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Textural assessment of clenbuterol treatment in beef
Meat Science 51 (1999) 297±303
Textural assessment of clenbuterol treatment in beef M. LunÄo a, J.A. BeltraÂn a, I. Jaime b, P. RoncaleÂs a,* Departamento de ProduccioÂn Animal y Ciencia de los Alimentos (TecnologõÂa de los Alimentos), Facultad de Veterinaria, Universidad de Zaragoza, C. Miguel Servet 177, 50013 Zaragoza, Spain b Departamento de BiotecnologõÂa y Ciencia de los Alimentos, Facultad de Ciencia y TecnologõÂa de los Alimentos y Ciencias QuõÂmicas, Universidad de Burgos, Pza. Misael BanÄuelos s/n, 09001 Burgos, Spain a
Received 8 November 1997; received in revised form 28 May 1998; accepted 18 July 1998
Abstract Eight Charolais heifers (ca. 300 kg) were fed a diet containing either 0 (control) or 1 ppm clenbuterol during 5 weeks. Daily live weight gain was higher ( p<0.01), by about 34%, in treated animals. They were slaughtered after a week of withdrawal. Activity of m-calpain of meat from clenbuterol-fed heifers was lower ( p<0.01) immediately post mortem, while activity of calpastatin was higher ( p<0.05) than in meat from controls. According to sensory and instrumental data, meat from clenbuterol-fed heifers did not tenderise during aging, and was tougher ( p<0.05) than control meat at 8th day post mortem. Both principal component analysis and multivariate discriminant analysis of a pool of data of the textural pro®le showed dierences due to clenbuterol treatment at 24 h. The slope after yield (calculated from the Warner±Bratzler shearing force±deformation curve), was the only textural parameter aected ( p<0.05) at 24 h by the treatment. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Texture; Tenderness; Calpains; Beef; Clenbuterol; Beta-adrenergic agonists
1. Introduction Several b-adrenergic agonists (BAA) have been evaluated for ecacy as repartitioning agents, as well as for safety, in a wide variety of animals. BAA were demonstrated to increase muscle mass of the carcass (Baker Dalrymple, Ingle, & Ricks, 1984). Nevertheless, BAA are not approved for use in animal production in many countries, including those of the European Union. Endogenous enzyme activity is largely aected by BAA. It is generally accepted that the activity of the calpain/calpastatin proteolytic system is modi®ed by BAA. However, there are some discrepancies regarding the eect on each form of calpain. Several authors have con®rmed that m-calpain activity is reduced in animals fed with BAA (Geesink et al., 1993), while other reports indicate that m-calpain activity is increased (Koohmaraie, Shackelford, Muggli-Cocket, & Stone, 1991; Kretchmar Hathaway, Epley, & Dayton, 1990). But there is a general agreement that these compounds bring * Corresponding author. Tel.: +34-976-761582; fax: +34-976761612; e-mail: [email protected]
about an increase in the inhibitory activity of calpastatin (Wheeler & Koohmaraie, 1992). Consistent with the role of calpains in meat tenderisationÐfor review, see RoncaleÂs et al. (1995)Ðpostmortem muscle proteolysis and tenderness have been shown to be decreased in meat from animals fed BAA (Koohmaraie et al., 1991; Kretchmar et al., 1990). In fact, some of the studies concerning BAA report increases in shear force of meat (Koohmaraie et al., 1991) or evidence demonstrating that such meats do not tenderise during aging (Wheeler & Koohmaraie, 1992), although a small number of reports (Jeremiah et al., 1994) have shown no eect on palatability or consumer acceptance. Therefore, there is experimental evidence to show that the use of any of the BAAs habitually used as growth promoters brings about tough meat. This is no doubt a negative eect on the sensory quality of meat, which can adversely aect consumer attitudes towards meat. The aim of this research was to characterise as accurately as possible the texture pro®le of the meat from clenbuterol-fed and control animals, based on enzyme (calpain) activities, sensory evaluation and instrumental
0309-1740/99/$Ðsee front matter # 1999 Elsevier Science Ltd. All rights reserved PII: S0309 -1 740(98)00117 -X
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assessment, to show discriminating dierences due to BAA clenbuterol treatment. 2. Materials and methods 2.1. Animals and muscle preparation During a 14-day adaptation period, eight Charolais heifers of about 8 months of age and weighing approximately 300 kg were individually bucket-fed ad libitum a standard growing-®nishing diet and straw. After this period, they were individually bucket-fed ad libitum the same diet containing either 0 ppm (four control animals) or 1 ppm clenbuterol (four treated animals) during 5 weeks. Throughout this period, they ate 9±10 kg per day of feed. Animal weight was measured at arrival and once a week until slaughter, and daily live weight gain (DLWG, g dayÿ1) calculated. Soon after slaughter (30±60 min), a small portion of m. Longissimus thoracis (6th rib) was excised from carcasses for determining enzyme activities as described below. The rest of m. Longissimus thoracis (7th to 12th ribs) was excised 24 h after death. It was divided in several sections; one was used for pH, fat and calpain/ calpastatin activity determination at this time, a second was vacuum packaged after cutting into 1.2 cm steaks and stored frozen until subsequent sensory analysis, the third was vacuum packaged whole and frozen until instrumental analysis was performed. The rest was vacuum packaged and stored at 4 C for 7 days to age, and was then divided and processed as on the ®rst day. Cross-sectional area (cm2) of m. Longissimus thoracis was measured at 12th rib. For pH determination, about 3 g of muscle were homogenised in 20 ml distilled water for 15 s. The measurement was carried out immediately using a Crison pH-meter with a combined glass electrode. Intramuscular fat (IF) was determined using the Soxhlet method. 2.2. Isolation and activity assay of calpains and calpastatin Partial puri®cation of calpains and calpastatin from muscle tissue was performed according to the method described by Ducastaing & Valin, Schollmeyer, and Cross (1985) and adapted to HPLC. Muscle tissue (20 g) was homogenised with an Ultraturrax in 3 vol. of 10 mM Tris±HCl buer, pH 7.5, containing 0.05 M NaCl, 4 mM EDTA, 2 mM 2-mercaptoethanol and 1 mM NaN3. After 1 hr extraction with magnetic stirring, the homogenate was centrifuged at 30 000 g for 30 min, the supernatant was ®ltered through cheese cloth and adjusted to pH 7.5. Precipitated material was eliminated by centrifugation at 50 000 g for 50 min. All operations were performed at 0±4 C with precooled solutions.
Portions of 50 000 g supernatant were ®ltered through a 0.22 mm millipore membrane and loaded on a mono Q HR 10/10 column (Pharmacia) equilibrated in 5 mM Tris±HCl buer, pH 7.5, 0.1 mM EDTA, 0.05 M NaCl and 2 mM 2-mercaptoethanol. Protein elution by a non-linear NaCl gradient (0.05±0.5 M) was performed at a ¯ow rate of 1 ml minÿ1 and fractions of 1 ml were collected. Calcium-dependent proteolytic activity was assayed, as described by Koohmaraie, Schollmeyer, and Dutson (1986), using casein (Hammerstein; 5 mg mlÿ1) as substrate. Activity of calpastatin was assayed by incubating inhibitor and enzyme at 25 C for 60 min in 1.5 ml reaction mixture, according to Koohmaraie, Seideman, Schollmeyer, Dutson, and Crousse (1987). 2.3. Sensory evaluation On the 1st and 8th days post mortem, ribs were cut into 1.2 cm thick steaks and frozen for subsequent taste panel evaluation. Steaks were thawed at 4 C for 24 h before cooking and serving. They were placed in a preheated double-plate grill at 160 C and removed when an internal temperature of 70 C was reached. Muscle steaks were served on preheated plates to a trained panel of 12 members. Tenderness at ®rst chew, overall tenderness, juiciness, ®brousness, residue and overall acceptability were evaluated as described by Cross, Moen, and Stan®eld (1978), and scored by placing a mark on a nonstructured 10 cm scale. Zero was extremely low (tough, dry, non ®brous, low residue, low quality), 10 was extremely high (tender, juicy, ®brous, high residue, high quality). 2.4. Objective texture measurements The texture parameters were measured on a Universal Testing Machine, Instron model 4301 (High Wycombe, UK), using a Warner±Bratzler shear blade. The parameters studied were: 1. Load: Load at Maximum and Load at Threshold Yield. 2. Energy: Energy to Yield Point and Energy to Break Point. 3. Slope: Slope after Yield. The parameter `Slope after Yield' was evaluated for the ®rst time in this study. The slope was de®ned as the characteristic slope of each curve immediately after the meat sample reached the yield point in the shear force± deformation curve. Calculation was made as depicted in Fig. 1: ®rst, the straight line which best ®tted (highest R2) to the immediate section of the curve after the yield point was drawn; then tga for this line was calculated. For the description of the other parameters, see Instron (1988).
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Meat samples, after thawing at 4 C for 24 h, were cut into 6 cm long1 cm high1 cm wide prismatic pieces. They were placed inside the Warner±Bratzler shear blade and sheared perpendicular to the muscle ®bre longitudinal axis.
component analysis and multivariate discriminant analysis were carried out using the GLM procedures of SAS (1985).
2.5. Statistical analysis
3.1. Daily live weight gain and muscle characteristics
The Student t test was used to assess the signi®cance of dierences between values. Correlation matrix, principal
Clenbuterol-fed heifers had a signi®cantly higher ( p<0.01) daily live weight gain (1.810.07 g dayÿ1)
3. Results and discussion
Fig. 1. Graphic representation of the calculation method for the parameter `slope after yield' (slope=tga=y/x) from a typical force±deformation curve obtained after shearing a meat sample with a Warner±Bratzler shear device mounted on an Instron universal testing machine.
Fig. 2. Representative replicate examples of typical force±deformation curves obtained after shearing with a Warner±Bratzler shear device meat samples from (A) control heifers, and (B) clenbuterol-fed animals.
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than control animals (1.350.11 g dayÿ1), that is, it was 34.1% higher in treated animals. These results agree with those reported by Schiavetta, Miller, Lunt, Davis, and Smith (1990), Berge, Culioli, and Ouali (1993) and Garssen, Geesink, Hoving-Bolink, and Verplanke (1995). However, a number of researchers (Miller et al., 1988; Wheeler & Koohmaraie, 1992) did not ®nd any signi®cant dierence. Final pH (5.54 and 5.57 for control and treated, respectively), cross-sectional area (97.7 and 111.3 cm2) and intramuscular fat (2.84 and 2.97 g 100 gÿ1) of m. Longissimus thoracis were not aected signi®cantly ( p>0.05) by clenbuterol treatment. 3.2. Calpains and calpastatin activity Table 1 gives the results regarding the activity of the two isoforms of calpain and their speci®c inhibitor calpastatin. It is worth mentioning the signi®cant dierences found as early as 3 h post mortem. Only m-calpain failed to show any signi®cant ( p>0.05) eect of clenbuterol treatment, probably due to animal variability. Micro-calpain activity was reduced by 31% ( p<0.05) after clenbuterol feeding, while calpastatin activity was increased by 49% ( p<0.05) in the same animals. As a consequence, the ratio m-calpain/calpastatin was highly decreased (53%; p<0.01), in good agreement with Table 1 Eect of clenbuterol administration on m-calpain, m-calpain and calpastatin activities (UI gÿ1) in m. Longissimus thoracis of heifers at 3 h and 1 day post mortem (pm)
m-calpain (3 h pm) m-calpain (1 d pm) m-calpain (3 h pm) m-calpain (1 d pm) calpastatin (3 h pm) calpastatin (1 d pm) m-calpain/calpastatin (3 h pm)
Control
Treated
0.0710.008 0.0350.004 0.0830.070 0.0190.014 0.0840.012 0.0300.003 0.840.052
0.0490.006*a 0.0160.004** 0.0550.024 0.0290.011 0.1250.015** 0.0710.042* 0.390.080**
a Means in the same row followed by asterisks are signi®cantly different (*p<0.05, **p<0.01).
Garssen et al. (1995). Therefore, the direct outcome of these changes in the calpain/calpastatin complex ought to be a reduced proteolytic capacity throughout aging. This, which has been shown to be closely correlated with eective post-mortem tenderisation of meat (RoncaleÂs et al., 1995), should lead to abnormally low tenderisation of meat from animals fed clenbuterol. Previous reports on the activity of the two isoforms of calpain and calpastatin are con¯icting, and most deal with only one of the enzyme forms. Our results oer a more comprehensive view of the eect of BAA on the calpain system, and essentially con®rm those reported by Geesink et al. (1993). They also agree with most reports regarding calpastatin activity (Garssen et al., 1995; Koohmaraie et al., 1991; Kretchmar et al., 1990; Wheeler & Koohmaraie, 1992; ), but disagree with many reports on m- and m-calpain (Garssen et al., 1995; Koohmaraie et al.; Kretchmar et al.; Wheeler & Koohmaraie, 1992). 3.3. Sensory evaluation of tenderness Table 2 shows the results obtained for the texture pro®le speci®cally designed for this experiment (see Materials and Methods). No signi®cant dierences ( p>0.05) between treatments were found on the ®rst day post mortem, but all texture parameters were signi®cantly dierent ( p<0.05) in meat from treated and untreated animals at 8th day post mortem. They demonstrate that meat from control animals was more tender, as seen by the dierence in tenderness at ®rst chew and overall tenderness. This also gave rise to a large dierence in overall acceptability in favour of untreated meat. Results for these three parameters, obtained at 1st and 8th day, showed a signi®cant dierence for control meat ( p<0.05), but not in meat from treated animals. Thus, the sensory consequence of feeding animals with clenbuterol was that meat was tough even after 8 days of aging, which usually leads to tender meat; that is to say, meat quality was greatly reduced. These results
Table 2 Sensory pro®le of meat texture from control and clenbuterol-treated heifers at 1 and 8 days post mortem, evaluated by a trained taste panel using a 10 cm nonstructured scale (0 was extremely low: tough, dry, non ®brous, low residue, low quality; 10 was extremely high: tender, juicy, ®brous, high residue, high quality) 1st day
Tenderness at ®rst chew Overall tenderness Juiciness Fibrousness Residue Overall acceptability a,b,c
8th day
Control
Treated
Control
Treated
4.880.77b 4.710.79b 4.730.44ab 5.450.36bc 5.070.99ab 4.790.96b
3.490.71b 3.140.84b 3.890.41b 6.290.53ab 6.240.39a 3.500.51b
6.310.41a 6.030.45a 5.200.36a 4.870.50c 4.020.46b 6.310.32a
4.160.22b 3.760.27b 4.180.41b 6.220.39a 6.020.37a 3.920.38b
Means in the same row followed by dierent superscripts are signi®cantly dierent ( p <0.05).
M. LunÄo et al./Meat Science 51 (1999) 297±303
seem to agree with those reported previously on the textural quality of meat assessed by instrumental techniques, as will be discussed later. However, there are a lack of studies dealing with the assessment of the texture of meat from clenbuterol treated animals using sensory evaluation. 3.4. Instrumental evaluation of tenderness Results corresponding to the texture parameters determined with a Warner±Bratzler shear blade are shown in Table 3. No signi®cant dierences ( p>0.05) were found in any of the parameters, measured on the ®rst day, between treated and untreated animals, with the exception of the `slope after yield' (Fig. 1). An example of some of the force±deformation curves, representative of replicates of one sample of meat from both control and treated animals, is shown in Fig. 2. As shown in Table 3 and Fig. 2, the slope after yield showed a clear and signi®cant dierence ( p<0.05) between control and treated animals. The latter having in all cases a steeper slope, with values always above 6, and a much lower standard deviation, i.e. less variability. Measurements at 8th day varied greatly with respect to 1st day. All parameters showed signi®cant dierences ( p<0.05) between treatments, with the exception of the energy to break point, which was not able to dis-
301
criminate between the two groups of samples. Eective discrimination by most textural parameters was related to the fact that meat from clenbuterol-fed animals did not tenderise during aging, as demonstrated by the absence of any signi®cant dierences with the 1st day. On the contrary, meat from control heifers was signi®cantly more tender ( p<0.05) after aging. This was demonstrated by the maximum load (shear force) results, which is the most widely used parameter used to assess tenderness. The shear force results essentially agree with those previously reported in animals treated with clenbuterol (Garssen et al., 1995; Miller et al., 1988; Schiavetta et al., 1990; ), L644,969 (Moloney, Allen, Joseph, Tarrant, & Convey, 1994; Wheeler & Koohmaraie, 1992) and cimaterol (Fiems, Buts, BoucqueÂ, Demeyer, & Cottyn, 1990). Thus according to our results and those of most other authors, instrumental parametersÐmainly shear force (maximum load)Ðare able to discriminate BAA-treated from untreated animals. However, discrimination is only possible after aging. We have though demonstrated that the slope after yield is capable of discriminating BAA treatment as early as 24 h after slaughter. The reason for that is not clear, but it must be related, at least in part, to the fact that, even if dierences for most parameters were not signi®cant, possibly due to the low number of animals studied, meat from treated animals
Table 3 Instrumental pro®le of meat texture from control and clenbuterol-treated heifers at 1 and 8 days post mortem, using a Warner±Bratzler shear blade mounted on an Instron universal testing machine 1st day Control Maximum load (N) Load at yield (N) Energy to yield (J) Energy to break (J) Slope after yield a,b
8th day Treated
a
89.118.2 88.412.1a 0.170.08ab 1.090.47a 3.691.66b
Control a
118.817.3 118.119.4a 0.360.18a 1.550.72a 8.000.37a
Treated b
51.29.51 49.410.4b 0.100.03b 0.810.21a 1.801.39b
109.112.1a 108.718.3a 0.260.10a 1.280.45a 7.740.70a
Means in the same row followed by dierent superscripts are signi®cantly dierent ( p<0.05).
Fig. 3. Principal components analysis of all texture parameters, both sensory and instrumental, of meat from control (*) and clenbuterol-treated heifers (*) at 1 day post mortem (A) and 8 days post mortem (B).
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Table 4 Correlation matrix of all texture parameters, both sensory and instrumental, of meat from control and clenbuterol-treated heifers at 1 day post mortem
Maximum load (M.L.) Load at yield (L.Y.) Energy to yield (E.Y.) Energy to break (E.B.) Slope after yield (S.Y.) Tenderness at ®rst chew (T.F.C.) Overall tenderness (O.T.) Residue (R.) Overall acceptability (O.A.) a
M.L.
L.Y.
E.Y.
E.B.
S.Y.
T.F.C
± 0.954***a 0.708* 0.632 0.566 ÿ0.740*
± 0.691 0.655 0.376 ÿ0.762*
± 0.891** 0.766* ÿ0.692
± 0.503 ÿ0.605
± ÿ0.481
±
ÿ0.782* 0.455 ÿ0.805*
ÿ0.817* 0.484 ÿ0.846**
ÿ0.738* 0.590 ÿ0.659
ÿ0.659 0.564 ÿ0.580
ÿ0.481 0.399 ÿ0.411
0.994*** 0.603 0.979***
O.T
R
O.A
± ÿ0.872** 0.985***
± ÿ0.840**
±
Correlation coecients followed by asterisks are signi®cant (*p<0.05; **p<0.01; ***p<0.001).
always had a higher shear force. Besides this, as seen in Fig. 2, clenbuterol treatment gives rise to a modi®cation of the ®nal part of the force±deformation curve (between the maximum load and the break point). This part, when recorded for untreated animals, usually gives a ¯atter and more variable slope than meat from treated animals, which gives a steeper and less variable slope. According to Voisey (1976), this part of the curve is related to the ®nal phase of cutting meat with the teeth, and might relate to the collagen characteristics of the sample. 3.5. Correlation matrix Table 4 shows the correlation matrix for all texture parameters, both sensory and instrumental, at the ®rst day post mortem. It is clear there are high and signi®cant correlations between most of the textural attributes of meat. Indeed, all sensory parameters were highly correlated ( p<0.01), and many instrumental parameters were also correlated ( p<0.05) both between themselves and with most of the sensory attributes. However, remarkably low correlations were found between the slope after yield and any other texture parameter. The reason for this is not clear, but agrees with the results shown above, suggesting that the slope Table 5 Results of multivariate discriminant analysis: canonical structure of all texture parameters, both sensory and instrumental, of meat from control and clenbuterol-treated heifers at 1 day post mortem; and canonical coecients distribution on a single axis of texture parameters and treatment groups Canonical structure Canonical coecients Maximum load Load at yield Energy to yield Energy to break Slope after yield Tenderness at ®rst chew Control group Treated group
0.555799 0.492400 0.915856 0.707177 0.853560 0.723747 ± ±
ÿ3.672710 1.243822 0.521930 ÿ0.820708 1.394431 0.475677 ÿ0.935414 0.935414
after yield may relate to the treatment of the animals with a BAA. Results (not shown) of the correlation matrix for texture parameters at 8th day con®rmed those of the 1st day post mortem; the only dierence being even higher correlation coecients than found on the 1st day. 3.6. Principal component analysis Fig. 3 shows the principal component analysis for all texture data, both sensory and instrumental, obtained at 1st (A) and 8th (B) day post mortem. At 1st day (A), the ®rst principal component explained 70% of the variation, while the second accounted for 15%; thus an accumulative 85% of total variation was explained by the ®rst two principal components. As seen in Fig. 3 (A), the ®rst component alone was able to separate meat of clenbuterol-fed animals from that of untreated animals. The contribution of each variable to the ®rst component (not shown) exhibited only small dierences, meaning that none of the texture parameters alone could reasonably explain dierences due to treatment, but all were necessary to eectively dierentiate treated from untreated animals. At the 8th day (B), the ®rst principal component explained 82% of the variation, while the second accounted for 11.5%; thus an accumulative 93.5% of total variation was explained by the ®rst two principal components. As stated above, the ®rst component alone was able to dierentiate clenbuterol treated animals from controls. Thus, although of limited value because of the high correlations found among most of the parameters, applying principal component analysis to all the sensory and instrumental data allowed discrimination of meat from BAA-treated and untreated animals in this experiment. 3.7. Multivariate discriminant analysis Results of multivariate discriminant analysis of the textural data at the 1st day post mortem are shown in
M. LunÄo et al./Meat Science 51 (1999) 297±303
Table 5. Three parameters were excluded in this analysis: overall tenderness, ®nal residue and overall acceptability, as they were not signi®cant in discriminating meat samples at either of sampling times. The canonical coecients for the rest of parameters, as well as for the treated and untreated groups, were located on a single axis. It is worth noting that meat from clenbuterol-fed and control animals was discriminated, showing the same absolute value with opposite sign. Thus discriminant analysis of the textural data obtained at the ®rst day after slaughter allowed, too, the dierentiation of meats from BAAtreated and untreated animals in this experiment. 4. Conclusions These results show a signi®cant lowering of meat tenderness on BAA treatment of animals, due to a decrease in eective calpain activity. However, this difference was manifest only after 8 days of post-mortem aging, if standard methods of measuring textural parameters, both sensory and instrumental, were used. A new parameter calculated from the Warner±Bratzler shear force±deformation curve appeared to show dierences due to clenbuterol treatment 24 h after slaughter ( p<0.05). In addition, both principal component analysis and multivariate discriminant analysis of all textural data, instrumental as well as sensory, showed up dierences due to BAA clenbuterol treatment in this experiment. Acknowledgements The authors wish to thank the ComisioÂn Interministerial de Ciencia y TecnologõÂa (CICYT; grant no. ALI92-0644) for support of this research, the DiputacioÂn General de AragoÂn for the fellowship of author M. LunÄo, and the personnel of Servicio de Apoyo a la ExperimentacioÂn Animal of the University of Zaragoza.
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activities of two Ca dependent neutral proteinases and their inhibitor in beef muscle. Meat Science, 15, 193±202. Fiems, L. O., Buts, B., BoucqueÂ, Ch.V., Demeyer, D. I., & Cottyn, B. G. (1990). Eect of a b-agonist on meat quality and myo®brillar protein fragmentation in bulls. Meat Science, 27, 29±39. Garssen, G. J., Geesink, G. H., Hoving-Bolink, A. H., & Verplanke, J. C. (1995). Eects of dietary clenbuterol and salbutamol on meat quality in veal calves. Meat Science, 40, 337±350. Geesink, G. H., Smulders, F. J. M., van Laack, H. L. J. M., van der Kolk, J. H., Wensing, Th., & Breukink, H. J. (1993). Eects on meat quality of the use of clenbuterol in veal calves. Journal of Animal Science, 71, 1161±1170. Instron, 1988. Reference manual, no. M12-2-152, Series IX Materials Testing System. Jeremiah, L. E., Ball, R. O., Merrill, J. K., Dick, P., Stobbs, L., Gibson, L. L., & Uttaro, B. (1994). Eects of feed treatment and gender on the ¯avour and texture pro®les of cured and uncured pork cuts. I. Ractopamine treatment and dietary protein level. Meat Science, 37, 1±20. Koohmaraie, M., Schollmeyer, J. E., & Dutson, T. R. (1986). Eect of low-calcium-requiring calcium activated factor on myo®brils under varying pH and temperature conditions. Journal of Food Science, 51, 28±32. Koohmaraie, M., Seideman, S. C., Schollmeyer, J. E., Dutson, T. R., & Crouse, J. D. (1987). Eect of post-mortem storage on Ca++dependent proteases, their inhibitor and myo®bril fragmentation. Meat Science, 19, 187±196. Koohmaraie, M., Shackelford, S. D., Muggli-Cockett, N. E., & Stone, R. T. (1991). Eect of the b-adrenergic agonist L644,969 on muscle growth, endogenous proteinase activities and postmortem proteolysis in wether lambs. Journal of Animal Science, 69, 4823± 4835. Kretchmar, D. H., Hathaway, M. R., Epley, R. J., & Dayton, W. R. (1990). Alterations in postmortem degradation of myo®brillar proteins in muscle of lambs fed a b-adrenergic agonist. Journal of Animal Science, 68, 1760±1772. Miller, M. F., GarcõÂa, D. K., Coleman, M. E., Ekeren, P. A., Lunt, D. K., Wagner, K. A., Procknor, M., Welsh, T. H., & Smith, S. B. (1988). Adipose tissue, longissimus muscle and anterior pituitary growth and function in clenbuterol-fed heifers. Journal of Animal Science, 66, 12±20. Moloney, A. P., Allen, P., Joseph, R. L., Tarrant, P. V., & Convey, E. M. (1994). Carcass and meat quality of ®nishing friesian steers fed the b-adrenergic agonist L644,969. Meat Science, 38, 419±432. RoncaleÂs, P., Geesink, G. H., van Laack, R. L. J. M., Jaime, I., BeltraÂn, J. A., Barnier, V. M. H., Smulders, F. J. M. (1995). Meat tenderisation: enzymatic mechanisms. In A. Ouali, F. J. M. Smulders, D. Demeyer, (Eds.), Expression of Tissue Proteinases and Regulation of Protein Degradation as Related to Meat Quality (pp. 311±332). Utrecht: ECCEAMST. SAS (1985). SAS user's guide: statistics. Cary, NC: SAS Institute, Schiavetta, A. M., Miller, M. F., Lunt, D. K., Davis, S. K., & Smith, S. B. (1990). Adipose tissue cellularity and muscle growth in young steers fed the b-adrenergic agonist clenbuterol for 50 days and after 78 days of withdrawal. Journal of Animal Science, 68, 3614±3623. Voisey, P. W. (1976). Engineering assessment and critique of instruments used for meat tenderness evaluation. Journal of Texture Studies, 7, 11±48. Wheeler, T. L., & Koohmaraie, M. (1992). Eects of the b-adrenergic agonist L644,969 on muscle protein turnover, endogenous proteinase activities, and meat tenderness in steers. Journal of Animal Science, 70, 3035±3043.
Textural assessment of clenbuterol treatment in beef M. LunÄo a, J.A. BeltraÂn a, I. Jaime b, P. RoncaleÂs a,* Departamento de ProduccioÂn Animal y Ciencia de los Alimentos (TecnologõÂa de los Alimentos), Facultad de Veterinaria, Universidad de Zaragoza, C. Miguel Servet 177, 50013 Zaragoza, Spain b Departamento de BiotecnologõÂa y Ciencia de los Alimentos, Facultad de Ciencia y TecnologõÂa de los Alimentos y Ciencias QuõÂmicas, Universidad de Burgos, Pza. Misael BanÄuelos s/n, 09001 Burgos, Spain a
Received 8 November 1997; received in revised form 28 May 1998; accepted 18 July 1998
Abstract Eight Charolais heifers (ca. 300 kg) were fed a diet containing either 0 (control) or 1 ppm clenbuterol during 5 weeks. Daily live weight gain was higher ( p<0.01), by about 34%, in treated animals. They were slaughtered after a week of withdrawal. Activity of m-calpain of meat from clenbuterol-fed heifers was lower ( p<0.01) immediately post mortem, while activity of calpastatin was higher ( p<0.05) than in meat from controls. According to sensory and instrumental data, meat from clenbuterol-fed heifers did not tenderise during aging, and was tougher ( p<0.05) than control meat at 8th day post mortem. Both principal component analysis and multivariate discriminant analysis of a pool of data of the textural pro®le showed dierences due to clenbuterol treatment at 24 h. The slope after yield (calculated from the Warner±Bratzler shearing force±deformation curve), was the only textural parameter aected ( p<0.05) at 24 h by the treatment. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Texture; Tenderness; Calpains; Beef; Clenbuterol; Beta-adrenergic agonists
1. Introduction Several b-adrenergic agonists (BAA) have been evaluated for ecacy as repartitioning agents, as well as for safety, in a wide variety of animals. BAA were demonstrated to increase muscle mass of the carcass (Baker Dalrymple, Ingle, & Ricks, 1984). Nevertheless, BAA are not approved for use in animal production in many countries, including those of the European Union. Endogenous enzyme activity is largely aected by BAA. It is generally accepted that the activity of the calpain/calpastatin proteolytic system is modi®ed by BAA. However, there are some discrepancies regarding the eect on each form of calpain. Several authors have con®rmed that m-calpain activity is reduced in animals fed with BAA (Geesink et al., 1993), while other reports indicate that m-calpain activity is increased (Koohmaraie, Shackelford, Muggli-Cocket, & Stone, 1991; Kretchmar Hathaway, Epley, & Dayton, 1990). But there is a general agreement that these compounds bring * Corresponding author. Tel.: +34-976-761582; fax: +34-976761612; e-mail: [email protected]
about an increase in the inhibitory activity of calpastatin (Wheeler & Koohmaraie, 1992). Consistent with the role of calpains in meat tenderisationÐfor review, see RoncaleÂs et al. (1995)Ðpostmortem muscle proteolysis and tenderness have been shown to be decreased in meat from animals fed BAA (Koohmaraie et al., 1991; Kretchmar et al., 1990). In fact, some of the studies concerning BAA report increases in shear force of meat (Koohmaraie et al., 1991) or evidence demonstrating that such meats do not tenderise during aging (Wheeler & Koohmaraie, 1992), although a small number of reports (Jeremiah et al., 1994) have shown no eect on palatability or consumer acceptance. Therefore, there is experimental evidence to show that the use of any of the BAAs habitually used as growth promoters brings about tough meat. This is no doubt a negative eect on the sensory quality of meat, which can adversely aect consumer attitudes towards meat. The aim of this research was to characterise as accurately as possible the texture pro®le of the meat from clenbuterol-fed and control animals, based on enzyme (calpain) activities, sensory evaluation and instrumental
0309-1740/99/$Ðsee front matter # 1999 Elsevier Science Ltd. All rights reserved PII: S0309 -1 740(98)00117 -X
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assessment, to show discriminating dierences due to BAA clenbuterol treatment. 2. Materials and methods 2.1. Animals and muscle preparation During a 14-day adaptation period, eight Charolais heifers of about 8 months of age and weighing approximately 300 kg were individually bucket-fed ad libitum a standard growing-®nishing diet and straw. After this period, they were individually bucket-fed ad libitum the same diet containing either 0 ppm (four control animals) or 1 ppm clenbuterol (four treated animals) during 5 weeks. Throughout this period, they ate 9±10 kg per day of feed. Animal weight was measured at arrival and once a week until slaughter, and daily live weight gain (DLWG, g dayÿ1) calculated. Soon after slaughter (30±60 min), a small portion of m. Longissimus thoracis (6th rib) was excised from carcasses for determining enzyme activities as described below. The rest of m. Longissimus thoracis (7th to 12th ribs) was excised 24 h after death. It was divided in several sections; one was used for pH, fat and calpain/ calpastatin activity determination at this time, a second was vacuum packaged after cutting into 1.2 cm steaks and stored frozen until subsequent sensory analysis, the third was vacuum packaged whole and frozen until instrumental analysis was performed. The rest was vacuum packaged and stored at 4 C for 7 days to age, and was then divided and processed as on the ®rst day. Cross-sectional area (cm2) of m. Longissimus thoracis was measured at 12th rib. For pH determination, about 3 g of muscle were homogenised in 20 ml distilled water for 15 s. The measurement was carried out immediately using a Crison pH-meter with a combined glass electrode. Intramuscular fat (IF) was determined using the Soxhlet method. 2.2. Isolation and activity assay of calpains and calpastatin Partial puri®cation of calpains and calpastatin from muscle tissue was performed according to the method described by Ducastaing & Valin, Schollmeyer, and Cross (1985) and adapted to HPLC. Muscle tissue (20 g) was homogenised with an Ultraturrax in 3 vol. of 10 mM Tris±HCl buer, pH 7.5, containing 0.05 M NaCl, 4 mM EDTA, 2 mM 2-mercaptoethanol and 1 mM NaN3. After 1 hr extraction with magnetic stirring, the homogenate was centrifuged at 30 000 g for 30 min, the supernatant was ®ltered through cheese cloth and adjusted to pH 7.5. Precipitated material was eliminated by centrifugation at 50 000 g for 50 min. All operations were performed at 0±4 C with precooled solutions.
Portions of 50 000 g supernatant were ®ltered through a 0.22 mm millipore membrane and loaded on a mono Q HR 10/10 column (Pharmacia) equilibrated in 5 mM Tris±HCl buer, pH 7.5, 0.1 mM EDTA, 0.05 M NaCl and 2 mM 2-mercaptoethanol. Protein elution by a non-linear NaCl gradient (0.05±0.5 M) was performed at a ¯ow rate of 1 ml minÿ1 and fractions of 1 ml were collected. Calcium-dependent proteolytic activity was assayed, as described by Koohmaraie, Schollmeyer, and Dutson (1986), using casein (Hammerstein; 5 mg mlÿ1) as substrate. Activity of calpastatin was assayed by incubating inhibitor and enzyme at 25 C for 60 min in 1.5 ml reaction mixture, according to Koohmaraie, Seideman, Schollmeyer, Dutson, and Crousse (1987). 2.3. Sensory evaluation On the 1st and 8th days post mortem, ribs were cut into 1.2 cm thick steaks and frozen for subsequent taste panel evaluation. Steaks were thawed at 4 C for 24 h before cooking and serving. They were placed in a preheated double-plate grill at 160 C and removed when an internal temperature of 70 C was reached. Muscle steaks were served on preheated plates to a trained panel of 12 members. Tenderness at ®rst chew, overall tenderness, juiciness, ®brousness, residue and overall acceptability were evaluated as described by Cross, Moen, and Stan®eld (1978), and scored by placing a mark on a nonstructured 10 cm scale. Zero was extremely low (tough, dry, non ®brous, low residue, low quality), 10 was extremely high (tender, juicy, ®brous, high residue, high quality). 2.4. Objective texture measurements The texture parameters were measured on a Universal Testing Machine, Instron model 4301 (High Wycombe, UK), using a Warner±Bratzler shear blade. The parameters studied were: 1. Load: Load at Maximum and Load at Threshold Yield. 2. Energy: Energy to Yield Point and Energy to Break Point. 3. Slope: Slope after Yield. The parameter `Slope after Yield' was evaluated for the ®rst time in this study. The slope was de®ned as the characteristic slope of each curve immediately after the meat sample reached the yield point in the shear force± deformation curve. Calculation was made as depicted in Fig. 1: ®rst, the straight line which best ®tted (highest R2) to the immediate section of the curve after the yield point was drawn; then tga for this line was calculated. For the description of the other parameters, see Instron (1988).
M. LunÄo et al./Meat Science 51 (1999) 297±303
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Meat samples, after thawing at 4 C for 24 h, were cut into 6 cm long1 cm high1 cm wide prismatic pieces. They were placed inside the Warner±Bratzler shear blade and sheared perpendicular to the muscle ®bre longitudinal axis.
component analysis and multivariate discriminant analysis were carried out using the GLM procedures of SAS (1985).
2.5. Statistical analysis
3.1. Daily live weight gain and muscle characteristics
The Student t test was used to assess the signi®cance of dierences between values. Correlation matrix, principal
Clenbuterol-fed heifers had a signi®cantly higher ( p<0.01) daily live weight gain (1.810.07 g dayÿ1)
3. Results and discussion
Fig. 1. Graphic representation of the calculation method for the parameter `slope after yield' (slope=tga=y/x) from a typical force±deformation curve obtained after shearing a meat sample with a Warner±Bratzler shear device mounted on an Instron universal testing machine.
Fig. 2. Representative replicate examples of typical force±deformation curves obtained after shearing with a Warner±Bratzler shear device meat samples from (A) control heifers, and (B) clenbuterol-fed animals.
300
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than control animals (1.350.11 g dayÿ1), that is, it was 34.1% higher in treated animals. These results agree with those reported by Schiavetta, Miller, Lunt, Davis, and Smith (1990), Berge, Culioli, and Ouali (1993) and Garssen, Geesink, Hoving-Bolink, and Verplanke (1995). However, a number of researchers (Miller et al., 1988; Wheeler & Koohmaraie, 1992) did not ®nd any signi®cant dierence. Final pH (5.54 and 5.57 for control and treated, respectively), cross-sectional area (97.7 and 111.3 cm2) and intramuscular fat (2.84 and 2.97 g 100 gÿ1) of m. Longissimus thoracis were not aected signi®cantly ( p>0.05) by clenbuterol treatment. 3.2. Calpains and calpastatin activity Table 1 gives the results regarding the activity of the two isoforms of calpain and their speci®c inhibitor calpastatin. It is worth mentioning the signi®cant dierences found as early as 3 h post mortem. Only m-calpain failed to show any signi®cant ( p>0.05) eect of clenbuterol treatment, probably due to animal variability. Micro-calpain activity was reduced by 31% ( p<0.05) after clenbuterol feeding, while calpastatin activity was increased by 49% ( p<0.05) in the same animals. As a consequence, the ratio m-calpain/calpastatin was highly decreased (53%; p<0.01), in good agreement with Table 1 Eect of clenbuterol administration on m-calpain, m-calpain and calpastatin activities (UI gÿ1) in m. Longissimus thoracis of heifers at 3 h and 1 day post mortem (pm)
m-calpain (3 h pm) m-calpain (1 d pm) m-calpain (3 h pm) m-calpain (1 d pm) calpastatin (3 h pm) calpastatin (1 d pm) m-calpain/calpastatin (3 h pm)
Control
Treated
0.0710.008 0.0350.004 0.0830.070 0.0190.014 0.0840.012 0.0300.003 0.840.052
0.0490.006*a 0.0160.004** 0.0550.024 0.0290.011 0.1250.015** 0.0710.042* 0.390.080**
a Means in the same row followed by asterisks are signi®cantly different (*p<0.05, **p<0.01).
Garssen et al. (1995). Therefore, the direct outcome of these changes in the calpain/calpastatin complex ought to be a reduced proteolytic capacity throughout aging. This, which has been shown to be closely correlated with eective post-mortem tenderisation of meat (RoncaleÂs et al., 1995), should lead to abnormally low tenderisation of meat from animals fed clenbuterol. Previous reports on the activity of the two isoforms of calpain and calpastatin are con¯icting, and most deal with only one of the enzyme forms. Our results oer a more comprehensive view of the eect of BAA on the calpain system, and essentially con®rm those reported by Geesink et al. (1993). They also agree with most reports regarding calpastatin activity (Garssen et al., 1995; Koohmaraie et al., 1991; Kretchmar et al., 1990; Wheeler & Koohmaraie, 1992; ), but disagree with many reports on m- and m-calpain (Garssen et al., 1995; Koohmaraie et al.; Kretchmar et al.; Wheeler & Koohmaraie, 1992). 3.3. Sensory evaluation of tenderness Table 2 shows the results obtained for the texture pro®le speci®cally designed for this experiment (see Materials and Methods). No signi®cant dierences ( p>0.05) between treatments were found on the ®rst day post mortem, but all texture parameters were signi®cantly dierent ( p<0.05) in meat from treated and untreated animals at 8th day post mortem. They demonstrate that meat from control animals was more tender, as seen by the dierence in tenderness at ®rst chew and overall tenderness. This also gave rise to a large dierence in overall acceptability in favour of untreated meat. Results for these three parameters, obtained at 1st and 8th day, showed a signi®cant dierence for control meat ( p<0.05), but not in meat from treated animals. Thus, the sensory consequence of feeding animals with clenbuterol was that meat was tough even after 8 days of aging, which usually leads to tender meat; that is to say, meat quality was greatly reduced. These results
Table 2 Sensory pro®le of meat texture from control and clenbuterol-treated heifers at 1 and 8 days post mortem, evaluated by a trained taste panel using a 10 cm nonstructured scale (0 was extremely low: tough, dry, non ®brous, low residue, low quality; 10 was extremely high: tender, juicy, ®brous, high residue, high quality) 1st day
Tenderness at ®rst chew Overall tenderness Juiciness Fibrousness Residue Overall acceptability a,b,c
8th day
Control
Treated
Control
Treated
4.880.77b 4.710.79b 4.730.44ab 5.450.36bc 5.070.99ab 4.790.96b
3.490.71b 3.140.84b 3.890.41b 6.290.53ab 6.240.39a 3.500.51b
6.310.41a 6.030.45a 5.200.36a 4.870.50c 4.020.46b 6.310.32a
4.160.22b 3.760.27b 4.180.41b 6.220.39a 6.020.37a 3.920.38b
Means in the same row followed by dierent superscripts are signi®cantly dierent ( p <0.05).
M. LunÄo et al./Meat Science 51 (1999) 297±303
seem to agree with those reported previously on the textural quality of meat assessed by instrumental techniques, as will be discussed later. However, there are a lack of studies dealing with the assessment of the texture of meat from clenbuterol treated animals using sensory evaluation. 3.4. Instrumental evaluation of tenderness Results corresponding to the texture parameters determined with a Warner±Bratzler shear blade are shown in Table 3. No signi®cant dierences ( p>0.05) were found in any of the parameters, measured on the ®rst day, between treated and untreated animals, with the exception of the `slope after yield' (Fig. 1). An example of some of the force±deformation curves, representative of replicates of one sample of meat from both control and treated animals, is shown in Fig. 2. As shown in Table 3 and Fig. 2, the slope after yield showed a clear and signi®cant dierence ( p<0.05) between control and treated animals. The latter having in all cases a steeper slope, with values always above 6, and a much lower standard deviation, i.e. less variability. Measurements at 8th day varied greatly with respect to 1st day. All parameters showed signi®cant dierences ( p<0.05) between treatments, with the exception of the energy to break point, which was not able to dis-
301
criminate between the two groups of samples. Eective discrimination by most textural parameters was related to the fact that meat from clenbuterol-fed animals did not tenderise during aging, as demonstrated by the absence of any signi®cant dierences with the 1st day. On the contrary, meat from control heifers was signi®cantly more tender ( p<0.05) after aging. This was demonstrated by the maximum load (shear force) results, which is the most widely used parameter used to assess tenderness. The shear force results essentially agree with those previously reported in animals treated with clenbuterol (Garssen et al., 1995; Miller et al., 1988; Schiavetta et al., 1990; ), L644,969 (Moloney, Allen, Joseph, Tarrant, & Convey, 1994; Wheeler & Koohmaraie, 1992) and cimaterol (Fiems, Buts, BoucqueÂ, Demeyer, & Cottyn, 1990). Thus according to our results and those of most other authors, instrumental parametersÐmainly shear force (maximum load)Ðare able to discriminate BAA-treated from untreated animals. However, discrimination is only possible after aging. We have though demonstrated that the slope after yield is capable of discriminating BAA treatment as early as 24 h after slaughter. The reason for that is not clear, but it must be related, at least in part, to the fact that, even if dierences for most parameters were not signi®cant, possibly due to the low number of animals studied, meat from treated animals
Table 3 Instrumental pro®le of meat texture from control and clenbuterol-treated heifers at 1 and 8 days post mortem, using a Warner±Bratzler shear blade mounted on an Instron universal testing machine 1st day Control Maximum load (N) Load at yield (N) Energy to yield (J) Energy to break (J) Slope after yield a,b
8th day Treated
a
89.118.2 88.412.1a 0.170.08ab 1.090.47a 3.691.66b
Control a
118.817.3 118.119.4a 0.360.18a 1.550.72a 8.000.37a
Treated b
51.29.51 49.410.4b 0.100.03b 0.810.21a 1.801.39b
109.112.1a 108.718.3a 0.260.10a 1.280.45a 7.740.70a
Means in the same row followed by dierent superscripts are signi®cantly dierent ( p<0.05).
Fig. 3. Principal components analysis of all texture parameters, both sensory and instrumental, of meat from control (*) and clenbuterol-treated heifers (*) at 1 day post mortem (A) and 8 days post mortem (B).
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M. LunÄo et al./Meat Science 51 (1999) 297±303
Table 4 Correlation matrix of all texture parameters, both sensory and instrumental, of meat from control and clenbuterol-treated heifers at 1 day post mortem
Maximum load (M.L.) Load at yield (L.Y.) Energy to yield (E.Y.) Energy to break (E.B.) Slope after yield (S.Y.) Tenderness at ®rst chew (T.F.C.) Overall tenderness (O.T.) Residue (R.) Overall acceptability (O.A.) a
M.L.
L.Y.
E.Y.
E.B.
S.Y.
T.F.C
± 0.954***a 0.708* 0.632 0.566 ÿ0.740*
± 0.691 0.655 0.376 ÿ0.762*
± 0.891** 0.766* ÿ0.692
± 0.503 ÿ0.605
± ÿ0.481
±
ÿ0.782* 0.455 ÿ0.805*
ÿ0.817* 0.484 ÿ0.846**
ÿ0.738* 0.590 ÿ0.659
ÿ0.659 0.564 ÿ0.580
ÿ0.481 0.399 ÿ0.411
0.994*** 0.603 0.979***
O.T
R
O.A
± ÿ0.872** 0.985***
± ÿ0.840**
±
Correlation coecients followed by asterisks are signi®cant (*p<0.05; **p<0.01; ***p<0.001).
always had a higher shear force. Besides this, as seen in Fig. 2, clenbuterol treatment gives rise to a modi®cation of the ®nal part of the force±deformation curve (between the maximum load and the break point). This part, when recorded for untreated animals, usually gives a ¯atter and more variable slope than meat from treated animals, which gives a steeper and less variable slope. According to Voisey (1976), this part of the curve is related to the ®nal phase of cutting meat with the teeth, and might relate to the collagen characteristics of the sample. 3.5. Correlation matrix Table 4 shows the correlation matrix for all texture parameters, both sensory and instrumental, at the ®rst day post mortem. It is clear there are high and signi®cant correlations between most of the textural attributes of meat. Indeed, all sensory parameters were highly correlated ( p<0.01), and many instrumental parameters were also correlated ( p<0.05) both between themselves and with most of the sensory attributes. However, remarkably low correlations were found between the slope after yield and any other texture parameter. The reason for this is not clear, but agrees with the results shown above, suggesting that the slope Table 5 Results of multivariate discriminant analysis: canonical structure of all texture parameters, both sensory and instrumental, of meat from control and clenbuterol-treated heifers at 1 day post mortem; and canonical coecients distribution on a single axis of texture parameters and treatment groups Canonical structure Canonical coecients Maximum load Load at yield Energy to yield Energy to break Slope after yield Tenderness at ®rst chew Control group Treated group
0.555799 0.492400 0.915856 0.707177 0.853560 0.723747 ± ±
ÿ3.672710 1.243822 0.521930 ÿ0.820708 1.394431 0.475677 ÿ0.935414 0.935414
after yield may relate to the treatment of the animals with a BAA. Results (not shown) of the correlation matrix for texture parameters at 8th day con®rmed those of the 1st day post mortem; the only dierence being even higher correlation coecients than found on the 1st day. 3.6. Principal component analysis Fig. 3 shows the principal component analysis for all texture data, both sensory and instrumental, obtained at 1st (A) and 8th (B) day post mortem. At 1st day (A), the ®rst principal component explained 70% of the variation, while the second accounted for 15%; thus an accumulative 85% of total variation was explained by the ®rst two principal components. As seen in Fig. 3 (A), the ®rst component alone was able to separate meat of clenbuterol-fed animals from that of untreated animals. The contribution of each variable to the ®rst component (not shown) exhibited only small dierences, meaning that none of the texture parameters alone could reasonably explain dierences due to treatment, but all were necessary to eectively dierentiate treated from untreated animals. At the 8th day (B), the ®rst principal component explained 82% of the variation, while the second accounted for 11.5%; thus an accumulative 93.5% of total variation was explained by the ®rst two principal components. As stated above, the ®rst component alone was able to dierentiate clenbuterol treated animals from controls. Thus, although of limited value because of the high correlations found among most of the parameters, applying principal component analysis to all the sensory and instrumental data allowed discrimination of meat from BAA-treated and untreated animals in this experiment. 3.7. Multivariate discriminant analysis Results of multivariate discriminant analysis of the textural data at the 1st day post mortem are shown in
M. LunÄo et al./Meat Science 51 (1999) 297±303
Table 5. Three parameters were excluded in this analysis: overall tenderness, ®nal residue and overall acceptability, as they were not signi®cant in discriminating meat samples at either of sampling times. The canonical coecients for the rest of parameters, as well as for the treated and untreated groups, were located on a single axis. It is worth noting that meat from clenbuterol-fed and control animals was discriminated, showing the same absolute value with opposite sign. Thus discriminant analysis of the textural data obtained at the ®rst day after slaughter allowed, too, the dierentiation of meats from BAAtreated and untreated animals in this experiment. 4. Conclusions These results show a signi®cant lowering of meat tenderness on BAA treatment of animals, due to a decrease in eective calpain activity. However, this difference was manifest only after 8 days of post-mortem aging, if standard methods of measuring textural parameters, both sensory and instrumental, were used. A new parameter calculated from the Warner±Bratzler shear force±deformation curve appeared to show dierences due to clenbuterol treatment 24 h after slaughter ( p<0.05). In addition, both principal component analysis and multivariate discriminant analysis of all textural data, instrumental as well as sensory, showed up dierences due to BAA clenbuterol treatment in this experiment. Acknowledgements The authors wish to thank the ComisioÂn Interministerial de Ciencia y TecnologõÂa (CICYT; grant no. ALI92-0644) for support of this research, the DiputacioÂn General de AragoÂn for the fellowship of author M. LunÄo, and the personnel of Servicio de Apoyo a la ExperimentacioÂn Animal of the University of Zaragoza.
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