The effect of meat quality, salt and ageing time on biochemical parameters of dry-cured Longissimus dorsi muscle

Meat Science 51 (1999) 329±337

The e€ect of meat quality, salt and ageing time on biochemical parameters of dry-cured Longissimus dorsi muscle Marta Gil, Luis Guerrero, Carmen SaÂrraga* Institut de Recerca i Tecnologia AgroalimentaÁries (IRTA), Centre de Tecnologia de la Carn, Granja Camps i Armet s/n, 17121 Monells, Girona, Spain Received 28 February 1998; received in revised form 25 May 1998; accepted 18 August 1998

Abstract Pieces of Longissimus muscle were used as models of dry-cured ham to study the e€ect of NaCl concentration, meat quality and ageing time on the biochemical parameters related to the ham curing process. Higher amounts of added salt did not result in an increase in the protein extractibility. The activity of the lysosomal enzymes cathepsins D and B showed dependence on the ageing time, whereas cathepsin B and cathepsin B + L activities depended on meat quality. Cathepsin B + L activity increased with the addition of salt. Meat quality had an in¯uence on all the parameters studied except on MFI, on cathepsin D activity and on the inhibitory activity on cathepsin B. Two protein bands likely to correspond to titin and nebulin were detected in the pherograms of myo®brillar proteins of muscle even those aged for as much as 48 h and irrespective of meat quality. Titin was also found in the sarcoplasmic protein fraction of salted samples from N and DFD meat, but not from PSE meat. # 1999 Elsevier Science Ltd. All rights reserved.

1. Introduction Meat product manufacture depends to a large extent on the quality of the raw material. The characteristics of the raw material vary in many ways, such as meat quality, fat quality and genetic line which a€ect the sensorial and technological attributes of the ®nal product (Arnau, 1991; Guerrero, Gou, Alonso, & Arnau, 1996; Oliver, Gispert, & Diestre, 1993). Meat of a pale, soft and exudative (PSE) nature and meat which is dark, ®rm and dry (DFD) present serious problems for the meat industry. The deviations from normal (N) meat that these types of meat present in pH and water holding capacity make a proper dry-curing process dif®cult and, thus, the desired levels in the quality of the ®nal product cannot be attained (Arnau, 1991). Cured meat products are based on the addition of salt which acts as a preserving agent and is also responsible for causing the physico-chemical and biochemical phenomena that contribute to the development of ¯avour. The di€usion of the salt is a key step in dry-cured ham processing. The dissolution of NaCl on the surface of

* Corresponding author. Tel.: +34-972-63-03-52; fax: +34-972-6303-73.

the meat is the principal factor regulating the penetration of the salt into the ham (SoÈrheim & Gumpen, 1986). The velocity of penetration of NaCl may be enhanced in PSE hams, and reduced in DFD hams. During the ham curing process an intense proteolysis takes place, which is mostly the result of the action of the muscle proteinases: calpains and cathepsins (HortoÂs & GarcõÂa-Regueiro, 1991; ParrenÄo, CussoÂ, Gil, & SaÂrraga, 1994; SaÂrraga, Gil, GarcõÂ-Regueiro, 1993; Toldra & Etherington, 1988). From these two groups of proteinases, calpains are quite unstable and their activity is not detected after the salting stage (ParrenÄo, SaÂrraga, Gil, & Cusso 1990; SaÂrraga et al., 1993), while cathepsins are active even at the end of the curing process (ToldraÂ, Flores, & Sanz, 1997; SaÂrraga et al.). Proteolysis is a key parameter for understanding some of the sensorial and technological problems in dry-cured ham processing, such as abnormal softness and the appearance of a white ®lm and white crystals of tyrosine on the surface of the ham (Arnau, Gou, & Guerrero, 1994; Virgili, Parolari, Schivazappa, Soresi Bordini, & Borri, 1995). A low salt-to-moisture ratio may favour softness (Parolari, Rivaldi, Leonelli, Bellati, & Bovis, 1988) because a higher residual moisture content in the muscle makes proteinases active for a longer period of time and this results in a higher degree of proteolysis.

0309-1740/99/$Ðsee front matter # 1999 Elsevier Science Ltd. All rights reserved PII: S0309 -1 740(98)00129 -6

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M. Gil et al./Meat Science 51 (1999) 329±337

Moreover, the current tendency towards low-sodium diets is promoting studies on meat products manufactured with lower amounts of sodium chloride (Gou, Guerrero, Gelabert, & Arnau, 1996), so that the problems arising out of excess proteolysis need to be controlled even more. The aim of this study was to evaluate the e€ect of NaCl concentration, meat quality and ageing time on acidic lysosomal cathepsins B, L and D activities, on the myo®brillar structure and on the protein degradation of the Longissimus dorsi muscle. We used this muscle as a model of the ®rst steps of dry-cured ham processing (salting and salt-equalization) in order to learn more about the mechanisms of muscle protein changes during these essential phases of curing. 2. Materials and methods Selection of the samples: Longissimus dorsi porcine muscles of normal (n=15) and PSE (n=15) meat were selected by measuring electric conductivity at the last rib level with a Quality Meater (Digi 550, Wissenschaftlichtechmishe, Weilheim, Germany) at 45 min postmortem (PSE > 10, N < 4.0) (Oliver, Gispert, Tibau, & Diestre, 1991). DFD samples (n=10) were selected by measuring pH at 24 h postmortem (pH > 6.1). The ageing times studied were t=0, 24 and 48 h for N and PSE meat, and 24 h and 48 h for DFD meat, because one cannot detect these characteristics prior to 24 h postmortem. Each loin adequately aged was trimmed of fat and connective tissue and then cut into ®ve pieces, which were put aside for one control and four treatments with increasing percentages (w/w) of NaCl (2, 4, 6 and 4% salt + 1% dextrose). The ®ve pieces were distributed in a balanced way to avoid the e€ect of anatomical location.The portion for control was packed under vacuum and kept at ÿ80 C until use. Five replicates were done for each ageing time and each type of meat. Salt treatment: The adequate amount of NaCl and dextrose to reach the desired percentage of the additive was added to each portion of loin (about 100 g) which were then vacuum packed in a dark plastic bag. The packed muscles were then hand-rubbed to facilitate the distribution of the salt and left for one week at 4 C. Afterwards each piece of loin was unpacked, washed in cold water and hung at 3±5 C in an atmosphere with a relative humidity of between 70 and 80% for 10 days. After this period, samples were kept at ÿ80 C until analysed. The following methods were carried out for controls and salt-treated samples. Physicochemical analysis: Meat pH was determined with 10 g of muscle in 90 ml of distilled water. Moisture was measured by weight loss at 103 ‹ 2 C (Presidencia

del Gobierno, 1979). Sodium chloride content was analyzed using the Charpentier±Volhard method (ISO, 1970). Preparation of myo®brils: Myo®brils were obtained according to the method of Bush, Stromer, Goll, and Susuki (1972) using 50 mM, pH 7.6, Tris±HCl bu€er, containing 5 mM EDTA and 100 mM KCl as the isolation medium. The myo®brils were suspended in 100 mM KCl, 1 mM NaN3. An aliquot was saved to perform the electrophoretic analysis. Myo®brillar fragmentation index (MFI) determination: An aliquot of the myo®bril suspension was diluted with the isolation medium to 0.5 g/l protein concentration and the absorbance of this suspension measured at 540 nm. MFI values were recorded as absorbance units per 0.5 g/l myo®bril protein concentration multiplied by 200 (Olson, Parrish, & Stromer, 1976). Myo®brillar protein solubility: Myo®brillar protein solubility was measured according to Ducastaing, Valin, Schollmeyer, and Cross (1985). Twenty to thirty mg of myo®brillar protein in a total volume of 6.5 ml of 4 mM ATP, 4 mM MgCl2, 0.2 mM CaCl2, adjusted to pH 7.7 with 2N KOH were incubated for 5 min at 25 C. The solubilized proteins were determined in the supernatant after centrifugation at 5000 g for 10 min according to Lowry, Rosebrough, Farr, and Randall (1951) and given as mg of protein solubilized per g of myo®brillar protein. Muscle protein extractibility: A portion of ground muscle was homogenised with an Ultra-Turrax T25 (13,500 rpm, 15 s) in 30 mM phosphate bu€er (1/10, w/ v) and allowed to stand for 1 h at 4 C. Sarcoplasmic proteins were recovered in the supernatant after a centrifugation at 10,000 g for 30 min at 4 C. The pellet resulting from the initial centrifugation process was resuspended in 100 mM phosphate bu€er, pH 7.4, 1.1 M KI (Helander, 1957), allowed to stand for 3 h and centrifuged again in the same way. The ®ltered supernatant contained the myo®brillar proteins. The resulting pellet was washed with CaCl2 to remove the KI and gently resuspended in a 1% SDS solution and stirred at room temperature overnight. The supernatant of the centrifugation (10,000 g, 30 min, 4 C) constituted the 1% SDS extractable proteins. Total protein of the supernatants was determined by the Biuret method (Gornall, Bardewill, & David, 1949) using bovine serum albumin as standard. An aliquot of each supernatant was saved to perform the electrophoretic analysis. Obtention of cathepsins and inhibitors: The preparation of enzymes and cystatins was based on the method described by Koohmaraie and Kretchmar (1990). A portion of ground muscle was homogenized with an Ultra-Turrax T25 (13,500 rpm, 15 s) in 4 vol. of ice-cold 50 mM sodium acetate bu€er, pH 5.0, containing 1 mM EDTA and 0.2% (v/v) Triton X-100. The homogenate

M. Gil et al./Meat Science 51 (1999) 329±337

was stirred for 1 h at 4 C and then centrifuged at 37,000 g for 30 min. The supernatant ± containing cathepsins and cystatins ± was ®ltered through glass wool and a 2-ml aliquot was allowed to react with 2 ml of S-carboxymethylated-papain-Sepharose in a column, for 2 h at 4 C and with constant end-over-end mixing. The CNBr-activated Sepharose gel (Pharmacia Biotech) was coupled to papain according to the procedure described by Anastasi et al. (1983). After mixing, the supernatant was eluted from the column and then a 6-ml wash with the homogenisation bu€er was done to recover all the enzyme activity. As the Ki value for papain is higher than the Ki value for cathepsins B or L (Barrett, 1987) these eluents were free of cystatins. The cystatins were recovered from the anity gel by elution with 6 ml of 50 mM trisodium phosphate bu€er, pH 11.5, containing 0.5 M NaCl. All the fractions collected were of 2 ml. Assay of cathepsin activity: Cysteine proteinases were assayed ¯uorimetrically with commercial substrates (Etherington & Wardale, 1982) in the eluted fractions from the S-carboxymethylated-papain-Sepharose column: Cathepsin B (EC 3.4.22.1) was assayed with NCBZ-l-arginyl-l-arginine 7-amido-4-methylcoumarin (Z-Arg-Arg-NHMec) and cathepsins B and L (EC 3.4.22.15) with the common substrate N-CBZ-l-phenylalanyl-l-arginine 7-amido-4-methylcoumarin (Z-PheArg-NHMec) (Bachem). One unit of activity was de®ned as the amount of enzyme hydrolysing 1 nmol of substrate per min at 37 C. The aspartyl proteinase cathepsin D (EC 3.4.23.5) was assayed against denatured bovine haemoglobin (Sigma) (Etherington, 1972) in the supernatant prior to the reaction with the Sepharose. The TCA-soluble peptides were measured using the method of Lowry et al. (1951) with l-tyrosine as standard. One unit of activity was de®ned as the amount of enzyme releasing 1 mg of tyrosine per min at 45 C. Activities were given in enzyme units per mg of extracted protein. Assay of inhibitor activity: Inhibitory activity on cathepsin B and cathepsin B + L activities was assessed under the same conditions as those used for the corresponding proteinase activities. A preincubation of the inhibitors with the enzyme for 10 min at 37 C was necessary before measuring the residual activity of the cathepsins in normal conditions. The inhibitory activity was calculated as the percentage of lost enzyme activity. One unit of inhibitor activity was de®ned as the amount that inhibited one unit of enzyme activity. Protein determination: Protein concentration of the isolated myo®bril suspension was determined by the method of Gornall et al. (1949). The protein concentration of the enzyme and inhibitor solutions was measured according to Lowry et al. (1951). In both cases, bovine serum albumin was used as standard.

331

SDS-PAGE Electrophoresis: The protein of each sample saved to perform the electrophoretic analysis was precipitated with acetone and the resulting pellet evaporated to dryness (Speed Vac Sc 210 A), and dissolved in rigor bu€er (75 mM KCl, 10 mM Tris, pH 7.0, 2 mM EGTA, 2 Mm MgCl2, 2 mM NaN3) and sample bu€er (8 M urea, 2 M thiourea, 3% SDS, 75 mM DTT, 25 mM Tris HCl, pH 6.8) (Fritz & Greaser, 1991), in (1:1) proportion, up to a 5 mg/ml protein concentration. The mixtures were then heated to 50 C for 20 min prior to processing. Phast Gel 12.5% (Pharmacia Biotech AB) was used to perform the electrophoresis in a horizontal system (Phast System, Pharmacia Biotech AB). Samples (5 mg) were run in the stacking gel at 1.0 mA for 1 V h and then in the separation gel at 3.0 mA, 80 V h at 15 C. Phast Gel 4±15% was necessary for the resolution of the high molecular weight myo®brillar proteins such as titin and nebulin. The separation gel was run at 10.0 mA, 110 V h at 15 C. The gels were stained with Coomassie Blue R-350. Statistical analysis: Analysis of data was performed using the ANOVA procedure from the Statistical Analysis System (SAS, 1988) using meat quality, salt, time and their double interactions as ®xed e€ects. 3. Results and discussion In this study, pieces of loin were used as models of the ®rst steps of the dry-cured ham processing, with percentages of added salt up to 6% (w/w), which is the highest amount of NaCl found in standard commercial hams (Arnau, 1991). The conditions of curing (temperature, relative humidity) simulated the salt-equalization step, which seems to be the most important step with regard to the changes a€ecting muscle proteins during the ham curing process. The study can be divided into four parts: (1) Physicochemical parameters determined as control parameters, (2) parameters related to the protein integrity, (3) enzyme and inhibitor activities and (4) electrophoresis. The statistical analysis of the parameters studied in parts 1±3 showed that meat quality had a marked e€ect on all the measured variables except the myo®brillar fragmentation index (MFI), cathepsin D activity and inhibitor activity on cathepsin B. The addition of salt had in¯uence on most of the variables, while ageing time a€ected mainly enzyme and inhibitor activities. All the double interactions were not signi®cant ( p > 0.05) and, therefore, meat quality, salt and time were analyzed as independent e€ects. Tables 1±3 show the values of least squares means obtained for all the parameters studied. Control parameters: As expected, pH depended on meat quality ( p < 0.001), moisture on meat quality

332

M. Gil et al./Meat Science 51 (1999) 329±337

consistent conclusions from them, though MFI did prove to be useful as an orientative parameter. MFI was found to depend only on salt ( p < 0.05). Table 3 shows the changes in MFI with the addition of salt. It seemed that high amounts of salt prevent the myo®brillar structure from fragmentation. Myo®brillar protein solubility depended on meat quality ( p < 0.001) and on salt ( p < 0.05), but not on the ageing time studied. Results from Table 1 show that it was higher in DFD meat with respect to N meat and lower in PSE meat. In

( p < 0.01) and salt ( p < 0.001), and NaCl on meat quality ( p < 0.001) and amount of added salt ( p < 0.001). The period of ageing studied (0±48 h) was not relevant on any of the control parameters. Through Clÿ determination it was con®rmed that NaCl penetration on the muscle was enhanced in PSE meat (Table 1) and that the maximum amount of salt di€used into the muscle was about 5% (Table 3). Parameters related to the protein integrity: MFI values showed high dispersion and it was dicult to obtain

Table 1 Physico-chemical composition, protein integrity and cathepsin activities of Longissimus muscle from N, PSE and DFD meat N

PSE

DFD

LS mean

Std error

LS mean

Std error

LS mean

Std error

pH Moisture (%) NaCl (%)

5.87 ba 68.34 b 2.90 b

0.032 0.256 0.056

5.56 c 68.26 b 3.19 a

0.032 0.256 0.056

6.33 a 69.67 a 2.88 b

0.042 0.339 0.075

MFI Myo®brillar protein solubility b Sarcoplasmic protein extractibility c Myo®brillar protein extractibility c 1% SDS-extractable protein c

95.95 20.11 b 6.87 b 13.53 a 6.26 b

3.609 1.083 0.107 0.260 0.231

96.62 14.06 c 5.83 c 11.21 b 8.09 a

3.609 1.083 0.107 0.260 0.231

98.98 30.21 a 7.93 a 13.03 a 5.46 c

4.774 1.432 0.142 0.344 0.305

Cathepsin D activity d Cathepsin B activity d Cathepsin B + L activity d Inhibition of Cathepsin B activity (%) Inhibition of Cathepsin B + L activity (%)

0.50 0.16 b 2.49 b 15.26 78.05 a

0.029 0.007 0.112 1.189 1.070

0.52 0.19 a 2.86 a 17.02 76.69 a

0.029 0.007 0.112 1.189 1.070

0.45 0.13 b 1.73 c 13.41 70.46 b

0.038 0.010 0.149 1.573 1.416

a b c d

Means with di€erent letters di€er ( p < 0.05). Values of myo®brillar protein solubility are given in mg of solubilized protein/g of myo®brillar protein. Results are given as mg of protein/ml. Values of enzyme activities are given in units/mg of protein.

Table 2 Physico-chemical composition, protein integrity and cathepsin activities of Longissimus muscle during ageing at 4 C 0 h Postmortem LS mean

Std error

24 h Postmortem LS mean

48 h Postmortem

Std error

LS mean

Std error

pH Moisture (%) NaCl (%)

5.94 69.20 3.12

0.042 0.339 0.075

5.87 68.25 2.92

0.032 0.256 0.056

5.95 68.82 2.94

0.032 0.256 0.056

MFI Myo®brillar protein solubility b Sarcoplasmic protein extractibility c Myo®brillar protein extractibility c 1% SDS-extractable protein c

96.76 21.40 7.07 11.43 ba 6.96

4.774 1.432 0.142 0.344 0.305

92.87 21.26 6.83 13.16 a 6.67

3.609 1.083 0.107 0.260 0.231

101.91 21.72 6.73 13.18 a 6.17

3.609 1.083 0.107 0.260 0.231

Cathepsin D activity d Cathepsin B activity d Cathepsin B + L activity d Inhibition of Cathepsin B activity (%) Inhibition of Cathepsin B + L activity (%)

0.39 b 0.15 b 2.39 13.43 75.24 ab

0.038 0.010 0.149 1.573 1.416

0.52 a 0.19 a 2.29 17.37 72.72 b

0.029 0.007 0.112 1.189 1.070

0.56 a 0.15 b 2.40 14.90 77.23 a

0.029 0.008 0.112 1.189 1.070

a b c d

Means with di€erent letters di€er ( p < 0.05). Values of myo®brillar protein solubility are given in mg of solubilized protein/g of myo®brillar protein. Results are given as mg of protein/ml. Values of enzyme activities are given in units/mg of protein.

M. Gil et al./Meat Science 51 (1999) 329±337

a previous study (HortoÂs, Gil, & SaÂrraga, 1994) MFI and myo®brillar protein solubility of Longissimus muscle aged from 0 to 14 days are, indeed, reported to depend on time; myo®brillar protein solubility is also reported to be a€ected by meat quality in accordance with the present results. It is well known that muscle protein extractibility is a€ected by meat quality and that the solubility of sarcoplasmic and myo®brillar proteins is lower in PSE meat than in N meat (LoÂpez-Bote, Warris, & Brown, 1989; Park, Ito, & Fukazawa, 1975; Sayre & Briskey, 1963), but few studies on the solubility of proteins in DFD meat are presently available (Warner, Kau€man, & Greaser, 1997). Table 1 shows the di€erences in protein extractibility between the di€erent kinds of meat. The results agree with Warner et al.: DFD samples showed the highest sarcoplasmic and myo®brillar protein values, whereas N samples showed intermediate values and PSE the lowest values. Accordingly, values of 1%SDS extractable protein Ð remaining protein after extraction of sarcoplasmic and myo®brillar protein Ð were higher in PSE samples and lower in DFD ones. The ageing time of the muscle only a€ected the extractibility of myo®brillar protein, which was higher in 24 h- and 48 h-aged muscles than in unaged samples (Table 2). Table 3 shows that whereas increasing the amount of added salt above 2% did not result in an increase in the myo®brillar protein extractibility, a 4% of salt was necessary to increase the extractibility of 1%SDS extractable protein. On the other hand, the sarcoplasmic protein extractibility was independent of the salt. The addition of dextrose did not bring about signi®cant changes in any of the parameters related to the protein integrity.

333

Enzyme and inhibitor activities: Cathepsin D activity was found to depend on the ageing time ( p < 0.05). Non-signi®cant di€erences of activity were found after 24 h postmortem (Table 2). Cathepsin B activity depended on meat quality ( p < 0.001) and on ageing time ( p < 0.01) and showed maximum values for PSE meat (Table 1) and at 24 h postmortem (Table 2). This result can partially explain the problems of excess of proteolysis that occur in PSE hams (Arnau, 1991). Parolari, Virgili, and Schivazappa (1994) recommend the use of green hams with a low level of cathepsin B activity in order to avoid texture problems due to excess of proteolysis and, thus, to improve the quality of the ®nal product. Previous results indicate that cathepsin B activity is the most stable activity during the ham curing process (ParrenÄo et al., 1994), probably due to the low inhibitory action of porcine cystatins on cathepsin B (Gil & SaÂrraga, 1997; Koohmaraie & Kretchmar, 1990). Cathepsin B + L activity was found to be a€ected by the meat quality as well ( p < 0.001), and indeed by the addition of salt ( p < 0.001) (Tables 1 and 3). DFD meat samples showed the lowest activity and PSE meat samples the highest. The presence of 2% salt favoured an increase in the activity of cathepsin B + L, which was not further a€ected by higher levels of salt or by dextrose. These results agree with those of Arnau, Guerrero, and SaÂrraga (in press) and Toldra and Etherington (1988) that suggest that the curing agents have a stabilizing e€ect on the activity of the proteinases. Although hams produced from DFD meat also present problems, these are not due to the excess of proteolysis but to easy spoilage by microorganisms because of the high pH of the meat. This agrees with the low

Table 3 Physico-chemical composition, protein integrity and cathepsin activities of Longissimus muscle related to added NaCl

pH Moisture (%) NaCl (%) MFI Myo®brillar protein solubility b Sarcoplasmic protein extractibility c Myo®brillar protein extractibility c 1% SDS-extractable protein c Cathepsin D activity d Cathepsin B activity d Cathepsin B + L activity d Inhibition of Cathepsin B activity (%) Inhibition of Cathepsin B + L activity (%) a b c d

0% NaCl

2% NaCl

4% NaCl

6% NaCl

4% NaCl + % dextrose

Std error

5.91 75.05 aa 0.04 d

6.00 68.06 b 2.12 c

5.90 67.39 b 3.89 b

5.88 66.21 c 5.16 a

5.90 67.08 bc 3.75 b

0.044 0.355 0.078

102.86 a 23.84 a 7.10 11.73 c 5.43 b

108.02 a 23.78 a 6.81 13.82 a 6.07 b

95.57 ab 20.63 ab 6.65 13.04 ab 7.05 a

84.82 b 17.93 b 6.95 11.86 c 7.30 a

94.63 ab 21.12 ab 6.86 12.51 bc 7.16 a

4.996 1.499 0.149 0.360 0.320

0.43 0.15 1.74 b 15.42 70.62 b

0.54 0.15 2.42 a 13.82 76.86 a

0.52 0.16 2.76 a 17.72 77.17 a

0.46 0.16 2.44 a 14.27 74.23 ab

0.51 0.18 2.45 a 14.94 76.45 a

0.040 0.010 0.156 1.64 1.482

Means with di€erent superscripts di€er ( p < 0.05). Values of myo®brillar protein solubility are given in mg of solubilized protein/g of myo®brillar protein. Results are given as mg of protein/ml. Values of enzyme activities are given in units/mg of protein.

334

M. Gil et al./Meat Science 51 (1999) 329±337

cathepsin B + L activity found in DFD meat (Table 1). Arnau et al. also report that samples of the Biceps femoris (BF) muscle from dry-cured hams with di€erent pH and aged for 6 months presented low lysosomal cystein proteinase activity in hams with high pH. The anity of muscular cystatins for cathepsin B is lower than their anity for cathepsin L (Gil & SaÂrraga, 1997; Koohmaraie & Kretchmar, 1990; Ouali et al., 1995). In accordance with this, inhibition of cathepsin B activity by cystatins was very low and showed constant values irrespective of salt, ageing time or meat quality. In contrast, inhibition of cathepsin B + L activity was higher and depended on the meat quality ( p < 0.001), the ageing time ( p < 0.05) and the addition of salt ( p < 0.01) (Tables 1±3). It is noteworthy (Table 1) that the inhibition in DFD meat was lower than the inhibition in N and PSE meat. The presence of 2% salt favoured inhibition, which was maintained when higher levels of salt were added (Table 3). The activity of cystatins during curing follows a similar pattern to the activity of cathepsin B and B + L (ParrenÄo et al., 1994). As in vitro studies (Gil & SaÂrraga, 1997; Ouali et al., 1995) showed that cystatin activityÐ unlike enzyme activityÐis not a€ected by pH, the lower inhibitory activity found in DFD meat would not be due to the higher pH of this kind of meat (Table 1). The control of cathepsins by their inhibitors during the curing process would probably depend on other factors such as the salt and the level of the enzyme activity. Further studies on cystatins are of interest because of their possible use as additives in meat products. Electrophoresis: Titin and nebulin are myo®brillar proteins with a very high molecular weight (1.0  106 and 5  105 Da, respectively) which constitute the major components of the gap ®laments (Locker, 1984). Because of the unusually large size of these proteins, the use of low concentrations of acrylamide gels becomes necessary to detect them by SDS-PAGE. Titin has generally been reported to migrate as a closely spaced doublet (T1 and T2) (Lusby, Ridpath, Parrish, & Robson, 1983; Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995), although some authors describe it as a single band at very early postmortem times (Fritz, Mitchell, Marsh, & Greaser, 1993), or as a triplet (Hu€Lonergan, Parrish, & Robson, 1995; Paterson & Parrish, 1987). Nebulin, detected as a single band below titin, is degraded faster than titin (Fritz & Greaser, 1991). Our results (Phast Gels 4±15%) showed two protein bands migrating in the high molecular weight zone of the gel likely to correspond to titin and nebulin (Fritz et al.). Both bands were still present in the muscle at 48 h of ageing, irrespective of meat quality. The rate of degradation of both proteins is related to species (Chou, Tseng, Lin, & Yeng, 1994; Paxhia & Parrish, 1988), age and sex (Hu€-Lonergan et al.) and to the ®nal pH of the meat (Watanabe & Devine, 1996; Warner et al., 1997).

Previous studies on pork muscle indicate that the bands of titin and nebulin are clearly detected at 7 days postmortem (Paxhia & Parrish, 1988), while Lusby et al. (1983); Taylor et al. (1995) in bovine muscles report that both proteins are completely broken down between 1 and 7 days postmortem. Warner et al. (1997), by using Western immunoblotting, ®nd that titin from pork meat is less degraded in PSE muscles than in N muscles, whereas nebulin is more degraded in PSE muscles; conversely, in DFD meat titin is degraded more rapidly than nebulin. The two bands observed in our pherograms on 4±15% Phast Gels were mainly detected in the myo®brillar protein fraction and myo®brils, but also in the 1%SDS extractable fraction [Fig. 1(A), lanes 2,3,4,6,7]. Titin was detected as a single band except on the myo®brils isolated from control samples in which it appeared as a doublet (T1 and T2). As stated by Fritz and Greaser (1991), the T2 band may arise during the isolation of the myo®brils. Nebulin was detected as a thin faint band. Neither of the proteins were found in the control sarcoplasmic protein fraction, but titin was detected in this fraction in the samples with NaCl in N and DFD meats [Fig. 1(B), lane 3]. In PSE meat, however, the addition of salt hardly enhanced the solubilization of titin to the sarcoplasmic fraction (Fig. 1(B), lanes 5,6], in line with the low protein solubility of this type of meat. A band in the zone of 100 kDa (just below a-actinin) appeared in the pherograms of the myo®brils isolated from salted samples [Fig. 1(A), lane 7] and also in those of the 1%SDS extractable protein fraction from salted samples [Fig. 1(A), lanes 3 and 4]. On the contrary, myo®brils and myo®brillar protein extracts obtained from control samples did not show the 100 kDa band [Fig. 1(A), lanes 2 and 6]. According to Warner et al. (1997), this band would correspond to phosphorylase b bound to the myo®brillar fraction. The pherograms of the sarcoplasmic fraction protein on 12.5% Phast Gels showed clearly two protein bands of approximately 100 and 40 kDa molecular weights in all the samples from DFD meat and N meat, their intensity being slightly lower in the samples with salt than in the controls [Fig. 2, lanes 3 and 4]. On the other hand, in PSE meat the 100 and 40 kDa bands were only found in the control samples (Fig. 2, lanes 1 and 2). These bands were likely to correspond to phosphorylase b and to aldolase respectively, and they could also be observed in the corresponding pherograms on 4±15% Phast Gels [Fig. 1(B), lanes 2,3 and 5]. During conditioning actin and myosin become progressively more soluble and, as a result, the protein extractibility rises to about 70±80% of the myo®brillar protein of muscles conditioned for about 7 days at 1± 4 C (Cheng & Parrish, 1978; Penny, 1970). In Table 2 it can be observed that myo®brillar protein extractibility at 24 h postmortem is higher than after slaughter (t=0).

M. Gil et al./Meat Science 51 (1999) 329±337

Fig. 1. SDS-PAGE of muscle protein extracts in a Phast Gel Gradient 4±15 (Pharmacia Biotech AB): (A) Myo®brillar proteins from control samples of 48 h aged meat (lane 2); 1%SDS extractable proteins from PSE (lane 3) and DFD meat (lane 4) from salted samples; myo®brils isolated from control (lane 6) and salted samples (lane 7). (B) Sarcoplasmic proteins in N or DFD meat from control (lane 2) and salted samples (lane 3); sarcoplasmic proteins in PSE meat from control (lane 5) and salted samples (lane 6). Lanes 1A and 1B: High molecular weight standards (Pharmacia Biotech AB) corresponding to 330 kDa (thyroglobulin), 220 kDa (ferritin), 67 kDa (albumin), 60 kDa (catalase), 36 kDa (lactate dehydrogenase), 18.5 kDa (ferritin) from top to bottom; lanes 5A and 4B: cross-linked phosphorylase b SDS molecular weight marker (Sigma) corresponding to 682, 584, 487, 390, 292, 195 and 97 kDa (heptamer to monomer forms of the protein) from top to bottom. Sample lanes were loaded with 4 mg of total protein. The arrows indicate the positions of the 1000, 500 and 97 kDa bands (A) and the 1000, 97 and 42 kDa bands (B) from top to bottom.

As conditioning proceeds there is an increase in the amount of actin and a-actinin extracted which contributes to the overall rise in protein extractibility. In spite of being a component of the myo®brillar structure, actin is partially soluble in solutions of low ionic strength. Our results showed that a large amount of protein migrating in the actin region (40 kDa) was

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Fig. 2. SDS-PAGE of muscle protein extracts in a Phast Gel Homogeneous 12.5 (Pharmacia Biotech AB): Sarcoplasmic proteins in PSE meat from control (lane 1) and salted samples (lane 2); sarcoplasmic proteins in N or DFD meat from control (lane 3) and salted samples (lane 4). Lane 5: Low molecular weight standards (Pharmacia Biotech AB) corresponding to 94 kDa (phosphorylase b), 67 kDa (bovine serum albumin), 43 kDa (ovalbumin), 30 kDa (carbonic anhydrase), 20.1 kDa (soybean trypsin inhibitor), 14.4 kDa (a-lactalbumin) from top to bottom. Sample lanes were loaded with 4 mg of total protein. The arrows indicate the positions of the 94 and 43 kDa bands from top to bottom.

solubilized in the sarcoplasmic fraction of all the samples [Fig. 1(B), lanes 2,3,5,6 and Fig. 2, lanes 1 to 4]. 4. Conclusions In summary, amounts of salt higher than 2% did not result in an increase in protein extractibility nor in changes in the cathepsin activity, and the addition of dextrose had no extra e€ects to the ones produced by the salt. This could suggest that most of the modi®cations produced by the curing salt are established at the early stages of salting and salt-equalization steps and maintainedÐbut probably not enhancedÐduring the remainder of the curing process. The cathepsin L enzyme system (activity + inhibition) was the most sensitive to di€erences in meat quality, salt and ageing time. DFD meat showed the lowest cathepsin B + L activity as well as the lowest inhibition by cystatins. In studies on the regulation of the NaCl content in hams by reduction or substitution of this salt, cathepsin L

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activity could be a useful parameter to indicate the extent of the changes occurring in the muscle. Irrespective of meat quality, titin and nebulin were hardly broken down during the ageing period studied, and titin was solubilized to the sarcoplasmic fraction in the salted samples from N and DFD meat. Further studies on the role of these two proteins in meat tenderization and in the ham curing process are needed. Acknowledgements This work was supported by CICYT project no. ALI94-0197. The authors thank Sr. NarcõÂs Sais for his technical assistance and Dr. Jacint Arnau for his helpful comments of the manuscript. References Anastasi, A., Brown, M. A., Kembhavi, A. A., Nicklin, M. J. H., Sayers, C. A., Sunter, D. C., & Barrett, A. J. (1983). Cystatin, a protein inhibitor of cysteine proteinases. Biochem. J., 211, 129±138. Arnau, J. (1991). Aportaciones a la calidad tecnoloÂgica del jamoÂn curado elaborado por procesos acelerados. Ph.D. thesis. Universitat AutoÁnoma, Barcelona, Spain Arnau, J., Gou, P., & Guerrero, L. (1994). The e€ects of freezing, meat pH and storage temperatures on the formation of white ®lm and tyrosine crystals in dry-cured hams. J. Sci. Food Agric., 66, 279± 282. Arnau, J., Guerrero, L., SaÂrraga, C. (in press). The e€ect of green ham pH and NaCl concentration on cathepsin activities and on the sensory characteristics of dry cured ham. J. Sci. Food Agric. Barrett, A. J. (1987). The cystatins: a new class of peptidase inhibitors. Trends Biochem. Sci., 12, 93. Bush, W. A., Stromer, M. H., Goll, D. E., & Susuki, A. (1972). Ca2+speci®c removal of Z lines from rabbit skeletal muscle. J. Cell Biol., 52, 367±381. Cheng, C.-S., Parrish Jr., F. C., 1978. Molecular changes in the saltsoluble myo®brillar proteins of bovine muscle. J. Food Sci. 43, 461 Chou, R.-G. R., Tseng, T.-F., Lin, K.-J., & Yang, J.-H. (1994). Postmortem changes in myo®brillar protein of breast and leg muscles from broilers, spent hens, and Taiwanese country chickens. J. Sci. Food Agric., 65, 297±302. Ducastaing, A., Valin, C., Schollmeyer, J., & Cross, R. (1985). E€ects of electrical stimulation on postmortem changes in the activities of two Ca-dependent neutral proteinases and their inhibitor in beef muscle. Meat Science, 15, 193±202. Etherington, D. J. (1972). The nature of the collagenolytic cathepsin of rat liver and its distribution in other rat tissues. Biochem. J., 127, 685±692. Etherington, D. J., & Wardale, J. (1982). The mononuclear cell population in rat leg muscle: its contribution to the lysosomal enzyme activities of whole muscle extracts. J. Cell Sci., 58, 139±148. Fritz, J. D., & Greaser, M. L. (1991). Changes in titin and nebulin in postmortem bovine muscle revealed by gel electrophoresis, western blotting and immuno¯uorescence microscopy. J. Food Sci., 56(3), 607±610, 615 Fritz, J. D., Mitchell, M. C., Marsh, B. B., & Greaser, M. L. (1993). Titin content of beef in relation to tenderness. Meat Science, 33, 41± 50. Gil, M., & SaÂrraga, C. (1997). Isolation and characteristics of a porcine muscle cysteine proteinase inhibitory fraction. Food Biotechnol., 11(1), 59±71.

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