Meat Science 100 (2015) 222–226
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Effect of pulsed electric field on the proteolysis of cold boned beef M. Longissimus lumborum and M. Semimembranosus Via Suwandy a, Alan Carne b, Remy van de Ven c, Alaa El-Din A. Bekhit a,⁎, David L. Hopkins d a
Department of Food Science, University of Otago, PO Box 56, Dunedin, New Zealand Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand NSW Department of Primary Industries, Orange Agricultural Institute, Forest Road, Orange, NSW 2800, Australia d NSW Department of Primary Industries, Centre for Red Meat and Sheep Development, PO Box 129, Cowra, NSW 2794, Australia b c
a r t i c l e
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Article history: Received 26 August 2014 Received in revised form 7 October 2014 Accepted 8 October 2014 Available online 29 October 2014 Keywords: desmin Troponin-T Frequency Voltage Ageing Meat
a b s t r a c t The effects of pulsed electric field (PEF) and ageing (3, 7, 14 and 21 days) on the shear force, protein profile, and post-mortem proteolysis of beef loins (M. Longissimus lumborum, LL) and topsides (M. Semimembranosus, SM) w ere investigated using a range of pulsed electric field treatments [voltages (5 and 10 kV) and frequencies (20, 50, and 90 Hz)]. PEF treatment decreased the shear force of beef LL and SM muscles by up to 19%. The reduction in the shear force in the LL was not affected by the treatment intensity whereas the reduction in the SM was dependent on PEF frequency. PEF treated beef loins showed increased proteolysis, both early post-mortem and during subsequent post-mortem storage reflected by increased degradation of troponin-T and desmin. The most prominent troponin-T degradation was found in samples treated with 5 kV–90 Hz, 10 kV–20 Hz at day 3 and day 7 post-treatment in addition to 10 kV–50 Hz in subsequent post-treatment times. The degradation of desmin in PEF treated beef loins increased with ageing time. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Meat is an important source of micronutrients (e.g. iron, selenium, zinc, vitamins A and B12) and is a rich source of high quality protein. Meat has been consumed widely around the world as a staple food and its consumption is important for optimal human growth and development (Biesalski, 2005; Higgs, 2000; Pereira & Vicente, 2013). Tenderness is considered to be the most important quality attribute for repeat purchasing decisions by the consumer (Bolumar, Enneking, Toepfl, & Heinz, 2013) since other eating quality attributes such as flavour and juiciness can be manipulated by the addition of ingredients during meal preparation. Pulsed electric field technology demonstrated great potential in liquid foods in relation to the inactivation of pathogenic microorganisms, spoilage microorganisms or enzymes associated with quality and safety issues in these foods (Elez-Martinez, Sobrino-Lopez, Soliva-Fortuny, & Martin-Belloso, 2012; Vega-Mercado et al., 1997). Recently, several studies have emerged investigating the use of PEF in solid foods with the aim of modifying their structure for various reasons (e.g. extraction of bioactive compounds or change of physical properties of plant material). However, there have only been very limited studies investigating the application of PEF in muscle foods (O'Dowd, Arimi, Noci, Cronin, &
⁎ Corresponding author. Tel.: +64 3 479 4994. E-mail address: [email protected] (A.E.-D.A. Bekhit).
http://dx.doi.org/10.1016/j.meatsci.2014.10.011 0309-1740/© 2014 Elsevier Ltd. All rights reserved.
Lyng, 2013). The application of PEF to meat may have multiple functions such as enhancing cell permeation by electroporation and consequently tenderness as well as possibly reducing the microbial load which would improve the shelf-life and the safety of the product (Jaeger, Balasa, & Knorr, 2008; Töpfl et al., 2007). Although there are limited studies on the effect of PEF on red meat, there are several possibilities in which PEF technology can accelerate the release of enzymes and the glycolysis process that are needed for early proteolysis generating optimum conditions for meat tenderisation. PEF technology offers the ability to optimize the technology input to provide optimum conditions for various meat cuts and quality upgrade for less tender meat cuts. The use of PEF for improving the quality of fresh beef M. Semitendinosus muscle was investigated by O'Dowd et al. (2013). The authors reported structural changes in the samples treated by PEF (1.1–2.8 kV cm−1, 5–200 Hz, 12.7–226 kJ kg−1) but there was no effect on the tenderness level of the beef M. Semitendinosus muscle. The study investigated the immediate effect of PEF treatment and did not consider the effect of ageing on tenderness. A recent study by Bekhit, van de Ven, Suwandy, Fahri, and Hopkins (2014) found a significant increase in the tenderness of beef Longissimus lumborum and Semimembranosus muscles due to PEF treatment (combinations of voltages (5 and 10 kV) and frequencies (20, 50 and 90 Hz)), but the mechanism of tenderization was not reported. Therefore, the present study aimed to investigate the effect of PEF treatment on the sarcoplasmic and myofibrillar proteins in LL and SM muscles as well as the proteolysis of the protein in beef LL during ageing.
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2. Materials and methods 2.1. Meat Loins (M. Longissimus lumborum) and topsides (M. Semimembranosus) were obtained from two different animal groups, each comprising 6 Hereford steers that had been raised on pasture. The animals were slaughtered by the Alliance Group (Pukeuri plant, Oamaru, New Zealand). The carcases were of grade P2 (prime steers with fat cover 3–10 mm) and the average hot carcass weights were 303.4 ± 23.3 kg and 299.2 ± 13.95 kg, for the loin and the topside animal groups respectively. The loins from both sides were excised at 24 h postmortem, vacuum-packed (VP) and treated within 6 h. The topsides from both sides were excised at 24 h post-mortem. The left topsides were treated at 24 h post-mortem similar to the loins while the right muscles were treated at 3 day post-mortem to examine the effect of PEF treatment on meat at different post-mortem times. 2.2. PEF treatments The samples were cut into blocks of 13 × 8 × 5 cm and randomly allocated to 7 treatment combinations; voltages (5 and 10 kV) and frequencies (20, 50 and 90 Hz) plus a non-treated control. The pulsed electric field treatment was carried out using Elcrack-HPV5 (DIL, Quakenburck, Germany) in batch mode as described in Bekhit et al. (2014) and the meat fibre direction was parallel to the electrodes. The PEF system consisted of a power generator, treatment chamber and an oscilloscope (Model UT2025C, Uni-Trend Group Ltd, Hong Kong, China) was used to monitor the pulse shape used (square wave bipolar). The PEF system has the ability to deliver a wide range of electrical inputs (voltage = 0–25 kV, frequency = 0–1000 Hz and pulse width = 4–32 μs). The samples were sliced into 4 pieces, weighed, vacuum packed and randomly assigned to 4 post-treatment ageing times (3, 7, 14, or 21 days). The samples were stored at 4 °C during ageing. 2.3. pH The pH was measured for each block directly before and after PEF treatment and for each sub-sample after the allocated storage time at 4 °C (i.e. 3, 7, 14 or 21 days). The pH difference from the initial pH before treatment was calculated at various measurement points. 2.4. Shear force The shear force was determined by the MIRINZ tenderometer test after cooking the samples individually in plastic bags immersed in a water bath at 80 °C until they reached an internal temperature of 75 °C (10–14 min) as described in Bekhit et al. (2014) and the values are reported in Newtons. 2.5. Sarcoplasmic and myofibril protein extraction Sacroplasmic and myofibrillar protein fractions were separated after PEF treatment and ageing according to the procedure described by (Han, Morton, Bekhit, & Sedcole, 2009). A 1.00 ± 0.01 gramme of meat was excised from each subsample and was cut into small pieces. A 5 μl aliquot of phenylmethylsulfonyl fluoride (PMSF) solution (17.42 mg of PMSF dissolved in 50 μl of ethanol and then the volume was made to 1 ml with Milli-Q water) and 5 ml homogenisation buffer (100 mM KCl, 2 mM MgCl2, 2 mM EGTA, 1 mM NaN3, 20 mM Na2HPO4, 20 mM NaH2PO4, pH 6.8) were added to the meat sample. The mixture was homogenised for 1 min to achieve fine particles. The mixture was centrifuged (CPR centrifuge, Beckman Coulter, Inc., California, USA) at 2800 × g at 4 °C for 10 min. A 1 ml aliquot of supernatant (sarcoplasmic extract) was transferred to a clean tube and stored at −20 °C until further processing. A 5 μl aliquot of PMSF solution, 5 μl 1% Triton X-100, and
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5 ml washing buffer (100 mM NaCl, 5 mM NaN3) were added to the remaining pellet. The mixture was homogenised for 30 s and centrifuged at 3200 ×g for 10 min at 4 °C. The resultant supernatant was discarded and 5 ml of SDS sample buffer (8.2 ml MilliQ-water, 1.25 ml stacking Tris buffer, 0.3 g SDS, 0.5 ml of β-mercaptoethanol) were added. The sample was homogenised for 30 s, heated at 90 °C for 5 min, and then centrifuged at 3200 ×g at 25 °C for 5 min. The supernatant was transferred to a clean tube and the extraction was repeated twice to make up a total volume of 15 ml supernatant (which was called the myofibrillar fragmentation index, MFI, protein extract). MFI protein extract was stored at −20 °C until further processing. 2.6. 1D-SDS-PAGE gel electrophoresis 1D-SDS-PAGE was used to examine the meat myofibril and sacroplasmic profiles. Myofibrillar and sarcoplasmic protein profiles were obtained according to the procedure described by Ha (2012) with modification. The myofibril and sarcoplasmic protein extracts were thawed and 30 μl aliquots were transferred to 600 μl microfuge tubes. 15 μl Milli-Q water, 17.1 μl BOLT™ sample buffer (4×) and 6.75 μl BOLT™ reducing agent (10 ×) were added to each extract to make a stock sample. The stock samples were heated at 90 °C for 5 min. A 15.3 μl aliquot of each stock sample was loaded onto a 15 well BOLT™ 4-12% Bis-Tris gel. Electrophoresis was performed in BOLT™ MES SDS running buffer (1×) at 164 V for 34 min at room temperature (21 °C ± 2.0). The gels were washed three times in Milli-Q water for 5 min each time and stained overnight in 20 ml Invitrogen SimplyBlue™ SafeStain with gentle shaking, or were further processed for western blotting. Stained gels were de-stained with Milli-Q water and scanned. 2.7. Western blotting Prepared gels were briefly washed in Towbin buffer (standard recipe for desmin transfer: 6.6 g Tris, 28.8 g glycine, 10% (v/v) methanol, made to a total volume of 2 l; for troponin transfer Tween detergent was also added: 6.6 g Tris, 28.8 g glycine, 10% (v/v) methanol and 0.05% (v/v) Tween 20 to a total volume of 2 l). Proteins were electro-transferred to nitrocellulose membrane using a Hoefer electroblot cell at 300 mA for 3 h with cold tap water (12 °C) circulated cooling. The electroblotted gels were stained overnight in 20 ml Invitrogen SimplyBlue™ SafeStain with gentle shaking. After electro-transfer nitrocellulose membranes were briefly washed with Milli-Q water prior to background blocking that was performed by adding 20 ml of 5% (w/v) non-fat milk powder in Tris-buffered saline (TBS) solution containing 0.1% (v/v) TWEEN 20 for 3 h with gentle shaking. Background block solution was discarded and 10 ml milk solution containing primary Ab (either 10 μl troponin or 5 μl desmin) was added to the nitrocellulose membrane and left overnight with gentle agitation at room temperature. Membranes were washed three times in TBS for 10 min each time. A 10 ml fresh milk solution containing 1 μl of secondary Ab was added and incubated for 3 h with gentle shaking at room temperature. Membranes were briefly washed three times in Milli-Q water. The membrane was washed in TBS solution twice for 30 min to remove background from the membrane. A 2.5 ml aliquot of ECL solution 1 [2.5 mM luminol, 1.36 mM p-coumaric acid, 1.5 M Tris (pH 6.8), Milli-Q water] and ECL solution 2 [2.5 ml of 1.5 M Tris (pH 6.8), containing 5 µl of 20% H2O2], Milli-Q water] were added to the membrane briefly and detected using a FUJI imager LAS-3000. 2.8. Statistical analysis The results, including shear force on the loge transformed scale, for SM and LL were analysed separately using linear mixed model (LMM) methods. Included in each LMM as random terms were effects associated with the split-plot nature of the experimental designs, these being
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animals; samples within animals assigned the initial ageing treatments (SM only); subsamples within samples (assigned the PEF treatments); and slices within sub-samples assigned the ageing periods post PEF treatments. Fixed effects included initial ageing period (SM only); PEF treatment; ageing period post PEF treatment; and interactions between these effects. Here the PEF treatment is further separated into frequency x voltage effects. Also included in the model as covariates were the pH of the samples, and for non-control treatments (i.e. frequency and voltage combinations other than (0, 0)) covariates pulse peak energy, pulse peak voltage, pulse peak current, calculated specific energy (kJ/kg), temperature change due to PEF treatment and the sample weight). 3. Results and discussion The processing parameters and the effects of PEF treatments on the changes in the samples pH, conductivity, and temperature, purge loss, cooking loss and modelling of the changes in shear force over ageing are reported in Bekhit et al. (2014). Only the predicted mean shear force values at 3, 7, 14 and 21 days of ageing, corresponding to the protein proteolysis results, are reported below. 3.1. Effect of PEF on the shear force of beef LL and SM muscles Tenderness of both LL and SM muscles were obtained in the present study by shear force measurement. A decrease in shear force value indicates an increase in tenderness. The shear force of the LL muscles was affected by PEF (P = 0.002) and by ageing (P b 0.001) (Fig. 1A). Lower shear force values were found in PEF treated LL samples
compared to control samples at the four ageing times. The standard errors for the predicted means were on average 3.22 (range 3.00–3.78) and 5.38 (range 4.91–6.19) for treated and controls, respectively. The shear force of LL samples was not affected by the treatment intensity. Shear force of SM muscle was decreased by increasing the frequency (P b 0.001) and ageing (P b 0.001) (Fig. 1B). The most significant reduction in the shear force was found in 90 Hz treated SM samples. O'Dowd et al. (2013) reported an increase in myofibrillar fragmentation in PEF treated beef ST muscle but the authors found no effect on beef shear force. The reported increase in myofibillar fragmentation is likely to be due to the result of proteolysis (Ducastaing, Valin, Schollmeyer, & Cross, 1985). However, this increase in myofibillar fragmentation did not translate into a reduction in the shear force as reported in that study. Pulsed electric field causes the generation of cell membrane electroporation which enhances the mobility of intracellular constituents. Therefore, the application of PEF may allow the possibility of earlier activation of biochemical events (e.g. release of calcium from sarcoplasmic reticulum and proteolysis). In the present study, we used an ageing step to examine if proteolysis progression and tenderness will be higher in the PEF treated-samples. 3.2. Effects of PEF treatment and post-treatment ageing on meat sarcoplasmic protein profiles The effects of PEF treatment on the sarcoplasmic proteins at 3, 7, 14 and 21 days of post-treatment of beef LL and SM are shown in Supplementary Fig. 1A and B. The profile of the sarcoplasmic proteins appears not affected by PEF treatment or by ageing, except for the glyceraldehyde phosphate dehydrogenase (GAPDH) 38 kDa band. The GAPDH band disappeared in the control, 5 kV–50 Hz and 10 kV–20 Hz samples at 21 day post-treatment time and at 14 day post-treatment time in 5 kV–20 Hz, 5 kV–90 Hz and 10 kV–50 Hz samples (Supplementary Fig. 1A). The disappearance of the GAPDH band during postmortem ageing was reported by (Bowker, Fahrenholz, Paroczay, & Solomon, 2008) for beef striploin. The intensity of bands N60 kDa in treated samples was decreased as the treatment intensity increased, with either increase in frequency at the same voltage or increase in voltage at the same frequency level, which may be related to increased drip loss and reduced levels of sarcoplasmic proteins. The early disappearance of the 38 kDa band as a result of PEF treatment was clearer in the sarcoplasmic protein profile in SM samples (Supplementary Fig. 1B). 3.3. Effects of PEF treatment and post-treatment ageing on meat myofibrillar protein profiles
Fig. 1. Predicted means for shear force at various ageing storage times (3–20 days) for PEF treated cold boned beef M. Longissimus lumborum (A) and M. Semimembranosus muscles (B). The shear force was determined by the MIRINZ tenderometer test as described in Bekhit et al. (2014) and the values are reported in Newtons.
The myofibrillar protein profiles for LL and SM muscles are shown in Supplementary Fig. 2A and B, respectively. The intensity of the 90 and 110 kDa bands was more intense in the 50 kV–50 Hz, 50 kV–90 Hz, and 10 kV–20 Hz treated samples at the 3 day post-treatment time (Supplementary Fig. 2A). All the treated samples, with the exception of 10 kV–90 Hz samples, had more intense 90 and 110 kDa bands at 7, 14 and 21 day post-treatment times. These bands are believed to originate from the proteolysis of myosin heavy chain (Yates, Dutson, Caldwell, & Carpenter, 1983). The band at 32 kDa was also more intense in the 5 kV–50 Hz, 5 kV–90 Hz and 10 kV–20 Hz treatments than the control at the 3 day post treatment time (Supplementary Fig. 2A). All the treated samples, with the exception of the 10 kV–90 Hz samples, had a more intense 32 kDa band compared with the corresponding controls at 7, 14 and 21 days of PEF post-treatment. A 30 kDa band had the same trend as the 32 kDa band but was far less obvious. The 30 and 32 kDa fragments belong to a group of polypeptides generated in meat termed the 27–32 kDa group (Han et al., 2009; Marino et al., 2013). The 30 kDa fragment is generated from the degradation of troponin-T (Ho, Stromer, & Robson, 1994; Olson & Parrish, 1977) and has been suggested to be an indicator of proteolysis and meat tenderness (Ho et al., 1994; McBride & Parrish, 1977; Yates et al., 1983).
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Fig. 2. Western blot analysis of troponin in meat myofibril extracts of PEF treated and non-treated beef M. Longissimus lumborum muscle at 3, 7, 14, and 21 days post-treatment.
However, the 30 kDa was reported to be present at death in lamb m. Longissimus thoracis et lumborum samples (Hopkins & Thompson, 2001), which was found in beef myofibrillar only when high μ-calpain was present (Geesink & Koohmaraie, 1999) or by use of electrical stimulation (Ducastaing et al., 1985). The 30 kDa band was reported to contribute to about 50 to 70% of the intensity of the 27–32 kDa bands, depending on the cattle breed (Marino et al., 2013). Visually, the intensity of that band in our study was much lower than the 32 kDa band, which may be related to the animal breed used in the present study (Hereford). The degradation of troponin-T was correlated with the shear force of Longissimus dorsi (LD) muscles from Friesian, Podolian, and Romagnola × Podolian crossbred young bulls
(r ranged from 0.62 to 0.81) and the 27–32 kDa bands (r ranged from −0.52 to −0.81) (Marino et al., 2013). 3.4. Effects of PEF treatment on post-mortem proteolysis of cold-boned beef LL muscle The proteolysis of troponin-T and desmin in LL muscles at 3, 7, 14 and 21 days of PEF post-treatment is shown in Figs. 2 and 3, respectively. Significant proteolysis of troponin-T was evident in the 5 kV–90 Hz and 10 kV–20 Hz treated samples compared with the rest of the treatments and the control at 3 days post-treatment. At 7 days post-treatment all PEF treated samples had higher troponin-T proteolysis compared with
Fig. 3. Western blot analysis of desmin in meat myofibril extracts of PEF treated and non-treated beef M. Longissimus lumborum muscle at 3, 7, 14, and 21 days post-treatment.
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control samples, which was most evident in the 5 kV–90 Hz, 10 kV–20 Hz and 10 kV–50 Hz treated samples. All the PEF treated samples, with the exception of the 10 kV–90 Hz samples, had more troponin-T proteolysis compared with control samples at the 14 and 21 days post-treatment times (Fig. 2). Troponin-T forms a complex with tropomyosin and its degradation has been used as a marker of myofibrillar protein degradation in aged beef (Sun et al., 2014). Increased proteolysis of troponin-T has been shown to be promoted under various tenderisation treatments (Claeys, Smet, Balcaen, Raes, & Demeyer, 2004; Han et al., 2009; Ho et al., 1994; McBride & Parrish, 1977) and significantly correlated to shear force values (Marino et al., 2013). Desmin proteolysis was higher in the 5 kV–50 Hz and 10 kV–20 Hz samples compared with controls at 3 and 7 days (Fig. 3). All the PEF treated samples, with the exception of the 10 kV–90 Hz treatment, had more desmin proteolysis compared with controls at 14 and 21 days post-treatment times. Desmin is a major component of the intermediate filaments that associate with the Z-line and its postmortem degradation has been implicated for the loss of myofibrillar integrity (Geesink, Bekhit, & Bickerstaffe, 2000; Ho, Stromer, & Robson, 1996). Thus, desmin degradation is used as a marker of post-mortem proteolysis and meat tenderisation (Koohmaraie & Shackelford, 1991; O'Halloran, Troy, Buckley, & Reville, 1997; Wheeler & Koohmaraie, 1999). Collectively, the above results provide evidence for increased proteolysis, both early post-mortem and during subsequent post-mortem storage, as a result of PEF treatment. This explains the observed lower shear force values found in these treatments. However, these findings do not explain the tenderising effect of high intensity PEF treatment found in the 10 kV–90 Hz. A physical stimulus may be responsible for this effect, i.e. vigorous contractions or rupture, which need to be confirmed by scanning electron microscopy studies in future work.
4. Conclusion The outcome of the present study indicates a promising potential for tenderisation of meat using pulsed electric field treatment (PEF) with a 19% reduction in the shear force of beef M. Longissimus lumborum (LL) and M. semimembranosus (SM) muscles. Western analysis correspondingly shows that there was a faster rate of protein degradation in PEF treated LL muscle than non-treated muscle. The two muscles behave differently towards PEF treatment. For SM muscle, shear force was dependent on the treatment frequency whereas LL muscle did not depend on the treatment frequency. As tenderness of SM muscle was not affected by the post-mortem time treatment, this has the advantage of allowing a greater flexibility in the use of the technology in addition to the other known benefits of the technology (fast treatment and green process).
Conflict of interest The authors declare no conflict of interest.
Acknowledgements The financial support by Meat and Livestock Australia and the Australian Meat Processor Corporation Ltd (project No. A.MQA.0005) is greatly acknowledged. The assistance of the management and staff of the Alliance Group and the team at the Pukeuri plant is acknowledged. The cardiac troponin-T antibody developed by Jim Jung-Ching Lin was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.
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Effect of pulsed electric field on the proteolysis of cold boned beef M. Longissimus lumborum and M. Semimembranosus Via Suwandy a, Alan Carne b, Remy van de Ven c, Alaa El-Din A. Bekhit a,⁎, David L. Hopkins d a
Department of Food Science, University of Otago, PO Box 56, Dunedin, New Zealand Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand NSW Department of Primary Industries, Orange Agricultural Institute, Forest Road, Orange, NSW 2800, Australia d NSW Department of Primary Industries, Centre for Red Meat and Sheep Development, PO Box 129, Cowra, NSW 2794, Australia b c
a r t i c l e
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Article history: Received 26 August 2014 Received in revised form 7 October 2014 Accepted 8 October 2014 Available online 29 October 2014 Keywords: desmin Troponin-T Frequency Voltage Ageing Meat
a b s t r a c t The effects of pulsed electric field (PEF) and ageing (3, 7, 14 and 21 days) on the shear force, protein profile, and post-mortem proteolysis of beef loins (M. Longissimus lumborum, LL) and topsides (M. Semimembranosus, SM) w ere investigated using a range of pulsed electric field treatments [voltages (5 and 10 kV) and frequencies (20, 50, and 90 Hz)]. PEF treatment decreased the shear force of beef LL and SM muscles by up to 19%. The reduction in the shear force in the LL was not affected by the treatment intensity whereas the reduction in the SM was dependent on PEF frequency. PEF treated beef loins showed increased proteolysis, both early post-mortem and during subsequent post-mortem storage reflected by increased degradation of troponin-T and desmin. The most prominent troponin-T degradation was found in samples treated with 5 kV–90 Hz, 10 kV–20 Hz at day 3 and day 7 post-treatment in addition to 10 kV–50 Hz in subsequent post-treatment times. The degradation of desmin in PEF treated beef loins increased with ageing time. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Meat is an important source of micronutrients (e.g. iron, selenium, zinc, vitamins A and B12) and is a rich source of high quality protein. Meat has been consumed widely around the world as a staple food and its consumption is important for optimal human growth and development (Biesalski, 2005; Higgs, 2000; Pereira & Vicente, 2013). Tenderness is considered to be the most important quality attribute for repeat purchasing decisions by the consumer (Bolumar, Enneking, Toepfl, & Heinz, 2013) since other eating quality attributes such as flavour and juiciness can be manipulated by the addition of ingredients during meal preparation. Pulsed electric field technology demonstrated great potential in liquid foods in relation to the inactivation of pathogenic microorganisms, spoilage microorganisms or enzymes associated with quality and safety issues in these foods (Elez-Martinez, Sobrino-Lopez, Soliva-Fortuny, & Martin-Belloso, 2012; Vega-Mercado et al., 1997). Recently, several studies have emerged investigating the use of PEF in solid foods with the aim of modifying their structure for various reasons (e.g. extraction of bioactive compounds or change of physical properties of plant material). However, there have only been very limited studies investigating the application of PEF in muscle foods (O'Dowd, Arimi, Noci, Cronin, &
⁎ Corresponding author. Tel.: +64 3 479 4994. E-mail address: [email protected] (A.E.-D.A. Bekhit).
http://dx.doi.org/10.1016/j.meatsci.2014.10.011 0309-1740/© 2014 Elsevier Ltd. All rights reserved.
Lyng, 2013). The application of PEF to meat may have multiple functions such as enhancing cell permeation by electroporation and consequently tenderness as well as possibly reducing the microbial load which would improve the shelf-life and the safety of the product (Jaeger, Balasa, & Knorr, 2008; Töpfl et al., 2007). Although there are limited studies on the effect of PEF on red meat, there are several possibilities in which PEF technology can accelerate the release of enzymes and the glycolysis process that are needed for early proteolysis generating optimum conditions for meat tenderisation. PEF technology offers the ability to optimize the technology input to provide optimum conditions for various meat cuts and quality upgrade for less tender meat cuts. The use of PEF for improving the quality of fresh beef M. Semitendinosus muscle was investigated by O'Dowd et al. (2013). The authors reported structural changes in the samples treated by PEF (1.1–2.8 kV cm−1, 5–200 Hz, 12.7–226 kJ kg−1) but there was no effect on the tenderness level of the beef M. Semitendinosus muscle. The study investigated the immediate effect of PEF treatment and did not consider the effect of ageing on tenderness. A recent study by Bekhit, van de Ven, Suwandy, Fahri, and Hopkins (2014) found a significant increase in the tenderness of beef Longissimus lumborum and Semimembranosus muscles due to PEF treatment (combinations of voltages (5 and 10 kV) and frequencies (20, 50 and 90 Hz)), but the mechanism of tenderization was not reported. Therefore, the present study aimed to investigate the effect of PEF treatment on the sarcoplasmic and myofibrillar proteins in LL and SM muscles as well as the proteolysis of the protein in beef LL during ageing.
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2. Materials and methods 2.1. Meat Loins (M. Longissimus lumborum) and topsides (M. Semimembranosus) were obtained from two different animal groups, each comprising 6 Hereford steers that had been raised on pasture. The animals were slaughtered by the Alliance Group (Pukeuri plant, Oamaru, New Zealand). The carcases were of grade P2 (prime steers with fat cover 3–10 mm) and the average hot carcass weights were 303.4 ± 23.3 kg and 299.2 ± 13.95 kg, for the loin and the topside animal groups respectively. The loins from both sides were excised at 24 h postmortem, vacuum-packed (VP) and treated within 6 h. The topsides from both sides were excised at 24 h post-mortem. The left topsides were treated at 24 h post-mortem similar to the loins while the right muscles were treated at 3 day post-mortem to examine the effect of PEF treatment on meat at different post-mortem times. 2.2. PEF treatments The samples were cut into blocks of 13 × 8 × 5 cm and randomly allocated to 7 treatment combinations; voltages (5 and 10 kV) and frequencies (20, 50 and 90 Hz) plus a non-treated control. The pulsed electric field treatment was carried out using Elcrack-HPV5 (DIL, Quakenburck, Germany) in batch mode as described in Bekhit et al. (2014) and the meat fibre direction was parallel to the electrodes. The PEF system consisted of a power generator, treatment chamber and an oscilloscope (Model UT2025C, Uni-Trend Group Ltd, Hong Kong, China) was used to monitor the pulse shape used (square wave bipolar). The PEF system has the ability to deliver a wide range of electrical inputs (voltage = 0–25 kV, frequency = 0–1000 Hz and pulse width = 4–32 μs). The samples were sliced into 4 pieces, weighed, vacuum packed and randomly assigned to 4 post-treatment ageing times (3, 7, 14, or 21 days). The samples were stored at 4 °C during ageing. 2.3. pH The pH was measured for each block directly before and after PEF treatment and for each sub-sample after the allocated storage time at 4 °C (i.e. 3, 7, 14 or 21 days). The pH difference from the initial pH before treatment was calculated at various measurement points. 2.4. Shear force The shear force was determined by the MIRINZ tenderometer test after cooking the samples individually in plastic bags immersed in a water bath at 80 °C until they reached an internal temperature of 75 °C (10–14 min) as described in Bekhit et al. (2014) and the values are reported in Newtons. 2.5. Sarcoplasmic and myofibril protein extraction Sacroplasmic and myofibrillar protein fractions were separated after PEF treatment and ageing according to the procedure described by (Han, Morton, Bekhit, & Sedcole, 2009). A 1.00 ± 0.01 gramme of meat was excised from each subsample and was cut into small pieces. A 5 μl aliquot of phenylmethylsulfonyl fluoride (PMSF) solution (17.42 mg of PMSF dissolved in 50 μl of ethanol and then the volume was made to 1 ml with Milli-Q water) and 5 ml homogenisation buffer (100 mM KCl, 2 mM MgCl2, 2 mM EGTA, 1 mM NaN3, 20 mM Na2HPO4, 20 mM NaH2PO4, pH 6.8) were added to the meat sample. The mixture was homogenised for 1 min to achieve fine particles. The mixture was centrifuged (CPR centrifuge, Beckman Coulter, Inc., California, USA) at 2800 × g at 4 °C for 10 min. A 1 ml aliquot of supernatant (sarcoplasmic extract) was transferred to a clean tube and stored at −20 °C until further processing. A 5 μl aliquot of PMSF solution, 5 μl 1% Triton X-100, and
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5 ml washing buffer (100 mM NaCl, 5 mM NaN3) were added to the remaining pellet. The mixture was homogenised for 30 s and centrifuged at 3200 ×g for 10 min at 4 °C. The resultant supernatant was discarded and 5 ml of SDS sample buffer (8.2 ml MilliQ-water, 1.25 ml stacking Tris buffer, 0.3 g SDS, 0.5 ml of β-mercaptoethanol) were added. The sample was homogenised for 30 s, heated at 90 °C for 5 min, and then centrifuged at 3200 ×g at 25 °C for 5 min. The supernatant was transferred to a clean tube and the extraction was repeated twice to make up a total volume of 15 ml supernatant (which was called the myofibrillar fragmentation index, MFI, protein extract). MFI protein extract was stored at −20 °C until further processing. 2.6. 1D-SDS-PAGE gel electrophoresis 1D-SDS-PAGE was used to examine the meat myofibril and sacroplasmic profiles. Myofibrillar and sarcoplasmic protein profiles were obtained according to the procedure described by Ha (2012) with modification. The myofibril and sarcoplasmic protein extracts were thawed and 30 μl aliquots were transferred to 600 μl microfuge tubes. 15 μl Milli-Q water, 17.1 μl BOLT™ sample buffer (4×) and 6.75 μl BOLT™ reducing agent (10 ×) were added to each extract to make a stock sample. The stock samples were heated at 90 °C for 5 min. A 15.3 μl aliquot of each stock sample was loaded onto a 15 well BOLT™ 4-12% Bis-Tris gel. Electrophoresis was performed in BOLT™ MES SDS running buffer (1×) at 164 V for 34 min at room temperature (21 °C ± 2.0). The gels were washed three times in Milli-Q water for 5 min each time and stained overnight in 20 ml Invitrogen SimplyBlue™ SafeStain with gentle shaking, or were further processed for western blotting. Stained gels were de-stained with Milli-Q water and scanned. 2.7. Western blotting Prepared gels were briefly washed in Towbin buffer (standard recipe for desmin transfer: 6.6 g Tris, 28.8 g glycine, 10% (v/v) methanol, made to a total volume of 2 l; for troponin transfer Tween detergent was also added: 6.6 g Tris, 28.8 g glycine, 10% (v/v) methanol and 0.05% (v/v) Tween 20 to a total volume of 2 l). Proteins were electro-transferred to nitrocellulose membrane using a Hoefer electroblot cell at 300 mA for 3 h with cold tap water (12 °C) circulated cooling. The electroblotted gels were stained overnight in 20 ml Invitrogen SimplyBlue™ SafeStain with gentle shaking. After electro-transfer nitrocellulose membranes were briefly washed with Milli-Q water prior to background blocking that was performed by adding 20 ml of 5% (w/v) non-fat milk powder in Tris-buffered saline (TBS) solution containing 0.1% (v/v) TWEEN 20 for 3 h with gentle shaking. Background block solution was discarded and 10 ml milk solution containing primary Ab (either 10 μl troponin or 5 μl desmin) was added to the nitrocellulose membrane and left overnight with gentle agitation at room temperature. Membranes were washed three times in TBS for 10 min each time. A 10 ml fresh milk solution containing 1 μl of secondary Ab was added and incubated for 3 h with gentle shaking at room temperature. Membranes were briefly washed three times in Milli-Q water. The membrane was washed in TBS solution twice for 30 min to remove background from the membrane. A 2.5 ml aliquot of ECL solution 1 [2.5 mM luminol, 1.36 mM p-coumaric acid, 1.5 M Tris (pH 6.8), Milli-Q water] and ECL solution 2 [2.5 ml of 1.5 M Tris (pH 6.8), containing 5 µl of 20% H2O2], Milli-Q water] were added to the membrane briefly and detected using a FUJI imager LAS-3000. 2.8. Statistical analysis The results, including shear force on the loge transformed scale, for SM and LL were analysed separately using linear mixed model (LMM) methods. Included in each LMM as random terms were effects associated with the split-plot nature of the experimental designs, these being
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animals; samples within animals assigned the initial ageing treatments (SM only); subsamples within samples (assigned the PEF treatments); and slices within sub-samples assigned the ageing periods post PEF treatments. Fixed effects included initial ageing period (SM only); PEF treatment; ageing period post PEF treatment; and interactions between these effects. Here the PEF treatment is further separated into frequency x voltage effects. Also included in the model as covariates were the pH of the samples, and for non-control treatments (i.e. frequency and voltage combinations other than (0, 0)) covariates pulse peak energy, pulse peak voltage, pulse peak current, calculated specific energy (kJ/kg), temperature change due to PEF treatment and the sample weight). 3. Results and discussion The processing parameters and the effects of PEF treatments on the changes in the samples pH, conductivity, and temperature, purge loss, cooking loss and modelling of the changes in shear force over ageing are reported in Bekhit et al. (2014). Only the predicted mean shear force values at 3, 7, 14 and 21 days of ageing, corresponding to the protein proteolysis results, are reported below. 3.1. Effect of PEF on the shear force of beef LL and SM muscles Tenderness of both LL and SM muscles were obtained in the present study by shear force measurement. A decrease in shear force value indicates an increase in tenderness. The shear force of the LL muscles was affected by PEF (P = 0.002) and by ageing (P b 0.001) (Fig. 1A). Lower shear force values were found in PEF treated LL samples
compared to control samples at the four ageing times. The standard errors for the predicted means were on average 3.22 (range 3.00–3.78) and 5.38 (range 4.91–6.19) for treated and controls, respectively. The shear force of LL samples was not affected by the treatment intensity. Shear force of SM muscle was decreased by increasing the frequency (P b 0.001) and ageing (P b 0.001) (Fig. 1B). The most significant reduction in the shear force was found in 90 Hz treated SM samples. O'Dowd et al. (2013) reported an increase in myofibrillar fragmentation in PEF treated beef ST muscle but the authors found no effect on beef shear force. The reported increase in myofibillar fragmentation is likely to be due to the result of proteolysis (Ducastaing, Valin, Schollmeyer, & Cross, 1985). However, this increase in myofibillar fragmentation did not translate into a reduction in the shear force as reported in that study. Pulsed electric field causes the generation of cell membrane electroporation which enhances the mobility of intracellular constituents. Therefore, the application of PEF may allow the possibility of earlier activation of biochemical events (e.g. release of calcium from sarcoplasmic reticulum and proteolysis). In the present study, we used an ageing step to examine if proteolysis progression and tenderness will be higher in the PEF treated-samples. 3.2. Effects of PEF treatment and post-treatment ageing on meat sarcoplasmic protein profiles The effects of PEF treatment on the sarcoplasmic proteins at 3, 7, 14 and 21 days of post-treatment of beef LL and SM are shown in Supplementary Fig. 1A and B. The profile of the sarcoplasmic proteins appears not affected by PEF treatment or by ageing, except for the glyceraldehyde phosphate dehydrogenase (GAPDH) 38 kDa band. The GAPDH band disappeared in the control, 5 kV–50 Hz and 10 kV–20 Hz samples at 21 day post-treatment time and at 14 day post-treatment time in 5 kV–20 Hz, 5 kV–90 Hz and 10 kV–50 Hz samples (Supplementary Fig. 1A). The disappearance of the GAPDH band during postmortem ageing was reported by (Bowker, Fahrenholz, Paroczay, & Solomon, 2008) for beef striploin. The intensity of bands N60 kDa in treated samples was decreased as the treatment intensity increased, with either increase in frequency at the same voltage or increase in voltage at the same frequency level, which may be related to increased drip loss and reduced levels of sarcoplasmic proteins. The early disappearance of the 38 kDa band as a result of PEF treatment was clearer in the sarcoplasmic protein profile in SM samples (Supplementary Fig. 1B). 3.3. Effects of PEF treatment and post-treatment ageing on meat myofibrillar protein profiles
Fig. 1. Predicted means for shear force at various ageing storage times (3–20 days) for PEF treated cold boned beef M. Longissimus lumborum (A) and M. Semimembranosus muscles (B). The shear force was determined by the MIRINZ tenderometer test as described in Bekhit et al. (2014) and the values are reported in Newtons.
The myofibrillar protein profiles for LL and SM muscles are shown in Supplementary Fig. 2A and B, respectively. The intensity of the 90 and 110 kDa bands was more intense in the 50 kV–50 Hz, 50 kV–90 Hz, and 10 kV–20 Hz treated samples at the 3 day post-treatment time (Supplementary Fig. 2A). All the treated samples, with the exception of 10 kV–90 Hz samples, had more intense 90 and 110 kDa bands at 7, 14 and 21 day post-treatment times. These bands are believed to originate from the proteolysis of myosin heavy chain (Yates, Dutson, Caldwell, & Carpenter, 1983). The band at 32 kDa was also more intense in the 5 kV–50 Hz, 5 kV–90 Hz and 10 kV–20 Hz treatments than the control at the 3 day post treatment time (Supplementary Fig. 2A). All the treated samples, with the exception of the 10 kV–90 Hz samples, had a more intense 32 kDa band compared with the corresponding controls at 7, 14 and 21 days of PEF post-treatment. A 30 kDa band had the same trend as the 32 kDa band but was far less obvious. The 30 and 32 kDa fragments belong to a group of polypeptides generated in meat termed the 27–32 kDa group (Han et al., 2009; Marino et al., 2013). The 30 kDa fragment is generated from the degradation of troponin-T (Ho, Stromer, & Robson, 1994; Olson & Parrish, 1977) and has been suggested to be an indicator of proteolysis and meat tenderness (Ho et al., 1994; McBride & Parrish, 1977; Yates et al., 1983).
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Fig. 2. Western blot analysis of troponin in meat myofibril extracts of PEF treated and non-treated beef M. Longissimus lumborum muscle at 3, 7, 14, and 21 days post-treatment.
However, the 30 kDa was reported to be present at death in lamb m. Longissimus thoracis et lumborum samples (Hopkins & Thompson, 2001), which was found in beef myofibrillar only when high μ-calpain was present (Geesink & Koohmaraie, 1999) or by use of electrical stimulation (Ducastaing et al., 1985). The 30 kDa band was reported to contribute to about 50 to 70% of the intensity of the 27–32 kDa bands, depending on the cattle breed (Marino et al., 2013). Visually, the intensity of that band in our study was much lower than the 32 kDa band, which may be related to the animal breed used in the present study (Hereford). The degradation of troponin-T was correlated with the shear force of Longissimus dorsi (LD) muscles from Friesian, Podolian, and Romagnola × Podolian crossbred young bulls
(r ranged from 0.62 to 0.81) and the 27–32 kDa bands (r ranged from −0.52 to −0.81) (Marino et al., 2013). 3.4. Effects of PEF treatment on post-mortem proteolysis of cold-boned beef LL muscle The proteolysis of troponin-T and desmin in LL muscles at 3, 7, 14 and 21 days of PEF post-treatment is shown in Figs. 2 and 3, respectively. Significant proteolysis of troponin-T was evident in the 5 kV–90 Hz and 10 kV–20 Hz treated samples compared with the rest of the treatments and the control at 3 days post-treatment. At 7 days post-treatment all PEF treated samples had higher troponin-T proteolysis compared with
Fig. 3. Western blot analysis of desmin in meat myofibril extracts of PEF treated and non-treated beef M. Longissimus lumborum muscle at 3, 7, 14, and 21 days post-treatment.
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control samples, which was most evident in the 5 kV–90 Hz, 10 kV–20 Hz and 10 kV–50 Hz treated samples. All the PEF treated samples, with the exception of the 10 kV–90 Hz samples, had more troponin-T proteolysis compared with control samples at the 14 and 21 days post-treatment times (Fig. 2). Troponin-T forms a complex with tropomyosin and its degradation has been used as a marker of myofibrillar protein degradation in aged beef (Sun et al., 2014). Increased proteolysis of troponin-T has been shown to be promoted under various tenderisation treatments (Claeys, Smet, Balcaen, Raes, & Demeyer, 2004; Han et al., 2009; Ho et al., 1994; McBride & Parrish, 1977) and significantly correlated to shear force values (Marino et al., 2013). Desmin proteolysis was higher in the 5 kV–50 Hz and 10 kV–20 Hz samples compared with controls at 3 and 7 days (Fig. 3). All the PEF treated samples, with the exception of the 10 kV–90 Hz treatment, had more desmin proteolysis compared with controls at 14 and 21 days post-treatment times. Desmin is a major component of the intermediate filaments that associate with the Z-line and its postmortem degradation has been implicated for the loss of myofibrillar integrity (Geesink, Bekhit, & Bickerstaffe, 2000; Ho, Stromer, & Robson, 1996). Thus, desmin degradation is used as a marker of post-mortem proteolysis and meat tenderisation (Koohmaraie & Shackelford, 1991; O'Halloran, Troy, Buckley, & Reville, 1997; Wheeler & Koohmaraie, 1999). Collectively, the above results provide evidence for increased proteolysis, both early post-mortem and during subsequent post-mortem storage, as a result of PEF treatment. This explains the observed lower shear force values found in these treatments. However, these findings do not explain the tenderising effect of high intensity PEF treatment found in the 10 kV–90 Hz. A physical stimulus may be responsible for this effect, i.e. vigorous contractions or rupture, which need to be confirmed by scanning electron microscopy studies in future work.
4. Conclusion The outcome of the present study indicates a promising potential for tenderisation of meat using pulsed electric field treatment (PEF) with a 19% reduction in the shear force of beef M. Longissimus lumborum (LL) and M. semimembranosus (SM) muscles. Western analysis correspondingly shows that there was a faster rate of protein degradation in PEF treated LL muscle than non-treated muscle. The two muscles behave differently towards PEF treatment. For SM muscle, shear force was dependent on the treatment frequency whereas LL muscle did not depend on the treatment frequency. As tenderness of SM muscle was not affected by the post-mortem time treatment, this has the advantage of allowing a greater flexibility in the use of the technology in addition to the other known benefits of the technology (fast treatment and green process).
Conflict of interest The authors declare no conflict of interest.
Acknowledgements The financial support by Meat and Livestock Australia and the Australian Meat Processor Corporation Ltd (project No. A.MQA.0005) is greatly acknowledged. The assistance of the management and staff of the Alliance Group and the team at the Pukeuri plant is acknowledged. The cardiac troponin-T antibody developed by Jim Jung-Ching Lin was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.
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