O†er and Knight (1988) suggested that storage temperature a†ects drip loss via an e†ect on the viscosity of the drip. At higher temperatures, viscosity will be lower resulting in a higher rate of migration of drip through the channels in and between the muscle Ðbres. In Fig 2 the relationship between viscosity of drip and temperature is shown. For comparison, the relationship between temperature and viscosity of water has been included. As di†erences in protein concentration at various temperatures were negligible, this relationship is only presented for drip with a protein content of 81É6 mg ml~1, the highest value found in the present experiment. At lower protein concentrations this relationship will follow a similar course, intermediate between this curve and the one for water. From Fig 2 it can be seen that between viscosity and temperature an exponential relationship existed, which can be described by the following equation : g \ 4É65 e~0Õ02T (1) where g is the viscosity (cP) and T is the temperature (¡C). In samples with transversal cut surfaces and longitudinal Ðbre axis (as in the present experiment), the rate at Fig 2. Relationship between temperature and viscosity of drip in a sample with a protein concentration of 81.6 mg ml~1 and between temperature and the viscosity of water. which Ñuid would Ñow from the meat channels can be described with the model given by O†er and Knight (1988) : t 1@2 \ 9gy2 2px2 (2) where t is the time in which half the Ñuid is squeezed 1@2 out ; g is the viscosity of the Ñuid ; y is the length of the channels ; p is the pressure exerted on the channels by the sample weight ; and x is the initial diameter of the meat channel. From eqn (2) it follows that the time in which half of the Ñuid in the meat channels is squeezed out (t ) 1@2 declines with the decrease in viscosity and thus, with an increase in storage temperature. However, upon replacing g by eqn (1), it becomes apparent that, according to the model of O†er and Knight (1988), drip loss should increase more rapidly at higher storage temperatures. In the present experiment, this would mean that the increase in drip loss should be smaller between 1 and 3¡C than between 7É5 and 10¡C. However, this was not the case. Hence, although increased drip loss at higher storage temperatures was indeed associated with decreased viscosity of the drip, this failed to explain the relationship between drip loss and temperature observed in our study. Another possible explanation for the non-linear relationship between storage temperature and drip loss may be based on the e†ect of ageing on water-holding capacity. Ageing during storage results in an increase of water-holding capacity, possibly as a consequence of disintegration of myoÐbrillar proteins by proteolytic enzymes (Hamm 1986 ; Paterson et al 1988 ; Boles et al 1992). Since enzymatic reactions proceed at faster rates as temperature increases, it was expected that ageing e†ects and thus improvement of water-holding capacity would occur more rapidly at higher storage temperatures. The total drip loss after storage at higher temperatures would then be determined by on the one hand the higher rate of drip formation (due to lowered viscosity) and the smaller amount of drip (due to improved water-holding capacity) on the other. To test this hypothesis, a small experiment on lab scale was conducted, in which degradation of myoÐbrillar proteins and myoÐbrillar water-holding capacity were studied as a function of storage temperature. The results of the drip loss measurements showed the same pattern as was found in Fig 1 : increasing storage temperatures resulted in elevated drip losses, but drip loss became less dependent on temperature as the latter was increased (data not shown). MyoÐbrillar water-holding capacity increased also with increasing storage temperature. In contrast to drip loss, myoÐbrillar water-holding capacity increased more rapidly with increasing storage temperature in the range from 5 to 10¡C than it did in the range from 1 to 5¡C 526 M J A den Hertog-Meischke, F J M Smulders, J G van L ogtestijn (Fig 3). The increase in myoÐbrillar water-holding capacity was not due to an increase in pH of the meat : the pH after storage did not di†er between temperature groups. Degradation of myoÐbrillar proteins during ageing is often characterised by SDS-gel electrophoresis. Bands with molecular weights of 27È30 kDa gradually appear and the Troponin T band disappears. SDSelectrophoresis of samples stored at 1, 3 and 5¡C showed little protein degradation, as the 30 kDa band was not measurable. Intensity of the Troponin T band did not di†er between samples stored at 1, 3, and 5¡C (intensity expressed as % relative to actin \13É0). However, in gels from samples stored at 7 and 10¡C, the 30 kDa band appeared (intensity expressed as % relative to actin 7É3 and 11É1, respectively), coinciding with disappearance of the Troponin T band (intensity expressed as % relative to actin 8É3 in both cases). This illustrates that higher storage temperatures lead to faster ageing of meat. The fact that an increase in storage temperature results in both faster ageing and increase in myoÐbrillar water-holding capacity suggests that the lower amount of drip at higher temperatures is due to the faster ageing. This hypothesis deserves further investigation. In summary, the results showed that an increase in the storage temperature of beef causes an increase in the amount of drip, probably as a result of the decrease in viscosity of the Ñuid within the meat. The observed relationship between storage temperature and drip loss is not linear and cannot be explained by a decline in viscosity alone. The increase in amount of drip is lower with increasing storage temperatures. This may be related to increased proteolysis at higher storage temperatures, causing a faster improvement of waterholding capacity. The e†ect of storage temperature on the total drip at a Ðxed storage time may therefore be determined by the e†ect of drip viscosity and the coinciding improvement of water-holding capacity induced by ageing at higher temperatures. Fig 3. E†ect of storage temperature on the myoÐbrillar waterholding capacity (M-WHC) of beef loin steaks after 3 days of storage. ACKNOWLEDGEMENTS The authors gratefully acknowledge The NetherlandsÏ Product Boards for Livestock, Meat and Eggs at Rijswijk and Edah Supermarkten B.V. at Helmond for supporting this study. Thanks are also due to A van Dijk, G Keizer and P Schippers for technical assistance. 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