Effect of low voltage electrical stimulation on protein and quality changes in bovine muscles during postmortem aging

Meat Science 94 (2013) 289–296

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Effect of low voltage electrical stimulation on protein and quality changes in bovine muscles during postmortem aging Y.H.B. Kim b,⁎, S.M. Lonergan a, J.K. Grubbs a, S.M. Cruzen a, A.N. Fritchen a, A. della Malva c, R. Marino c, E. Huff-Lonergan a a b c

Muscle Biology Group, Department of Animal Science, Iowa State University, Ames, IA 50011, United States AgResearch Ltd., Ruakura Research Centre, Private Bag 3123, Hamilton 3240, New Zealand University of Foggia, Department of Production and Innovation in Mediterranean Agriculture and Food System, Foggia, Italy

a r t i c l e

i n f o

Article history: Received 9 November 2012 Received in revised form 19 February 2013 Accepted 21 February 2013 Keywords: Electrical stimulation Beef Round muscles Tenderness Proteolysis

a b s t r a c t This experiment was conducted to determine the influence of low voltage electrical stimulation (ES) on the tenderness development of beef round muscles. Eight steers were slaughtered, and ES applied to one side of each carcass within 90 min of exsanguination. Steaks from M. longissimus dorsi, semimembranosus, adductor, and gracilis were vacuum packaged and aged at 4 °C for 9 d. Star probe, sensory evaluation, Western blot assays of troponin-T and μ-calpain autolysis and 2D-DIGE were conducted. ES resulted in accelerated (P b 0.05) pH decline of the longissimus in the first 24 h postmortem. ES did not influence (P > 0.05) proteolysis and tenderness, but did alter the predominance of metabolic proteins in the soluble fraction of muscle. Aging for 9 d improved tenderness (P b 0.05). The data confirmed that low voltage ES at 90 min of exsanguination had no effect on proteolysis and tenderness development in the longissimus dorsi, semimembranosus, adductor or gracilis in beef. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Tenderness ranks as a primary quality concern among beef consumers (Platter et al., 2003; Tatum, Gruber, & Schneider, 2007). The muscles of the round are prone to being less tender than the greater value cuts of the strip loin and ribeye, limiting the merchandizing of the round. Platter et al. (2005) suggested an increase in tenderness could translate into an increase in consumers' willingness to purchase beef. One method to minimize inconsistent meat tenderness is the use of electrical stimulation (Ferguson, Jiang, Hearnshaw, Rymill, & Thompson, 2000; Strydom, Frylinck, & Smith, 2005). Postmortem electrical stimulation of carcasses involves running an electrical current through the carcass. It was originally adopted to prevent cold shortening and toughening (Locker & Daines, 1975). Electrical stimulation speeds up the use of glycogen and accelerates pH decline by producing more lactic acid through glycolysis. While an early postmortem pH below 6.2–6.3 has been shown to alleviate the number of cold shortening incidents (Simmons et al., 2008), it has also been associated with improvements in tenderness during aging. Some studies suggested that electrical stimulation creates conditions that are ideal for activating the calpain enzymes in early postmortem muscle (Ferguson et al., 2000; Hwang, Devine, & Hopkins, 2003; Rhee & Kim, 2001; Rhee, Ryu, Imm, & Kim, 2000). Calpains

⁎ Corresponding author. Tel.: +64 7 838 5152; fax: +64 7 838 5012. E-mail address: [email protected] (Y.H.B. Kim). 0309-1740/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2013.02.013

are responsible for a majority of proteolysis and tenderization in the first 72 h postmortem (Geesink, Kuchay, Chishti, & Koohmaraie, 2006). Autolysis of μ-calpain, often associated with accelerated pH decline, is considered to be the hallmark for activation of μ-calpain in postmortem muscle. Autolysis of μ-calpain reduces the Ca2+ requirement for calpain activity (Li, Thompson, & Goll, 2004; Mellgren, 1987). Accelerated pH decline in the longissimus dorsi (LD) is associated with earlier autolysis of μ-calpain (Melody et al., 2004; Rowe, Maddock, Lonergan, & Huff-Lonergan, 2004) and subsequently more rapid tenderization. A greater proportion of μ-calpain catalytic subunit present as the 76-kDa autolysis product indicates a greater proportion of μ-calpain has been active. The opposite is true with a greater proportion of the unautolyzed 80-kDa subunit, which can be interpreted as less calpain being autolyzed and thus less calpain has been activated. Acceleration of pH decline in the LD, which would accelerate μ-calpain autolysis and activity, occurs in electrically stimulated carcasses. If ES has the same effect in the round muscles as in the LD, the rate of tenderization of round muscles could be improved. A considerable amount of variation exists in ultimate pH, the rate of tenderization, and the rate of autolysis of μ-calpain in the muscles of the round (Anderson et al., 2012). Rate of pH decline may also vary significantly among muscles of the round, since different chilling rate can occur at different locations (e.g. the interior and superficial portion) of the round muscle due to its size and thickness (Tarrant & Mothersill, 1977). Application of electrical stimulation could optimize pH decline and aid in improving the uniformity of tenderness of muscles from

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the round. However, it is not known how or if electrical stimulation early postmortem influences the tenderness and proteolytic activity of the round muscles (particularly semimembranosus (SM), adductor (AD) and gracillis (GR) muscles). The primary objective of this study was to determine the effects of electrical stimulation on postmortem protein changes and tenderness of beef.

2. Materials and methods 2.1. Project description Eight market weight beef steers were harvested at the Iowa State University Meat Laboratory. The carcasses were split and one half of each carcass electrically stimulated (ES; 100 V, 60 Hz for 30 s; Model BV-80 Low Voltage Beef Stimulator, Jarvis Products Corporation, Middletown, CT) within 90 min (average 81 min) of exsanguination while the other half remained as a non-stimulated control (NON-ES). Thus, each carcass served as its own control. At 24 h postmortem, four muscles (LD, SM, AD and GR) from each side of the carcass were removed. Muscles were cut into 2.54 cm and 0.64 cm thick steaks. The 2.54 cm steaks were used for sensory and instrumental tenderness and the 0.64 cm steaks were used for biochemical analysis. Steaks were cut perpendicular to the long axis of the muscle and individually vacuum packaged. Within the SM, the steaks were separated to deep portion (DSM; the medial inner 1/3 closest to the femur) and superficial portion (SSM; the lateral outer 1/3 closest to the surface of the carcass) (Kim, Lonergan, & Huff-Lonergan, 2010), and were analyzed as a separate muscle for each trait. Steaks were aged at 4 °C for either 24 h or 9 d. Because the LD is commonly used as a whole muscle retail cut (steaks) and because it is known to respond to electrical stimulation (Savell, McKeith, & Smith, 1981) it was used as the reference sample and served as an internal control for other muscles and the electrical stimulation for each side of each carcass. Sensory and instrumental tenderness, pH, and protein degradation were measured on steaks that were aged 24 h and 9 d postmortem.

2.2. Temperature and pH Postmortem temperature decline was monitored for 24 h using temperature loggers (Barnant; Model 600-1040, Barrington, IL, USA) inserted into the round muscles — DSM and SSM, and LD. Postmortem pH was recorded at 1.5, 3.5, 5.5, 8.5, 24 h and 9 d postmortem in the SSM and LD using a Hanna 9025 pH/ORP pH meter with a FC 200 (Kynar® body) probe (Hanna Instruments, Woonsocket, RI) calibrated to pH range 4–7 between each time point at carcass temperature.

2.3. Sensory and instrumental tenderness Steaks aged 1 d and 9 d were cooked on clamshell grills to an internal temperature of 71 °C. Star probe force (Anderson et al., 2012a) of cooked steaks was measured. A trained sensory panel (n = 6) evaluated sensory characteristics of steaks (Lonergan et al., 2007). Sensory traits including tenderness, chewiness and juiciness were evaluated using a 15-cm line scale (0 = not tender, not chewy, not juicy, low beef flavor, no off-flavor; 15 = very tender, very chewy, very juicy, high beef flavor, high off-flavor). Sensory data were recorded using a computerized sensory software system (Compusense five 4.6, Compusense, Inc., Guelph, Ontario, Canada). This panel is a standing sensory panel which routinely carries out sensory evaluates of fresh beef. Panelists had three 30 min orientation sessions that included the diversity of cuts in this experiment. During these training sessions, panelists were familiarized with the computer software scoring system. During each session, four panelists evaluated each steak.

2.4. SDS-PAGE and Western blotting The 0.64 cm thick steaks were used immediately for biochemical analysis. Steaks were minced and frozen in liquid nitrogen and powdered using a commercial blender. Whole muscle protein samples were prepared from each of the powdered samples and stored at − 80 °C (Melody et al., 2004). Samples from each steak were analyzed for titin degradation using a 5% polyacrylamide continuous gel (acrylamide:N, N′-bis-methylene acrylamide = 100:1 [wt./wt.],0.1% [wt./vol.] SDS, 0.05% [vol./vol.] TEMED, 0.05% [wt./vol.] APS, and 0.5 M Tris- HCl, pH 8.8). Gels (14 cm wide by 15 cm tall) were loaded with 160 μg protein and run for 48 h in Hoefer SE 400 electrophoresis units at 5 mA before staining with colloidal Coomassie dye (34% methanol, 17% ammonium sulfate (wt./vol.), 3% phosphoric acid, 0.1% Coomassie G-250 (wt./vol.)). Immunoblotting assay of troponin-T degradation and μ-calpain autolysis was examined on the samples from each steak taken at 1 and 9 d postmortem as described by Melody et al. (2004) with modifications. An 8% polyacrylamide separating gel was used for determination of μ-calpain autolysis. All gels (10 cm wide × 8 cm tall) were run in SE 260 Hoefer Mighty Small II electrophoresis units. Gels for troponin-T analysis were loaded with 40 μg of protein and run at 20 V overnight. Gels for μ-calpain were loaded with 80 μg of protein and run at 120 V. Gels were transferred to polyvinylidene difluoride (PVDF) membrane as described by Melody et al. (2004). Western blotting was performed as described by Huff-Lonergan et al. (1996). Membrane blocking was performed using 5% non-fat dry milk in a PBS solution containing 0.1% Tween-20 for 1 h at room temperature. Primary antibodies, dilutions, and incubations were anti troponin-T (JLT-12; Sigma, St Louis, MO) diluted 1:40,000, incubated overnight at 4 °C and anti μ-calpain (MA3-940, Thermo Scientific, Rockford, IL) diluted 1:5000, and incubated overnight at 4 °C. Membranes were incubated in secondary antibody for 1 h at room temperature (Goat anti-mouse-HRP, No 2554, Sigma, diluted 1:30,000 and 1:20,000 for troponin-T and μ-calpain respectively). Blots were developed using ECL Plus Western Blotting Detection System (GE Healthcare, Piscataway, NJ). Images were captured using a ChemiImager 5500 (Alpha Innotech, San Leandro, CA) and Alpha Ease FC software (v 3.03 Alpha Innotech). Bands were quantified using densitometry; titin T1 and T2 (T2 is the degradation product of titin), as well as the 30 kDa troponin-T degradation product were analyzed and reported as a percentage of a reference sample (titin: 0 d postmortem LD; troponin-T: 9 d postmortem LD; μ-calpain: 1 d postmortem LD, respectively) run on each gel. The intact catalytic subunit of μ-calpain (80 kDa) as well as the autolysis products of that subunit (78 and 76 kDa) were each reported as a percentage of the total of all three bands for each sample. 2.5. 2D-difference in gel electrophoresis 2.5.1. Protein extraction Samples from the LD aged 1 or 9 d postmortem were frozen and pulverized in liquid nitrogen. Pulverized samples (1.5 g) were homogenized with 4.5 ml of cold sarcoplasmic extraction buffer (50 mM Tris, 1 mM EDTA; pH 8.5). The homogenate was separated into sarcoplasmic and myofibrillar fractions by centrifugation (40,000 ×g for 20 min at 4 °C). After centrifugation the supernatant was filtered through cheesecloth and recovered as sarcoplasmic protein fraction. The remaining pellet was washed 3 times with standard salt solution (100 mM KCl, 20 mM potassium phosphate (K2HPO4 and KH2PO4), 2 mM MgCl2, 2 mM EGTA, 1 mM NaN3) and 3 times with 5 mM Tris–HCl pH 8.0. After washing, 1 ml of myofibrillar extraction buffer (8.3 M Urea, 2 M Thiourea, 2% Chaps; 1% DTT; pH 8.5) was added per 100 mg of pellet and rocked at 4 °C for 30 min, and samples were then centrifuged (10,000 ×g for 30 min at 4 °C) to extract the myofibrillar proteins. The resulting supernatant was kept as the myofibrillar portion of the muscle. The protein concentration of sarcoplasmic fraction was determined by the Lowry protein assay (Lowry, Rosebrough, Farr, & Randall, 1951) using bovine

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2.5.2. Two-dimensional difference in-gel electrophoresis Fluorescent two-dimensional difference in-gel electrophoresis (2D-DIGE) was performed on protein extracts from the LD aged 1 or 9 d postmortem and both as soluble and insoluble fractions as described by Anderson, Lonergan, and Huff-Lonergan (2012). Protein extracts (50 μg of each) were labeled with 400 pmol of CyDye Fluor Cy3 or Cy5 (GE Healthcare, Piscataway, NJ) (Table 1). All 2D-DIGE gels included the reference, created by mixing equal amounts of samples from each experimental condition (n = 16) allowing both ES and NON-ES samples in each gel. This pooled protein was labeled with Cy2. Labeling was performed by incubating each sample with its respective CyDye for 30 min on ice in the dark. Labeling reactions were stopped with the addition of 1 μL of 10 mM lysine and incubating for 10 min on ice in the dark. Samples were passively rehydrated into immobilized pH gradient (IPG) strips overnight in the dark at room temperature; two pH ranges were used (pH 3–10, 11 cm and pH 4–7, 13 cm; GE Healthcare). Isoelectric focusing was performed with an isoelectrofocusing system (Ettan IPGphor, GE Healthcare) with a maximum current setting of 50 mA/strip for a total of 11,501Vh for the 11 cm gels and 75 mA/ strip for 18,500 Vh for the 13 cm gels. During the run the temperature was maintained at 20 °C. Prior to the second dimension, IPG strips were equilibrated at room temperature with gentle rocking for 15 min in SDS Equilibration/ Reduction buffer (50 mM Tris, pH 8.8, 6 M urea, 30% v/v glycerol, 2% wt./vol. SDS, bromophenol blue) supplemented with 1% DTT, followed by the same buffer containing 2.5% iodoacetamide instead of DTT for an additional 15 min at room temperature. The IPG strips were placed on top of 12.5% acrylamide separating gels (100:1 ratio of acrylamide to bis-acrylamide) and sealed with 0.5% (wt./vol.) agarose containing bromophenol blue (BioRad, Hercules, CA). The second dimension separation was performed on a Hoefer SE 600 Ruby vertical system (Amersham Biosciences) at a constant voltage of 60 V/gel with SDS running buffer (250 mM Tris, 1.92 M Glycine, 1% SDS, 2 mM EDTA). All electrophoresis procedures were performed in the dark.

(which includes automatic background correction, spot volume normalization, and volume ratio calculation), the images from separate gels were loaded and compared using the BVA module of the DeCyder software (GE Healthcare, Piscataway, NJ) to match gels using the Cy2 pooled standard on each gel to normalize the images. The BVA matches the different gel images across groups (ES vs. NON-ES) and provides statistical data on differential protein abundance levels between groups. The statistical significance of each spot abundance difference was calculated using Student's t-test.

2.5.4. Protein identification Preparative gels were loaded with unlabeled sample (300 μg of proteins per strip). Electrophoretic conditions were as for 2-D DIGE. Proteins were visualized by colloidal Coomassie staining to pick spots of interest. Excised gel spots were destained and proteins were digested with trypsin and identified with Electrospray Mass Spectrometry (ESI/MS) (Stewart, 1999). Identification was conducted using a mass spectrometer Q-Star XL quadrupole-TOF (Applied Biosystems) equipped with ESI (previously described by Anderson et al., 2012b). For confirmation of identifications, spectra were also searched against the database using MASCOT (http://www.matrixscience.com/).

A

6.8 ES NON-ES

6.6 6.4 6.2

pH

serum albumin (BSA) as a standard, while myofibrillar protein concentration was measured with 2-D Quant Kit (GE Healthcare, Piscataway, NJ). Sarcoplasmic and myofibrillar extracts were visualized on 15% SDS-PAGE gels to confirm concentrations.

6.0 5.8 5.6 5.4 1.5

Table 1 Experimental design of the differential expression of the skeletal muscle proteome as affected by two different treatments (ES = electrical stimulated vs NON-ES = not electrical stimulated). Gel 1 2 3 4

Cy3 (400 pmol) ES animal 1 + 2 NON-ES animal 3 + 4 ES animal 5 + 6 NON-ES animal 7 + 8

Cy5 (400 pmol) NON-ES animal 1 + 2 ES animal 3 + 4 NON-ES animal 5 + 6 ES animal 7 + 8

Reference Reference Reference Reference

(n (n (n (n

= = = =

B

16) 16) 16) 16)

The same labeling was used for the myofibrillar and sarcoplasmic fractions at 1 and 9 days postmortem. The reference sample includes a pool of all samples in each experiment.

5.5

8.5

24.0

6.6 ES NON-ES

6.4

6.2

6.0

5.8

5.6

5.4 1.5

Cy2 (400 pmol)

3.5

Time postmortem (hours)

pH

2.5.3. Image acquisition and data analysis Immediately following electrophoresis, fluorescent images were scanned directly using the Ettan DIGE Imager (GE Healthcare). The excitation/emission wavelengths for Cy3, Cy5 and Cy2 were 540 nm/ 595 nm, 635/680 nm and 480 nm/530 nm respectively. The images were processed using the DeCyder 2D software 7.0 (GE Healthcare, Piscataway, NJ) and used for simultaneous comparison of abundance changes across all 8 animals and ES/NON-ES treatment. Each gel image was analyzed using the DeCyder Differential in-gel Analysis (DIA software, GE Healthcare, Piscataway, NJ) with the optimal settings for spot detection and exclusion (estimated spot number set to 1500 spots). All gels were processed and analyzed under the same parameters and automatically matched. After spot detection

291

3.5

5.5

8.5

24.0

Time postmortem (hours) Fig. 1. A: Postmortem pH decline of M. longissimus dorsi of electrically stimulated (ES) or non-stimulated (NON-ES) beef carcass sides. Values at each time postmortem with different letters (a–b) are different (P b 0.05). B: Postmortem pH decline of superficial M. semimembranosus muscle of electrically stimulated (ES) or non-stimulated (NON-ES) beef carcass sides.

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2.6. Data analysis The experimental data were analyzed as a randomized block design with carcass serving as a random effect. Type-3 tests of fixed effects for ES treatment, aging time, muscle and their interaction, and random effects for animal and animal by ES treatment were analyzed by using the Mixed Model procedure of SAS for ANOVA (SAS, 2007). Least squares means for all traits of interest were separated (F test, P b 0.05) by using least significant differences generated by the PDIFF option. 3. Results and discussion 3.1. pH and temperature decline The application of ES to beef carcasses resulted in a more rapid pH decline of the LD during the first 24 h of postmortem chilling compared to LD of the NON-ES beef carcass side (P b 0.05; Fig. 1A). A difference in pH between the ES LD and NON-ES LD was found at 3.5 h postmortem (P b 0.05). At 5.5 h postmortem, the pH of ES LD was below 6, whereas the NON-ES LD had a pH value above 6. However, the ultimate pH measurement at 24 h postmortem showed no difference between the ES and NON-ES treatment (P > 0.05). In contrast to LD, the ES treatment did not influence the pH decline rate of the SSM muscle compared to the NON-ES SSM, which suggests that different muscles and perhaps fiber types react differently to the ES treatment in the rate of postmortem

Temperature (°C)

3.2. μ-Calpain autolysis and protein degradation Immunoblotting for μ-calpain autolysis indicated an increase (P b 0.05) in the presence of the 76 kDa autolysis product in ES LD (Fig. 3) and no differences in appearance of the 76 kDa autolysis product in the AD and GR (Table 2). Comparison of ES and NON-ES DSM showed a decrease (P b 0.05) in the un-autolyzed 80 kDa form of μ-calpain in ES DSM, indicating an increase in activated μ-calpain in the ES DSM. In the ES SSM, more μ-calpain was found to have undergone

A 40 ES NON-ES

30

glycolysis (Fig. 1B). At 3.5 h postmortem, the SSM already reached a pH value below 6 regardless of the treatment. The more rapid pH decline rate of SM compared to LD could be attributed to the slower temperature decline rate of SM than that of LD as shown in Fig. 2. At 3.5 h postmortem, SSM was around 35 °C (Fig. 2B), whereas LD was just above 25 °C (Fig. 2A). Thus there was an almost 10 °C difference regardless of the ES treatment (P b 0.05). Accelerated glycolysis and subsequent lactic acid production of muscle at high pre rigor temperatures (>35 °C) have been reported by several studies (Hertzman, Olsson, & Tornberg, 1993; Kim, Stuart, Nygaard, & Rosenvold, 2012; Rosenvold et al., 2008). There was a significant difference in the postmortem temperature decline rate between the DSM and SSM muscles during the first 24 h postmortem (Fig. 2B). The DSM had a slower temperature decline than the SSM throughout this period regardless of the ES treatment (P b 0.05). At 3.5 h postmortem, the temperature of DSM was still above 40 °C. The slower chilling rate of the DSM is due to the size and thickness of the whole SM, making the cooling of the inner portion of the muscle less efficient. The slower chilling rate of DSM has been reported in several studies, and can ultimately result in less metmyoglobin reducing ability, water-holding capacity, color stability, μ-calpain autolysis and protein degradation. This is due to protein denaturing caused by a combined effect of high temperature and rapid pH decline (Kim et al., 2010; Kim et al., 2012; Sammel et al., 2002; Seyfert et al., 2004; Tarrant, 1977).

20

10

0

1.5 3.5 5.5

8.5

24.0

Time postmortem (hours)

B 45 ES DSM ES SSM NON-ES DSM NON-ES SSM

Temperature (°C)

40 35 30 25 20 15 10 5 1.5

3.5

5.5

8.5

24.0

Time postmortem (hours) Fig. 2. A: Postmortem temperature decline of M. longissimus dorsi of electrically stimulated (ES) or non-stimulated (NON-ES) beef carcass sides. B: Postmortem temperature decline of deep M. semimembranosus (DSM) and superficial M. semimembranosus (SSM) muscle of electrically stimulated (ES) or non-stimulated (NON-ES) beef carcass. Values at each time postmortem with different letters (a–b) are different (P b 0.05).

Fig. 3. Representative Western blots and gels for (A) μ-calpain autolysis, (B) troponin -T degradation, and (C) titin degradation (T1 and T2). In all blots and gels, samples are from the longissimus dorsi. Samples include E1: electrically stimulated day 1, N1: nonstimulated day 1, E9: electrically stimulated day 9, and N9: non-stimulated day 9.

Y.H.B. Kim et al. / Meat Science 94 (2013) 289–296 Table 2 Least squares means for μ-calpain autolysis of beef steaks of electrically stimulated (ES) or non-stimulated (NON-ES) beef carcass sides aged for 1 d at 1 °C. The intact catalytic subunit of μ-calpain (80 kDa) as well as the autolysis products of that subunit (78 and 76 kDa) were each reported as a percentage of the total of all three bands for each lane. Muscle

μ-Calpain subunit

ES

Non-ES

PES-value

Longissimus

80 78 76 80 78 76 80 78 76 80 78 76 80 78 76

24 42 34 19 52 29 41 35 24 21 51 28 10 35 55 3

36 40 24 33 48 19 37 35 28 31 46 23 17 38 45 3

0.01 NS 0.04 NS NS NS NS NS NS 0.04 NS NS NS NS 0.03

Adductor

Gracilis

Deep semimembranosus

Superficial semimembranosus

SEMa a

Standard errors of means.

autolysis than in the NON-ES SSM (P b 0.05). When directly comparing the DSM and SSM, the DSM had more un-autolyzed μ-calpain then the SSM (P b 0.05) regardless of treatment. Kim et al. (2010) showed similar results to the current study with DSM containing more un-autolyzed μ-calpain than the SSM. They concluded that the protein denaturing condition of DSM might result in less μ-calpain autolysis and subsequently less protein degradation compared to SSM. In general, low voltage ES did not influence titin (T) degradation from T1 to T2 (Fig. 3 and Table 3). However, there was a tendency for T1 to be greater in ES muscles at day 1 (P = 0.09); while opposite results might have been expected, it is possible that protein solubility was decreased in the ES muscles as a result of faster pH decline combined with high temperatures in some muscles. This would potentially cause the rate of protein degradation to be slower. In general, aging treatment decreased the amount of T1 present, while increasing the amount of T2 (P b 0.001). While the LD had the least T2 at day 1 (P b 0.05), by day 9 the LD had experienced the greatest titin degradation, along with the SSM and gracilis, as evidenced by the least presence of T1 (P b 0.05). Low voltage ES treatment did not influence troponin-T degradation (P > 0.05; Table 4 and Fig. 3). In general, postmortem aging increased troponin-T degradation based on the ratio of bands corresponding to that product to a reference sample (day 1 ratio = 0.64; day 9 ratio = 1.78; standard error of difference = 0.48; P b 0.05). This response is consistent with previous reports (Koohmaraie, 1996; Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995). When comparing DSM

293

and SSM, the DSM had more troponin-T degradation product than SSM at 1 d postmortem (P b 0.05), which is contradictory to the μ-calpain autolysis outcomes discussed above. However, no significant difference was observed in the NON-ES counterparts although a numerical difference was observed. At 9 d postmortem both the ES and NON-ES DSM had more (P b 0.05) troponin-T degradation product present than the SSM. This could be due to the continual degradation of troponin-T as the aging process continues. As troponin-T is further degraded, immunoblotting may no longer be able to identify the degradation products. 3.3. Instrumental and sensory tenderness A significant muscle by aging interaction, but no ES treatment effect (P > 0.05) was detected for star probe values (Table 5). In meat aged 9 d, the star probe values of LD, AD, and DSM steaks were decreased compared to meat aged 1 d (P b 0.05) but no aging effect was observed for star probe values of the GR and SSM steaks. The DSM steaks had greater star probe values than the SSM steaks at 1 d regardless of the ES treatment (P b 0.05), which indicates the existence of a location difference in instrumental tenderness values within the same SM muscle, confirming the results seen with μ-calpain autolysis and contradictory to the troponin-T degradation. The DSM had the greatest star probe values among all muscles at 1 d postmortem (P b 0.05). This could be attributed to myosin denaturation and poorer water-holding capacity of DSM due to the high temperature in the early postmortem environment (Bekhit, Farouk, Cassidy, & Gilbert, 2007; den Hertog-Meischke, Smulders, Van Logtestijn, & van Knapen, 1997; Kim et al., 2012). Sensory analysis revealed no beneficial ES impact on tenderness of beef muscles throughout the whole aging period (Table 6). In fact, the non-stimulated LD and AD showed greater tenderness scores than their counterpart at 1 d postmortem. By 9 d of aging, no difference existed between the ES LD and the NON-ES LD (P > 0.05), while aging had a significant effect on tenderness regardless of treatment. No ES treatment effects were observed for juiciness and chewiness either. These results agree with the findings from other studies (Hildrum, Solvang, Nilsen, Frøystein, & Berg, 1999; Rødbotten, Lea, & Hildrum, 2001; Unruh, Kastner, Kropf, Dikeman, & Hunt, 1986). Rødbotten et al. (2001) reported that low-voltage ES had no significant effect on tenderness of beef LD, while aging significantly influenced meat tenderness development. Furthermore, Unruh et al. (1986) found adverse effects of low-voltage ES on beef tenderness and juiciness assessed at 6 d postmortem. They postulated that moderate chilling conditions coupled with low-voltage ES would not result in muscle tissue rupture and coldshortening would not be the issue at the moderate chilling temperature. However, too rapid acidification while temperature of prerigor muscle is still hot could result in meat quality deterioration such as increased purge loss, paler color and myofibrillar lattice shrinkage by the denaturation of structural and sarcoplasmic meat proteins (Bekhit et al., 2007;

Table 3 Least squares means for titin (T1) and its degradation product (T2) in beef steaks of electrically stimulated (ES) or non-stimulated (NON-ES) beef carcasses aged for 1 or 9 d at 1 °C. Means are presented as a ratio of intensity to a reference sample run on each gel. Muscle

T1/T2

Longissimus

T1 T2 T1 T2 T1 T2 T1 T2 T1 T2

Adductor Gracilis Deep semimembranosus Superficial semimembranosus a

Standard errors of means.

Day 1 ES

Non-ES

1.22 0.58 0.89 0.98 1.09 0.85 0.93 0.92 1.05 0.96

1.03 0.64 0.85 0.93 0.99 0.88 0.86 0.84 0.91 0.88

SEMa

PES-value

0.15 0.12 0.15 0.12 0.15 0.12 0.15 0.12 0.14 0.12

NS NS NS NS NS NS NS NS NS NS

Day 9 ES

Non-ES

0.09 1.10 0.68 0.93 0.23 1.30 0.38 0.91 0.26 1.03

0.15 1.18 0.57 1.08 0.30 1.23 0.57 1.00 0.25 1.07

SEM

PES-value

0.12 0.08 0.12 0.08 0.12 0.08 0.12 0.08 0.12 0.08

NS NS NS 0.06 NS NS 0.10 NS NS NS

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Table 4 Least squares means for troponin-T degradation product (30 kDa) in beef steaks from electrically stimulated (ES) or non-stimulated (NON-ES) beef carcass sides aged for 9 d at 1 °C. Means are presented as a ratio of intensity to a reference sample run on each gel. Muscle

Longissimus Adductor Gracilis Deep semimembranosus Superficial semimembranosus SEMa a

ES

Non-ES

PES-value

Day 1

Day 9

Day 1

Day 9

0.15 0.30 0.07 3.33 0.30 1.52

0.79 1.35 3.47 3.51 0.78 1.52

0.05 0.32 0.11 1.45 0.37 1.52

0.78 1.04 2.77 2.46 0.89 1.52

NS NS NS NS NS

Standard errors of difference between means.

den Hertog-Meischke et al., 1997; Simmons et al., 2008; Unruh et al., 1986). 3.4. Two dimensional difference in-gel electrophoresis 3.4.1. Soluble fraction Twenty-eight protein spots were found to change significantly in abundance after ES treatment at 1 d and 9 d postmortem. A total of 19 (12 spots pH range 3–10; 7 spots pH range 4–7) spots were successfully identified and these are listed in Table 7. Nine spots were not extracted from the preparative gels due to low abundance. Relative abundance of the identified proteins is shown in the last column of the table. In samples aged 1 d, 5 spots showed less intensity in the ES group than control (pH range 3–10). Two spots were identified as glyceraldehyde3-phosphate dehydrogenase (P b 0.05) and the other three as fructosebisphosphate aldolase A (P b 0.05), phosphoglycerate kinase 1 (P b 0.05) and pyruvate kinase isozyme M1 (P b 0.05). All these spots are classified as glycolytic enzymes (Bjarnadottir, Hollung, Faergestad, & Veiseth-Kent, 2010; Laville et al., 2009) and are involved in the glycolytic pathway. These changes were caused by the action of the ES that results in an acceleration of post-mortem glycolysis. In particular, after 9 d postmortem, 6 of the 7 spots that showed lesser intensity in the ES group (to P b 0.05 from P b 0.001) were identified as glyceraldehyde-3phosphate dehydrogenase (GAPDH); this result may reflect different isoforms of this protein or that the protein is fragmented or otherwise modified due to treatment. This result is consistent with the GAPDH results reported by Polati et al. (2012). In most cases several protein spots present at different positions on the gel were identified as the same protein; these protein spots have the same molecular weight but different isoelectric points. In samples aged 9 d, creatine kinase M-type was more abundant (P b 0.05) in the soluble fraction from the ES samples. Previous studies on proteomics showed that creatine kinase increases during the first 48 h postmortem in bovine LD (Bjarnadottir et al., 2010) and during postmortem storage of pork (Lametsch, Roepstorff, & Bendixen, 2002). Creatine kinase M-type was less abundant in the insoluble fraction of beef LD muscle in response to ES (Bjarnadottir et al., 2010). Finally, several forms of creatine kinase M-type were shown to change in

abundance during aging of beef LD (Polati et al., 2012). Taken together with the current results, there is a strong suggestion that ES can change the relative solubility of this enzyme. Even in the absence of ES, creatine kinase abundance has been linked to variation in beef tenderness (D'Alessandro et al., 2012). Creatine kinase is an enzyme essential for the control of intracellular ATP levels. Okumura et al. (2005) found that the appearance of creatine kinase was strongly expressed in different glycolytic muscles. The amount of creatine kinase in m. longissimus lumborum has been reported to have a positive correlation with pH (Kwasiborski et al., 2008). The current study also observed a more rapid pH decline in the LD of an ES group compared with a NON-ES group, confirming the relationship between creatine kinase and pH decline. Using the pH range 4–7, 7 protein spots changed in intensity between ES and NON-ES groups (Table 7). Soluble extracts from samples aged 1 d had 5 spots that responded to ES. Three spots of glycerol-3 phosphate dehydrogenase (P b 0.05) showed lesser intensity and one of pyruvate dehydrogenase (P b 0.05) and Annexin A5 (P b 0.05), respectively, showed greater intensity in the ES group compared with the control. Annexin A5 is a protein included in the annexin group. The annexins are a family of Ca2+ dependent protein that binds to membrane phospholipids (Laville et al., 2009). This protein is associated with sarcoplasmic reticulum membranes in skeletal muscle and may play a regulatory role in the Ca 2+release/uptake cycle. The presence of the greatest level of this protein in the ES group could be associated with the calpain system and activation of calcium-dependent proteolysis. Indeed, Hwang et al. (2003) reported electrical stimulation early postmortem may be ideal for activating the meat endogenous enzymes that are involved in the tenderness improvement during aging. After 9 d postmortem only two spots showed changes in intensity — GAPDH was less intense in ES compared to the NON-ES counterpart (P b 0.05).

3.4.2. Insoluble fraction Nine protein spots were found to change significantly in abundance after ES treatment at the two different aging times. Six protein spots identified (3 spots pH range 3–10; 3 spots pH range 4–7) in the myofibrillar protein fraction are listed in Table 8. Three spots were not extracted from the preparative gels due to low abundance. Relative abundance of the identified proteins is shown in the last column of the table.

Table 6 Least squares means for sensory traits of beef steaks of electrically stimulated (ES) or non-stimulated (NON-ES) beef carcass sides aged for 1 and 9 d at 1 °C. Sensory traits including tenderness, chewiness, and juiciness were evaluated using a 15-cm line scale (1 = not tender, chewy and juicy; 15 = very tender, chewy and juicy). Muscle

Trait

Longissimus

Tenderness Chewiness Juiciness Tenderness Chewiness Juiciness Tenderness Chewiness Juiciness Tenderness Chewiness Juiciness Tenderness Chewiness Juiciness

Adductor Table 5 Least squares means for star probe values (kg) of beef steaks of electrically stimulated (ES) or non-stimulated (NON-ES) beef carcass sides aged for 9 d at 1 °C. Muscle

Longissimus Adductor Gracilis Deep semimembranosus Superficial semimembranosus a

ES

Non-ES

SEM

Day 1

Day 9

Day 1

Day 9

6.2 5.7 5.3 7.9 6.0

4.9 5.3 5.5 6.6 5.8

6.0 6.1 5.2 7.4 5.9

4. 8 5.0 4.8 5.8 6.0

Standard errors of difference between means.

a

Gracilis

PES-value Deep semimembranosus

0.48 0.48 0.48 0.48 0.48

NS NS NS NS NS

Superficial semimembranosus

ES

SEMA

Non-ES

Day 1

Day 9

Day 1

Day 9

6.1a 7.6a 10.2a 7.0a 6.4a 7.1a 7.5ab 7.9a 8.7a 4.4a 8.7a 9.1 5.1ab 8.3a 10.1

9.3b 4.6ab 11.5b 7.7a 6.0ab 7.5a 7.8ab 7.2ab 9.3ab 7.4b 6.6b 8.4 6.4a 7.3a 9.6

7.6c 6.0c 10.4ab 8.3a 6.0ab 7.2a 7.1a 7.8a 8.3a 4.9a 8.8a 9.1 4.4b 9.8b 9.6

9.3b 4.9cb 10.1a 9.2b 4.8b 8.6b 8.7b 6.3b 9.8b 7.9b 5.6b 9.6 6.3a 7.9a 9.5

Means in a row with different letters (a–b) are different (P b 0.05). a Standard errors of means.

0.489 0.478 0.405 0.489 0.478 0.405 0.489 0.478 0.405 0.505 0.493 0.419 0.511 0.498 0.423

Y.H.B. Kim et al. / Meat Science 94 (2013) 289–296

295

Table 7 Identified proteins of the soluble fraction from M. longissimus dorsi that changed in abundance between ES and NON-ES groups. Day

Sequence coverage

Score

Theoretical pI/Mr (Da)

Fold changeb

P- value

sp|P00883| (Rabbit) sp|P10096| (Bovine) sp|P10096| (Bovine) sp|Q3T0P6| (Bovine) sp|P00548| (Chicken) sp|P10096| (Bovine) sp|P10096| (Bovine) sp|P10096| (Bovine) sp|P10096| (Bovine) sp|P10096| (Bovine) sp|P10096| (Bovine) sp|Q9XSC6| (Bovine)

17% 17% 8% 20% 16% 17% 15% 15% 17% 17% 17% 21%

398 320 108 412 372 274 285 302 319 310 310 460

8.31/39774 8.50/36073 8.50/36073 8.48/44908 7.24/58522 8.50/36073 8.50/36073 8.50/36073 8.50/36073 8.50/36073 8.50/36073 6.63/43190

+1.12 +1.13 +1.11 +1.08 +1.10 +1.20 +1.20 +1.18 +1.19 +1.17 +1.18 −1.08

P P P P P P P P P P P P

b b b b b b b b b b b b

0.05 0.05 0.05 0.05 0.05 0.01 0.001 0.05 0.001 0.05 0.01 0.05

sp|Q5EA88| (Bovine) sp|Q5EA88| (Bovine) sp|Q5EA88| (Bovine) sp|P11966| (Bovine) sp|P81287| (Bovine) sp|P10096| (Bovine) sp|P10096| (Bovine)

22% 24% 20% 16% 28% 17% 17%

305 423 333 253 432 289 313

6.42/38194 6.42/38194 6.42/38194 6.21/39443 4.86/36124 8.50/36073 8.50/36073

+1.20 +1.18 +1.16 −1.19 −1.44 +1.30 +1.23

P P P P P P P

b b b b b b b

0.05 0.05 0.05 0.05 0.05 0.05 0.01

Identified proteins

SWISS-PROT

pH 3–10 1 370 1 426 1 361 1 344 1 248 9 358 9 351 9 350 9 347 9 345 9 343 9 294

Fructose-bisphosphate aldolase A Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Phosphoglycerate kinase 1 Pyruvate kinase isozyme M1 Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Creatine kinase M-type

pH 4–7 1 480 1 475 1 478 1 461 1 473 9 448 9 449

Glycerol-3 phosphate dehydrogenase Glycerol-3 phosphate dehydrogenase Glycerol-3 phosphate dehydrogenase Pyruvate dehydrogenase Annexin Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase

a b

Spots

a

Primary accession number in the SWISS-PROT database. + is more abundant in NON-ES than in ES and − less abundant in NON-ES than in ES.

Significantly greater intensity of GAPDH and fructose-bisphosphate aldolase A protein spots were found in ES group at pH 3–10 after 9 d postmortem. Changes in abundance of GAPDH in both the soluble and insoluble fractions in this experiment suggest that ES has an impact on protein solubility. Previous studies have also identified GAPDH after postmortem storage in the myofibrillar fraction (Bjarnadottir et al., 2010; Boles, Parrish, Huiatt, & Robson, 1992; Laville et al., 2009) suggesting that this enzyme changes in solubility during postmortem storage. Bjarnadottir et al. (2010) hypothesized the change in solubility could be due to precipitation or aggregation. Boles et al. (1992) also suggested that high temperature with a drop in pH caused a denaturation of proteins that aggregated and precipitated onto myofibrils. On the other hand, it has been hypothesized that the modification of the solubility of some enzymes may be related to their susceptibility to oxidation (Laville et al., 2009). The myofibrillar fraction analyzed with the pH 4–7 range showed that after 1 d post-mortem, 3 spots with significant differences between the two treatments: myosin regulatory light chain 2, tropomyosin b chain and tropomyosin a chain. All these spots had greater abundance in the ES group. Myosin regulatory light chain 2 was reportedly greater in abundance in more tender top loin steaks at 1 h postmortem (Bjarnadóttir et al., 2012) suggesting that ES might influence myosin regulatory light chain 2 early postmortem that may be linked to tenderness.

No spots showed significant differences after 9 d postmortem in the myofibrillar fraction separated with the pH 4–7 range between the ES and NON-ES groups. In the present study, only 3 spots of the structural proteins changed significantly between ES and NON-ES groups, suggesting that maybe the low ES applied at the carcasses did not affect the breakdown of the myofibril but did accelerate postmortem glycolysis. 4. Conclusion The application of low voltage ES to beef carcasses at 90 min of exsanguination did not affect instrumental tenderness values and proteolysis (troponin-T degradation) of beef muscles (P > 0.05). The ES treatment resulted in a more rapid pH decline of LD during the first 24 h postmortem compared to NON-ES LD (P b 0.05). However, the extent of the pH difference (about 0.2 at 3.5 h and 0.3 at 5.5 h postmortem) between ES- and NON-ES-treated LD may not be sufficient to induce the metabolic change needed to differentiate tenderness and proteolysis of LD. Further, the low voltage ES treatment did not affect the pH decline rate of the SSM (P > 0.05). Thus, it is suggested that different muscles and different muscle fiber types react differently to the low voltage ES application in their rate of postmortem glycolysis. The present data support the conclusion that low voltage ES had no effect on proteolysis and instrumental

Table 8 Identified proteins of the insoluble fraction from M. longissimus dorsi that changed in abundance between ES and NON-ES groups. Identified proteins

SWISS-PROTa

Sequence coverage

Score

Theoretical pI/Mr (Da)

Fold changeb

P- value

pH 3–10 1 348 9 259 9 260 9 263 9 282

Myosin regulatory light chain 2 Glyceraldehyde-3-phosphate dehydrogenase Fructose-bisphosphate aldolase A Fructose-bisphosphate aldolase A Glyceraldehyde-3-phosphate dehydrogenase

sp|Q3SZE5| (Bovine) sp|P10096| (Bovine) sp|P00883| (Rabbit) sp|P00883| (Rabbit) sp|P10096| (Bovine)

31% 6% 16% 7% 22%

223 48 232 149 379

4.86/18968 8.50/36073 8.31/39774 8.31/39774 8.50/36073

+2.10 −1.16 −1.14 −1.18 +1.08

0.08 P b 0.05 P b 0.05 P b 0.05 0.29

pH 4–7 1 374 1 317 1 330

Myosin regulatory light chain 2 Tropomyosin beta chain Tropomyosin alpha chain

sp|Q0P571| (Bovine) sp|Q5KR48| (Bovine) sp|Q5KR49| (Bovine)

38% 20% 23%

419 431 499

4.91/19114 4.66/32931 4.69/32732

−1.13 −1.14 −1.17

P b 0.05 P b 0.05 P b 0.05

Day

a b

Spots

Primary accession number in the SWISS-PROT database. + is more abundant in NES than in ES and − less abundant in NES than in ES.

296

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